Daria J. Hazuda

Integrase is essential for human immunodeficiency virus–type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.

Although combination therapy for HIV infection represents a triumph for modern medicine, chronic suppressive therapy is required to contain persistent infection in reservoirs such as latently infected CD4 + lymphocytes and cells of the macrophage-monocyte lineage. Despite its success, chronic suppressive therapy is limited by its cost, the requirement of lifelong adherence, and the unknown effects of long-term treatment. This review discusses our current understanding of suppressive antiretroviral therapy, the latent viral reservoir, and the needs for and challenges of attacking this reservoir to achieve a cure.

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

The most significant advance in the medical management of HIV-1 infection has been the treatment of patients with antiviral drugs, which can suppress HIV-1 replication to undetectable levels.The discovery of HIV-1 as the causative agent of AIDS together with an everincreasing understanding of the virus replication cycle have been instrumental in this effort by providing researchers with the knowledge and tools required to prosecute drug discovery efforts focused on targeted inhibition with specific pharmacological agents.To date, an arsenal of 24 Food and Drug Administration (FDA)-approved drugs are available for treatment of HIV-1 infections.These drugs are distributed into six distinct classes based on their molecular mechanism and resistance profiles: (1) nucleoside-analog reverse transcriptase inhibitors (NNRTIs), (2) non-nucleoside reverse transcriptase inhibitors (NNRTIs), (3) integrase inhibitors, (4) protease inhibitors (PIs), ( 5) fusion inhibitors, and (6) coreceptor antagonists.In this article, we will review the basic principles of antiretroviral drug therapy, the mode of drug action, and the factors leading to treatment failure (i.e., drug resistance).

Raltegravir (MK-0518) is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase active against HIV-1 susceptible or resistant to older antiretroviral drugs.We conducted two identical trials in different geographic regions to evaluate the safety and efficacy of raltegravir, as compared with placebo, in combination with optimized background therapy, in patients infected with HIV-1 that has triple-class drug resistance in whom antiretroviral therapy had failed. Patients were randomly assigned to raltegravir or placebo in a 2:1 ratio.In the combined studies, 699 of 703 randomized patients (462 and 237 in the raltegravir and placebo groups, respectively) received the study drug. Seventeen of the 699 patients (2.4%) discontinued the study before week 16. Discontinuation was related to the study treatment in 13 of these 17 patients: 7 of the 462 raltegravir recipients (1.5%) and 6 of the 237 placebo recipients (2.5%). The results of the two studies were consistent. At week 16, counting noncompletion as treatment failure, 355 of 458 raltegravir recipients (77.5%) had HIV-1 RNA levels below 400 copies per milliliter, as compared with 99 of 236 placebo recipients (41.9%, P<0.001). Suppression of HIV-1 RNA to a level below 50 copies per milliliter was achieved at week 16 in 61.8% of the raltegravir recipients, as compared with 34.7% of placebo recipients, and at week 48 in 62.1% as compared with 32.9% (P<0.001 for both comparisons). Without adjustment for the length of follow-up, cancers were detected in 3.5% of raltegravir recipients and in 1.7% of placebo recipients. The overall frequencies of drug-related adverse events were similar in the raltegravir and placebo groups.In HIV-infected patients with limited treatment options, raltegravir plus optimized background therapy provided better viral suppression than optimized background therapy alone for at least 48 weeks. (ClinicalTrials.gov numbers, NCT00293267 and NCT00293254.)

Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.

To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.

We evaluated the efficacy of raltegravir and the development of viral resistance in two identical trials involving patients who were infected with human immunodeficiency virus type 1 (HIV-1) with triple-class drug resistance and in whom antiretroviral therapy had failed.

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.ClinicalTrials.gov NCT01365065.

We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an "encyclopedia" of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Transcription factor A from immature Xenopus oocytes is found associated with 5 S RNA in a 7 S nucleoprotein complex. Atomic absorption analysis of EDTA-dialyzed 7 S particles reveals 2 mol of zinc/mol of particle. Factor A obtained from EDTA-dialyzed particles binds specifically to the 5 S RNA gene as determined by DNase I footprinting. Factor A alone, obtained by RNase digestion of the 7 S particle, contains zinc when dialyzed in the absence of EDTA. However, the zinc bound to free factor A is removed by dialysis against a buffer containing EDTA. The apoprotein does not bind to the 5 S RNA gene. Inhibition of footprinting is also effected by addition of EDTA to factor A without prolonged dialysis. Under these conditions, specific DNA binding ability is restored following addition of zinc. 1,10-Phenanthroline also inhibits binding of factor A to the intragenic control region of the 5 S RNA gene. In addition, this reagent specifically inhibits factor A-dependent synthesis of 5 S RNA but not factor A-independent tRNA synthesis in a HeLa cell in vitro transcription system.

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.In suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Research toward a cure for human immunodeficiency virus type 1 (HIV-1) infection has joined prevention and treatment efforts in the global public health agenda. A major approach to HIV eradication envisions antiretroviral suppression, paired with targeted therapies to enforce the expression of viral antigen from quiescent HIV-1 genomes, and immunotherapies to clear latent infection. These strategies are targeted to lead to viral eradication--a cure for AIDS. Paired testing of latency reversal and clearance strategies has begun, but additional obstacles to HIV eradication may emerge. Nevertheless, there is reason for optimism that advances in long-acting antiretroviral therapy and HIV prevention strategies will contribute to efforts in HIV cure research and that the implementation of these efforts will synergize to markedly blunt the effect of the HIV pandemic on society.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

Abstract Natural products represent a rich reservoir of small molecule drug candidates utilized as antimicrobial drugs, anticancer therapies, and immunomodulatory agents. These molecules are microbial secondary metabolites synthesized by co-localized genes termed Biosynthetic Gene Clusters (BGCs). The increase in full microbial genomes and similar resources has led to development of BGC prediction algorithms, although their precision and ability to identify novel BGC classes could be improved. Here we present a deep learning strategy (DeepBGC) that offers reduced false positive rates in BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing machine-learning tools. We supplemented this with random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable putative BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a major addition to in-silico BGC identification.

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption.

Abstract The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2′-<i>C</i>-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-α results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2′-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2′-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

We describe the efficacy of L-870812, an inhibitor of HIV-1 and SIV integrase, in rhesus macaques infected with the simian-human immunodeficiency virus (SHIV) 89.6P. When initiated before CD4 cell depletion, L-870812 therapy mediated a sustained suppression of viremia, preserving CD4 levels and permitting the induction of virus-specific cellular immunity. L-870812 was also active in chronic infection; however, the magnitude and durability of the effect varied in conjunction with the pretreatment immune response and viral load. These studies demonstrate integrase inhibitor activity in vivo and suggest that cellular immunity facilitates chemotherapeutic efficacy in retroviral infections.

Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.

We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).

ABSTRACT Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2′- C -methyl-adenosine and 2′- C -methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2′- C -methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5′-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2′- C -methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2′- C -methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2′- C -methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.

Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4(+) T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

Objective: Raltegravir (MK-0518) belongs to the new class of HIV integrase inhibitors. To date, there have been no reports investigating the potential for differential effects on viral dynamics with integrase inhibitors relative to current antiretroviral drugs. Methods: Patients in this phase II study (P004) were antiretroviral treatment naive. Part 1 of this study compared monotherapy with raltegravir (100 mg, 200 mg, 400 mg, or 600 mg twice daily) with placebo over 10 days. In part 2, patients were enrolled for 48 weeks of combination therapy, with randomization to one of the four dosages of raltegravir or to efavirenz, in addition to tenofovir and lamivudine. Mathematical models were used to investigate processes underlying viral dynamics. Results: From day 15 through to day 57, individuals in the raltegravir arm were significantly more likely to have HIV RNA < 50 copies/ml (P ≤ 0.047). Plasma viral loads were 70% lower at initiation of second-phase decay for individuals taking raltegravir than for those taking efavirenz (P < 0.0001). This challenges the current hypothesis that second-phase virus originates from infected long-lived cells, as an integrase inhibitor should not impact on viral production from this cell population. Mathematical modeling supported two hypotheses as consistent with these observations: (i) that second-phase virus arises from cells newly infected by long-lived infected cells and (2) that it arises from activation of latently infected cells with full-length unintegrated HIV DNA. Conclusions: These observations challenge the current understanding of HIV-1 turnover and compartmentalization. They also indicate the promise of this new integrase inhibitor raltegravir.

ABSTRACT Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4 + T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4 + T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4 + T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

ADVERTISEMENT RETURN TO ISSUELetterNEXT4-Aryl-2,4-dioxobutanoic Acid Inhibitors of HIV-1 Integrase and Viral Replication in CellsJohn S. Wai, Melissa S. Egbertson, Linda S. Payne, Thorsten E. Fisher, Mark W. Embrey, Lekhanh O. Tran, Jeffrey Y. Melamed, H. Marie Langford, James P. Guare,, Linghang Zhuang, Vanessa E. Grey, Joseph P. Vacca, M. Katharine Holloway, Adel M. Naylor-Olsen, Daria J. Hazuda, Peter J. Felock, Abigail L. Wolfe, Kara A. Stillmock, William A. Schleif, Lori J. Gabryelski, and Steven D. YoungView Author Information Departments of Medicinal Chemistry, Molecular Systems, and Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486 Cite this: J. Med. Chem. 2000, 43, 26, 4923–4926Publication Date (Web):November 28, 2000Publication History Received19 April 2000Published online28 November 2000Published inissue 1 December 2000https://doi.org/10.1021/jm000176bCopyright © 2000 American Chemical SocietyRequest reuse permissions Your access to this publication has been provided by Learn MoreArticle Views1526Altmetric-Citations171LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (57 KB) open URLGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Cell culture,DNA replication,Genetics,Inhibition,Inhibitors Get e-Alerts

Background Twice-daily raltegravir with once-daily tenofovir-emtricitabine is an effective initial antiretroviral regimen for patients with HIV-1. On the basis of pharmacokinetic data suggesting efficacy of once-daily raltegravir and because adherence is often improved with once-daily dosing, we aimed to compare these dosing schedules. Methods In our international, double-blind, randomised, phase 3 non-inferiority study, we enrolled antiretroviral-naive patients with HIV RNA loads of more than 5000 copies per mL and no baseline resistance to tenofovir or emtricitabine at 83 centres worldwide. We randomly allocated patients (1:1) by use of a computer-generated sequence to receive raltegravir once daily (two 400 mg tablets taken together every 24 h), or twice daily (one 400 mg tablet every 12 h), both in combination with once-daily co-formulated tenofovir 300 mg plus emtricitabine 150 mg. The primary outcome was virological response at 48 weeks (viral RNA loads <50 copies per mL) in patients who received at least one dose of study drug, counting non-completers as failure. We assessed non-inferiority in terms of the proportion of patients in both treatment groups who achieved the primary outcome, with a non-inferiority margin of −10%. This study is registered with ClinicalTrials.gov, number NCT00745823. Findings From Oct 15, 2008, to Nov 2, 2009, we randomly allocated 775 patients, of whom 382 (99%) of 386 patients in the once-daily group and 388 (99%) of 389 in the twice-daily group received at least one dose of study drug. At baseline, 304 (39%) of 770 treated patients had viral loads of more than 100 000 copies per mL and 188 (24%) had CD4 cell counts of fewer than 200 cells per μL. 318 (83%) of 382 patients in the once-daily group had virological response compared with 343 (89%) of 386 in the twice-daily group (difference −5·7%, 95% CI −10·7 to −0·83; p=0·044). Serious adverse events were reported in 26 (7%) of 382 once-daily recipients and 40 (10%) of 388 twice-daily recipients, and adverse events leading to discontinuation occurred in four (1%) patients in each group. Interpretation Despite high response rates with both regimens, once-daily raltegravir cannot be recommended in place of twice-daily dosing. Funding Merck.

Synthesis of influenza virus mRNA is primed by capped and methylated (cap 1, m7GpppXm) RNAs which the virus derives by endonucleolytic cleavage from RNA polymerase II transcripts in host cells. The conserved nature of the endonucleolytic processing provides a unique target for the development of antiviral agents for influenza viruses. A series of 4-substituted 2,4-dioxobutanoic acid compounds has been identified as selective inhibitors of this activity in both influenza A and B viruses. These inhibitors exhibited 50% inhibitory concentrations in the range of 0.2 to 29.0 microM for cap-dependent influenza virus transcription and had no effect on the activity of other viral and cellular polymerases when tested at 100- to 500-fold higher concentrations. The compounds did not inhibit the initiation or elongation of influenza virus mRNA synthesis but specifically inhibited the cleavage of capped RNAs by the influenza virus endonuclease and were not inhibitory to the activities of other nucleases. Additionally, the compounds specifically inhibited replication of influenza A and B viruses in cell culture with potencies comparable to the 50% inhibitory concentrations obtained for transcription.

The human immunodeficiency virus type 1 (HIV-1) integrase mutations N155H and Q148R(H)(K) that reduce susceptibility to the integrase inhibitor raltegravir have been identified in patients failing treatment regimens containing raltegravir. Whether these resistance mutations occur individually or in combination within a single virus genome has not been defined, nor do we fully understand the impact of these primary mutations and other secondary mutations on raltegravir susceptibility and viral replication capacity. To address these important questions, we investigated the raltegravir susceptibility and replication capacity of viruses containing mutations at positions 155 and 148 separately or in combination with secondary mutations selected in subjects failing treatment regimens containing raltegravir. Clonal analysis demonstrated that N155H and Q148R(H)(K) occur independently, not in combination. Viruses containing a Q148R(H)(K) mutation generally displayed larger reductions in raltegravir susceptibility than viruses with an N155H mutation. Analysis of site-directed mutants indicated that E92Q in combination with N155H resulted in a higher level of resistance to raltegravir than N155H alone. Viruses containing a Q148R(H) mutation together with a G140S mutation were more resistant to raltegravir than viruses containing a Q148R(H) mutation alone; however, viruses containing G140S and Q148K were more susceptible to raltegravir than viruses containing a Q148K mutation alone. Both N155H and Q148R(H)(K) mutations reduced the replication capacity, while the addition of secondary mutations either improved or reduced the replication capacity depending on the primary mutation. This study demonstrates distinct genetic pathways to resistance in subjects failing raltegravir regimens and defines the effects of primary and secondary resistance mutations on raltegravir susceptibility and replication capacity.

A family of histone deacetylases (HDACs) mediates chromatin remodeling, and repression of gene expression. Deacetylation of histones within the HIV-1 long terminal repeat (LTR) by HDACs plays a key role in the maintenance of latency, whereas acetylation of histones about the LTR is linked to proviral expression and escape of HIV from latency. Global HDAC inhibition may adversely affect host gene expression, leading to cellular toxicities. Potent inhibitors selective for HDACs that maintain LTR repression could be ideal antilatency therapeutics.We investigated the ability of selective HDAC inhibitors to de-repress the HIV-1 LTR in both a cell line model of latency and in resting CD4 T cells isolated from patients who were aviremic on antiretroviral therapy (ART).We found that inhibition of class I HDACs increased acetylation of histones at the LTR, but that LTR chromatin was unaffected by class II HDAC inhibitors. In a latently infected cell line, inhibitors selective for class I HDACs were more efficient activators of the LTR than inhibitors that target class II HDACs. Class I HDAC inhibitors were strikingly efficient inducers of virus outgrowth from resting CD4 T cells of aviremic patients, whereas HIV was rarely recovered from patient's cells exposed to class II HDAC inhibitors.Further development of selective HDAC inhibitors as part of a clinical strategy to target persistent HIV infection is warranted.

ABSTRACT The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.

HIV infection results in gastrointestinal (GI) tract damage, microbial translocation, and immune activation, which are not completely ameliorated with suppression of viremia by antiretroviral (ARV) therapy. Furthermore, increased morbidity and mortality of ARV-treated HIV-infected individuals is associated with these dysfunctions. Thus, to enhance GI tract physiology, we treated SIV-infected pigtail macaques with ARVs, probiotics, and prebiotics or with ARVs alone. This synbiotic treatment resulted in increased frequency and functionality of GI tract APCs, enhanced reconstitution and functionality of CD4+ T cells, and reduced fibrosis of lymphoid follicles in the colon. Thus, ARV synbiotic supplementation in HIV-infected individuals may improve GI tract immunity and thereby mitigate inflammatory sequelae, ultimately improving prognosis.

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC 50 ) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC 95 ) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC 95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of &lt;3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials.

Abstract Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N -methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total HIV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.

ABSTRACT Doravirine (DOR, formerly known as MK-1439) is a human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is currently in phase 2b clinical trials. In vitro resistance selection of subtype B virus (MT4-green fluorescent protein [GFP] cells), as well as subtype A and C viruses (MT4-GFP/CCR5 cells) was conducted with DOR, rilpivirine (RPV), and efavirine (EFV) under low-multiplicity-of-infection conditions in a 96-well format. Resistance selection was performed with escalating concentrations of the NNRTIs ranging from the 95% effective concentration (1× EC 95 ) to 1,000× EC 95 in the presence of 10% fetal bovine serum. In the resistance selection of subtype B virus with DOR, a V106A mutant virus led to two mutation pathways, followed by the emergence separately of either F227L or L234I. In the resistance selection of subtype A and C viruses, similar mutation development pathways were detected, in which a V106A or V106M mutant was also the starting virus in the pathways. Mutations that are commonly associated with RPV and EFV in clinical settings were also identified in subtype B viruses such as the E138K and K103N mutants, respectively, in this in vitro resistance selection study. The susceptibility of subtype B mutant viruses selected by DOR, RPV, and EFV to NNRTIs was evaluated. Results suggest that mutant viruses selected by DOR are susceptible to RPV and EFV and mutants selected by RPV and EFV are susceptible to DOR. When the replication capacity of the V106A mutant was compared with that of the wild-type (WT) virus, the mutant virus was 4-fold less fit than the WT virus.

Doravirine (DOR), which is currently in a phase 3 clinical trial, is a novel human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI). DOR exhibits potent antiviral activity against wild-type virus and K103N, Y181C, and K103N/Y181C mutant viruses, with 50% inhibitory concentrations (IC50s) of 12, 21, 31, and 33 nM, respectively, when measured in 100% normal human serum (NHS). To assess the potential for DOR to suppress NNRTI-associated and rilpivirine (RPV)-specific mutants at concentrations achieved in the clinic setting, inhibitory quotients (IQs) were calculated by determining the ratio of the clinical trough concentration over the antiviral IC50for each virus with DOR and RPV and efavirenz (EFV). DOR displayed IQs of 39, 27, and 25 against the K103N, Y181C, and K103N/Y181C mutants, respectively. In contrast, RPV exhibited IQs of 4.6, 1.4, and 0.8, and EFV showed IQs of 2.5, 60, and 1.9 against these viruses, respectively. DOR also displayed higher IQs than those of RPV and EFV against other prevalent NNRTI-associated mutants, with the exception of Y188L. Both DOR and EFV exhibited higher IQs than RPV when analyzed with RPV-associated mutants. Resistance selections were conducted with K103N, Y181C, G190A, and K103N/Y181C mutants at clinically relevant concentrations of DOR, RPV, and EFV. No viral breakthrough was observed with DOR, whereas breakthrough viruses were readily detected with RPV and EFV against Y181C and K103N viruses, respectively. These data suggest that DOR should impose a higher barrier to the development of resistance than RPV and EFV at the concentrations achieved in the clinic setting.

By performing phenotypic analysis of latency reversal in CD4 + T cells from virally suppressed individuals, we identify the T EM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4 + T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4 + T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of T EM -associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4 + T cells.

Significance The urgency of curbing the COVID-19 pandemic has motivated many investigators to pivot their research to understand the basic biology of SARS-CoV-2 and to search for new pharmaceutical compounds for potential COVID-19 treatment. However, most SARS-CoV-2 studies require biosafety level 3 facilities, which are in high demand, costly, and difficult to access. To overcome these limitations, we engineered a SARS-CoV-2 replicon, which is a modified virus subgenome capable of self-replicating without producing infectious virus, allowing the viral replication to be studied in a conventional biomedical laboratory setting. The replicon system also provides a valuable tool to screen and test antiviral compounds in biologically relevant cells. Successful implementation of the technology will accelerate the development of effective treatment for SARS-CoV-2 infection.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1- and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1⁺^/^–-PS2^–^/^– and PS1^–^/^–PS2⁺^/⁺ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by <i>in vitro</i> transcription and translation. Cellular membranes from wild type, PS1^–^/^–PS2⁺^/⁺, and PS1⁺^/^–-PS2^–^/^– but not PS1^–^/^–PS2^–^/^– BD cells generated the Aβ40 and Aβ42 products from the C100FLAG substrate. PS1-associated γ-secretase displays considerably higher specific activity than PS2-associated γ-secretase. Moreover, the PS1⁺^/^–PS2^–^/^– BD cells and corresponding membranes exhibited much higher γ-secretase activity as compared with other BD cells and membranes. The PS1-mediated γ-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active site-directed γ-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active γ-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its γ-secretase activity. Furthermore, the PS1 γ-secretase complex and the PS2 γ-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent γ-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 γ-secretase complex and the PS2 γ-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes.

Integration is an essential step in HIV replication and is catalyzed by an enzyme called integrase. We have isolated equisetin (1a), and a novel opposite stereochemical homolog, phomasetin (2a), from Fusarium heterosporum and a Phoma sp. respectively. They inhibit the invitro recombinant integrase enzyme with IC50 values of 7–20 μM. Unlike known inhibitors, these compounds also inhibit the integration reactions catalyzed by preintegration complexes isolated from HIV-1 infected cells.

Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC₅₀ = 3.2 μm) but has no effect on DNA polymerization (IC₅₀ > 50 μm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the α-C helix from <i>Escherichia coli</i> RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn²⁺implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro . D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a “hot spot” for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

A series of 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists containing a variety of fused heterocycles at the 4-position of the piperidine side chain has been discovered, which are orally bioavailable with potent anti-HIV activity.

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are highly specific and potent allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase.NNRTIs inhibit reverse transcription in a substrate lengthdependent manner in biochemical assays and in cell-based HIV-1 replication assays, suggesting a stochastic inhibitory mechanism.Surprisingly, we observed that NNRTIs potently inhibited plus-strand initiation in vitro under conditions in which little or no inhibition of minus-strand DNA synthesis was observed.In assays that recapitulated the initiation of plus-strand DNA synthesis, greater inhibition was observed with an RNA PPT primer than with a DNA primer of corresponding sequence and with wild-type reverse transcriptase but not with NNRTI-resistant enzymes.Structural elements that dictate sensitivity to NNRTIs were revealed using modified plus-strand initiation substrates.The data presented here suggest that specific inhibition of plus-strand initiation may be an important mechanism by which NNRTIs block HIV-1 replication.

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimer9s disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC₅₀ ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC₅₀ of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to approximately 13% of wt levels and increased resistance to MK-2048 to approximately 8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg(2+) binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2'-C-methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC(50)) of 0.3 microM (EC(90) = 1.3 microM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log(10) IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log(10), and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.

We investigated the frequency of RAVs among patients failing to achieve SVR in two clinical trials. We also investigated the impact of interferon responsiveness on RAVs and specific baseline RAVs relationship with boceprevir treatment failure.Data are from 1020 patients enrolled into either SPRINT-2 or RESPOND-2; patients received a 4-week PR lead-in prior to receiving boceprevir or placebo. RAVs were analyzed via population-based sequence analysis of the NS3 protease gene (success rate of >90% at a virus level of ≥ 10,000IU/mL) RESULTS: The high SVR rate in patients who received boceprevir resulted in a low rate of RAVs; 7% was detected at baseline in all patients, which rose to 15% after treatment. However, RAVs were detected in 53% of patients that failed to achieve SVR, which declined to 22.8% 6-14 months following cessation of boceprevir therapy. Baseline RAVs alone were not predictive of virologic outcome; poor interferon responsiveness was highly predictive of non-SVR. RAVs were more frequently detected in poor interferon responders.We detected no association between the presence of baseline amino acid variants at boceprevir resistance-associated loci and outcome in the context of good IFN response.

Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3 bispecific antibody. These findings extend beyond classical nucleic acid endpoints, which are confounded by the predominance of mutated, defective proviruses and, of paramount importance, enable assessment of cells making HIV protein that can now be targeted by immunological approaches.

The latent reservoir in resting CD4(+) T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents.Here, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4(+) T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4(+) T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells.We have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Shingles is a painful, blistering rash caused by reactivation of latent varicella-zoster virus (VZV) and most frequently occurs in elderly and immunocompromised individuals. Currently, two approved vaccines for the prevention of shingles are on the market, a live attenuated virus vaccine ZOSTAVAX® (Merck & Co., Inc., Kenilworth, NJ, USA) and an AS01B adjuvanted subunit protein vaccine Shingrix™ (Glaxo Smith Kline, Rockville, MD, USA). Human clinical immunogenicity and vaccine efficacy data is available for these two benchmark vaccines, offering a unique opportunity for comparative analyses with novel vaccine platforms and animal model translatability studies. The studies presented here utilized non-human primates (NHP) to evaluate humoral and cellular immune response by three vaccine modalities: the new platform of lipid nanoparticle (LNP) formulated mRNA encoding VZV gE antigen (VZV gE mRNA/LNP) as compared with well-established platforms of live attenuated VZV (VZV LAV) and adjuvanted VZV gE subunit protein (VZV gE protein/adjuvant). The magnitude of response to vaccination with a single 100–200 μg mRNA dose or two 50 μg mRNA doses of VZV gE mRNA/LNP were comparable to two 50 μg protein doses of VZV gE protein/adjuvant, suggesting the VZV gE mRNA/LNP platform has the potential to elicit a robust immune response, and both modalities generated markedly higher responses than VZV LAV. Additionally, the slopes of decay for VZV-specific antibody titers were roughly similar across all three vaccines, indicating the magnitude of peak immunogenicity was the driving force in determining immune response longevity. Finally, vaccine-induced immunogenicity with VZV LAV and VZV gE protein/adjuvant in NHP closely resembled human clinical trials immune response data for ZOSTAVAX® and Shingrix™, helping to validate NHP as an appropriate preclinical model for evaluating these vaccines.

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

Covering: up to the end of 2020. The machine learning field can be defined as the study and application of algorithms that perform classification and prediction tasks through pattern recognition instead of explicitly defined rules. Among other areas, machine learning has excelled in natural language processing. As such methods have excelled at understanding written languages (e.g. English), they are also being applied to biological problems to better understand the "genomic language". In this review we focus on recent advances in applying machine learning to natural products and genomics, and how those advances are improving our understanding of natural product biology, chemistry, and drug discovery. We discuss machine learning applications in genome mining (identifying biosynthetic signatures in genomic data), predictions of what structures will be created from those genomic signatures, and the types of activity we might expect from those molecules. We further explore the application of these approaches to data derived from complex microbiomes, with a focus on the human microbiome. We also review challenges in leveraging machine learning approaches in the field, and how the availability of other "omics" data layers provides value. Finally, we provide insights into the challenges associated with interpreting machine learning models and the underlying biology and promises of applying machine learning to natural product drug discovery. We believe that the application of machine learning methods to natural product research is poised to accelerate the identification of new molecular entities that may be used to treat a variety of disease indications.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies. In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described. These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity. The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid. These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M. The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.

A macrocyclic inhibitor of beta-secretase was designed by covalently cross-linking the P1 and P3 side chains of an isophthalamide-based inhibitor. Macrocyclization resulted in significantly improved potency and physical properties when compared to the initial lead structures. More importantly, these macrocyclic inhibitors also displayed in vivo amyloid lowering when dosed in a murine model.

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.

We describe the discovery and optimization of tertiary carbinamine derived inhibitors of the enzyme β-secretase (BACE-1). These novel non-transition-state-derived ligands incorporate a single primary amine to interact with the catalytic aspartates of the target enzyme. Optimization of this series provided inhibitors with intrinsic and functional potency comparable to evolved transition state isostere derived inhibitors of BACE-1.

The first integrase inhibitor licensed to treat HIV-1 infection was approved in late 2007, more than a decade after the introduction of the first inhibitors of the HIV-1 reverse transcriptase and protease. The unique biochemical and molecular mechanism of action of this novel class of antiretroviral drugs is the fundamental basis for their activity in treating multidrug-resistant HIV-1 infection and is important for understanding both the cellular and in vivo pharmacology and metabolism of these agents. In addition, available pharmacokinetic and drug interaction data for raltegravir and elvitegravir, the two integrase inhibitors that are the most advanced in clinical development to date, are reviewed.

Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC95 of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.

Purpose of review Integrase strand transfer inhibitors are the most recent class of antiretroviral agents to be introduced into clinical practice. This review describes the discovery of the first inhibitors and insights into their distinct mechanism of action with potential translational implications. Recent findings HIV replication depends on the successful integration of the viral genetic material into the host cell chromosome. The virally encoded enzyme integrase mediates both the DNA cutting and strand transfer or DNA joining steps which are required for this process. Understanding the mechanistic aspects of integration was critical for the initial discovery of integrase strand transfer inhibitors and the advancement of clinical candidates. The recent adoption of these inhibitors into clinic practice has now proven the therapeutic utility of the class. Integrase inhibitors are characterized by a more rapid decrease in viral load in HIV-1-infected patients initiating therapy and possess prolonged window for intervention in the viral life cycle, thus offering an advantage in the setting of HIV-1 chemoprevention. Summary The distinct biochemical and antiviral mechanism of action of integrase strand transfer inhibitors are directly relevant to understanding the clinical properties which are characteristic of the class and their potential significance for the use of these agents in both treatment and prevention.

Purpose of review A serious effort has begun to develop therapies that may be capable of eradicating established HIV infection in man. Because of the biological complexity of HIV infection that persists despite potent antiretroviral therapy, it is widely believed that if such therapies can be developed they will involve complex, multimodality approaches. We highlight some of the recent studies in this effort. Recent findings An inhibitor of histone deacetylase has been demonstrated to disrupt latency in man, and new histone deacetylase inhibitors have been identified. Other potential targets, such as histone methyltransferase, protein kinase C, and BRD4, have been recently studied. Model systems, both in primary cells and in animal models, are beginning to be validated. In the clinic, immune-based therapies to aid in the clearance of persistent infection are also being tested. Summary It is too early to know what combination eradication therapies for HIV infection will look like in the future, but candidate therapies and model systems to perform preclinical validation are beginning to take shape.

Raltegravir is highly efficacious in the treatment of HIV-1 infection. The prevalence and impact on virologic outcome of low-frequency resistant mutations among HIV-1-infected patients not previously treated with raltegravir have not been fully established. Samples from HIV treatment-experienced patients entering a clinical trial of raltegravir treatment were analyzed using a parallel allele-specific sequencing (PASS) assay that assessed six primary and six secondary integrase mutations. Patients who achieved and sustained virologic suppression (success patients, n = 36) and those who experienced virologic rebound (failure patients, n = 35) were compared. Patients who experienced treatment failure had twice as many raltegravir-associated resistance mutations prior to initiating treatment as those who achieved sustained virologic success, but the difference was not statistically significant. The frequency of nearly all detected resistance mutations was less than 1% of viral population, and the frequencies of mutations between the success and failure groups were similar. Expansion of pre-existing mutations (one primary and five secondary) was observed in 16 treatment failure patients in whom minority resistant mutations were detected at baseline, suggesting that they might play a role in the development of drug resistance. Two or more mutations were found in 13 patients (18.3%), but multiple mutations were not present in any single viral genome by linkage analysis. Our study demonstrates that low-frequency primary RAL-resistant mutations were uncommon, while minority secondary RAL-resistant mutations were more frequently detected in patients naïve to raltegravir. Additional studies in larger populations are warranted to fully understand the clinical implications of these mutations.

The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.

Long-acting/extended-release drug formulations have proved very successful in diverse areas of medicine, including contraception, psychiatry and, most recently, human immunodeficiency virus (HIV) disease. Though challenging, application of this technology to anti-tuberculosis treatment could have substantial impact. The duration of treatment required for all forms of tuberculosis (TB) put existing regimens at risk of failure because of early discontinuations and treatment loss to follow-up. Long-acting injections, for example, administered every month, could improve patient adherence and treatment outcomes. We review the state of the science for potential long-acting formulations of existing tuberculosis drugs, and propose a target product profile for new formulations to treat latent tuberculous infection (LTBI). The physicochemical properties of some anti-tuberculosis drugs make them unsuitable for long-acting formulation, but there are promising candidates that have been identified through modeling and simulation, as well as other novel agents and formulations in preclinical testing. An efficacious long-acting treatment for LTBI, particularly for those co-infected with HIV, and if coupled with a biomarker to target those at highest risk for disease progression, would be an important tool to accelerate progress towards TB elimination.

We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.

(2S)-2-(3-Chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (1b) has been identified as a potent CCR5 antagonist having an IC50=10 nM. Herein, structure-activity relationship studies of non-spiro piperidines are described, which led to the discovery of 4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidine derivatives (3-5) as potent CCR5 antagonists.

From the screening of a microbial extract library, isocomplestatin (1), a new axial-chiral isomer of complestatin (2) which is a known rigid bicyclic hexapeptide, was identified as a potent natural product inhibitor of HIV-1 integrase, a unique enzyme responsible for viral replication. Isocomplestatin showed inhibitory activities (IC(50)) in coupled 3'-end processing/strand transfer (200 nM), strand transfer (4 microM), and HIV-1 replication (200 nM) in virus-infected cells. Attempted large-scale isolation of 1 by the literature method, used for the isolation of complestatin, led to lower yield and limited availability. We have developed several new, two-step, high-yielding absorption/elution methods of isolation based on reverse-phase chromatography at pH 8 that are applicable to scales from one gram to potential industrial quantities. We have also discovered and determined the structure of two new congeners of 1, namely, complestatins A (4) and B (5), with almost equal HIV-1 integrase activity. They differ from 1 at C2' and C3' of the tryptophan moiety (residue F). Selective acid hydrolysis of chloropeptin I (3), itself a known acid-catalyzed rearranged isomer of 1 and 2 (8'- vs 7'-substitution in tryptophan residue F, respectively), an isomer of complestatin, and isocomplestatin resulted in a number of fragments (6-10) with retention of most of the HIV-1 integrase activity. The structure-activity relationship as revealed by these compounds could possibly lead to the design of better inhibitors or understanding of the HIV-1 integrase target.

HIV-1 integrase is critical for viral replication and is absent in the host, and therefore is a potential target for the development of non-toxic antiviral therapy. From the screening of natural product libraries we have discovered integric acid, a novel eremophilane sesquiterpenoid, from a Xylaria sp. It inhibited 3′ -end processing, strand transfer and disintegration reactions catalyzed by HIV-1 integrase with IC50 values of 3–10 μM. The isolation, structure elucidation, relative, and absolute stereochemistry of integric acid are described.

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

The human immunodeficiency virus type 1 (HIV-1) integrase protein is required for the productive infection of T-lymphoid cells in culture (R. L. LaFemina, C. L. Schneider, H. L. Robbins, P. L. Callahan, K. LeGrow, E. Roth, W. A. Schleif, and E. A. Emini, J. Virol. 66:7414-7419, 1992). This observation suggests that chemical inhibitors of integrase may prevent the spread of HIV in infected individuals. In our search for such potential chemotherapeutic agents, we observed that beta-conidendrol inhibits both the sequence-dependent and sequence-independent endonucleolytic activities of integrase with comparable potencies in vitro (50% inhibitory concentration, 500 nM). Structurally related compounds tested for their abilities to inhibit integrase generated a limited structure-activity analysis which demonstrated that potency is associated with the bis-catechol structure: two pairs of adjacent hydroxyls on separate benzene rings. beta-Conidendrol did not inhibit several other endonucleases and/or phosphoryltransferases. Although beta-conidendrol was not effective in preventing HIV-1 infection in cell culture, the in vitro data demonstrate that it is possible to identify selective agents targeted against this essential HIV-1 function.

The amyloid β peptides (Aβ) are the major components of the senile plaques characteristic of Alzheimer's disease. Aβ peptides are generated from the cleavage of amyloid precursor protein (APP) by β- and γ-secretases. β-Secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Aβ peptides. γ-Secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Aβ peptides. The production of Aβ peptides is, therefore, dependent on the activities of both BACE and γ-secretase. The cleavage of APP by BACE is believed to be a prerequisite for γ-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Aβ-34 site) in the Aβ region is dependent on γ-secretase activity. In vitro, the Aβ-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Aβ-34 site by BACE1 or BACE2 occurred only when Aβ 1– 40 peptide, a γ-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Aβ 1–34 species. More importantly, the cellular production of Aβ 1–34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known γ-secretase inhibitors in a concentration-dependent manner. These γ-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that γ-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Aβ-34 site. Therefore, BACE and γ-secretase activity can be mutually dependent. The amyloid β peptides (Aβ) are the major components of the senile plaques characteristic of Alzheimer's disease. Aβ peptides are generated from the cleavage of amyloid precursor protein (APP) by β- and γ-secretases. β-Secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Aβ peptides. γ-Secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Aβ peptides. The production of Aβ peptides is, therefore, dependent on the activities of both BACE and γ-secretase. The cleavage of APP by BACE is believed to be a prerequisite for γ-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Aβ-34 site) in the Aβ region is dependent on γ-secretase activity. In vitro, the Aβ-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Aβ-34 site by BACE1 or BACE2 occurred only when Aβ 1– 40 peptide, a γ-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Aβ 1–34 species. More importantly, the cellular production of Aβ 1–34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known γ-secretase inhibitors in a concentration-dependent manner. These γ-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that γ-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Aβ-34 site. Therefore, BACE and γ-secretase activity can be mutually dependent. Amyloid β (Aβ) 1The abbreviations used are: Aβ, β amyloid peptide; AD, Alzheimer's disease; BACE, β site APP-cleaving enzyme; APP, amyloid precursor protein; CT99, C-terminal; HEK, human embryonic kidney; MALDI, matrix-assisted laser desorption/ionization; SELDI, surface-enhanced laser desorption/ionization; TOF, time of flight; ELISA, enzyme-linked immunosorbent assay. 1The abbreviations used are: Aβ, β amyloid peptide; AD, Alzheimer's disease; BACE, β site APP-cleaving enzyme; APP, amyloid precursor protein; CT99, C-terminal; HEK, human embryonic kidney; MALDI, matrix-assisted laser desorption/ionization; SELDI, surface-enhanced laser desorption/ionization; TOF, time of flight; ELISA, enzyme-linked immunosorbent assay. peptides are principal components of the neuritic plaques that represent one of the hallmarks of AD pathology. Production of Aβ peptides is initiated first by activities of BACE1, which cleaves APP at the β site to yield a membrane-associated APP C-terminal fragment of 99 amino acid residues (CT99) (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar, 2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.-C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3235) Google Scholar, 3Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1465) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1321) Google Scholar, 5Cai H. Wang Y. McCarthey D. Wen H. Borchelt D. Price D. Wong P. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (940) Google Scholar, 6Luo Y. Bolon B. Kahn S. Bennett B. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Matin L. Louis J.-C. Yan Q. Richards W. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (935) Google Scholar). A subsequent cleavage within the transmembrane domain of CT99 by γ-secretase then releases Aβ peptides of 39–42 amino acid residues (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar, 2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.-C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3235) Google Scholar, 3Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1465) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1321) Google Scholar, 5Cai H. Wang Y. McCarthey D. Wen H. Borchelt D. Price D. Wong P. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (940) Google Scholar, 6Luo Y. Bolon B. Kahn S. Bennett B. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Matin L. Louis J.-C. Yan Q. Richards W. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (935) Google Scholar). APP can also be cleaved within its Aβ region by α-secretase(s) to generate CT83 (5Cai H. Wang Y. McCarthey D. Wen H. Borchelt D. Price D. Wong P. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (940) Google Scholar), which is also a substrate for γ-secretase (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar, 7Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (626) Google Scholar). In cells, the activity of BACE and γ-secretase to process APP has been observed in the membrane fractions, particularly in the trans-Golgi network (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar, 8Kamal A. Almenar-Queralt A. LeBlanc J.F. Roberts E.A. Goldstein L.S.B. Nature. 2001; 414: 643-648Crossref PubMed Scopus (494) Google Scholar, 9Citron M. Teplow D.B. Selkoe D.J. Neuron. 1995; 14: 661-670Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 10Huse J.T. Liu K. Pijak D.S. Carlin D. Lee V.M.Y. Doms R.W. J. Biol. Chem. 2002; 277: 16278-16284Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chretien M. Nabil G.S. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Production of Aβ has also been detected in both the endoplasmic reticulum and the trans-Golgi network (12Yan R. Han P. Miao H. Greengard P. Xu H. J. Biol. Chem. 2001; 276: 36788-36796Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (341) Google Scholar, 14Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (423) Google Scholar, 15Greenfield J.P. Tsai J. Gouras G.K. Hai B. Thinakaran G. Checler F. Sisodia S.S. Greengard P. Xu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 742-747Crossref PubMed Scopus (326) Google Scholar). Several lines of experimental evidence suggest that cellular APP processing is sequential and that cleavage of APP by α- or β-secretase is a prerequisite for γ-secretase-mediated processing. First, only two species of APP N-terminal products cleaved by endogenous secretases were reportedly detected in cells: the BACE-cleaved product sAPPβ and the α-secretase-cleaved product sAPPα (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar, 2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.-C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3235) Google Scholar, 3Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1465) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1321) Google Scholar, 5Cai H. Wang Y. McCarthey D. Wen H. Borchelt D. Price D. Wong P. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (940) Google Scholar, 6Luo Y. Bolon B. Kahn S. Bennett B. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Matin L. Louis J.-C. Yan Q. Richards W. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (935) Google Scholar, 7Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (626) Google Scholar). No γ-secretase cleaved N-terminal APP, or so-called sAPPγ, without prior α- or β-secretase cleavage, was ever detected or reported. Second, in an in vitro cell-free assay system, the N-terminally truncated form of APP, CT99 (or CT100), has been demonstrated as an efficient substrate for γ-secretase (16Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar, 17Li Y.-M. Xu M. Lai M.-T. Huang Q. Castro J. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M. Shearman M.K. Smith A.L. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (857) Google Scholar). In contrast, the full-length APP is a very poor substrate for γ-secretase. 2Y.-M. Li, M. Xu, Q. Huang, and S. J. Gardell, unpublished results. 2Y.-M. Li, M. Xu, Q. Huang, and S. J. Gardell, unpublished results. Therefore, removal of the N-terminal region of APP seems to be essential for γ-secretase-mediated cleavage. In the present study, we found that BACE-mediated cleavage at the Aβ-34 site is dependent on the γ-secretase activity. BACE cleaves the Aβ-34 site in vitro only within Aβ 1– 40 peptide, a product from γ-secretase, not the site in the non-cleaved CT99 fragment. Moreover, the cellular production of Aβ 1–34 species induced by overexpression of BACE1 or BACE2 was blocked by known γ-secretase inhibitors in a concentration-dependent manner, whereas the γ-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our results suggest that the cleavage activity of BACE at the Aβ-34 site, both in vitro and in cells, is dependent on γ-secretase activity. Therefore, BACE and γ-secretase activity can be mutually dependent. BACE1 and BACE2 Expression and Purification—Cloning of human BACE1 and BACE2 cDNA and purification of human BACE1 and BACE2 full-length protein from transiently transfected human embryonic kidney 293T cells have been described previously (18Shi X.-P. Wu G.-X. Tugusheva K. Register R.B. Chen-Dodson E. Price E. Sardana M. Lucka A. Bruce J.E. Hazuda D.J. Neurobiol Aging. 2002; 23: S180Google Scholar). Expression and purification of truncated version of BACE1 from a recombinant baculovirus expression system was performed as described previously (19Shi X.-P. Chen E. Yin K.-C. Na S. Garsky V.M. Lai M.-T. Li Y.-M. Platchek M. Register R.B. Sradana M. Tang M.-J. Thiebeau J. Wood T. Shafer J.A. Gardell S.J. J. Biol. Chem. 2001; 276: 10366-10373Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). BACE Activity in Vitro Assay—Aβ 1– 40 was purchased from Enzyme Systems Products (Livermore, CA). Human cathepsin D was purchased from Calbiochem. The production of recombinant APP-CT100-Flag was described previously (16Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar). Treatment of CT100-Flag with aminopeptidase (Sigma) resulted in a CT99-Flag with the amino acid residue Asp at its N terminus. CT99-flag was confirmed by mass spectrometric analysis and by its reactivity with a neo-epitope-specific antibody, FCA-18, purchased from Dr. F. Checler (20Barelli H. Lebeau A. Vizzavona J. Delaere P. Chevallier N. Drouot C. Marambaud P. Ancolio K. Buxbaum J.D. Khorkova O. Heroux J. Sahasrabudhe S. Martinez J. Warter J.-M. Mohr M. Checler F. Mol. Med. 1997; 3: 695-707Crossref PubMed Google Scholar). Assays were performed for the indicated time in the presence of 50 mm ammonium acetate, 0.15 m NaCl, pH 4.5 or 5.0, and 0.1 mg/ml BSA at 37 °C. The reaction was terminated by heating samples at 75 °C for 5 min. Samples were analyzed by mass spectrometry or/and by reversed-phase high pressure liquid chromatography. BACE1 or BACE2 activity in vitro was also assessed by cleavage of a FRET substrate encompassing P8-P4′ of the APPsw β site, TAMRA-5-CO-EEISEVNLDAEF-NH-QSY7, similar to the one described by Ermolieff et al. (21Ermolieff J. Loy J.A. Koelsch G. Tang J. Biochemistry. 2000; 39: 12450-12456Crossref PubMed Scopus (133) Google Scholar). The reaction condition is the same as described above and the cleavage product was measured by a LJL Analyst AD instrument (LJL BioSystems, Sunnyvale, CA) with excitation at 530 nm and emission at 580 nm. Cell Culture, Transfection, and Analysis of APP Processing Products—HEK293T cells stably expressing human APP695 were seeded in 100-mm dishes and transfected with either BACE1 or BACE2 DNA as described above. Transfection was performed using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. Media were changed on the next day, and either Me2SO control or compound-1 was added to the new media. After an additional 24 h of incubation at 37° C, the media and cells were harvested. Aβ level in media was assayed by Origen system, as described previously, using biotinylated 6E10 antibody (Senetek, Maryland Heights, MO) and ruthenylated G2–10 antibody (16Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar) licensed from the University of Heidelberg. Mass Spectrometric Analysis of BACE-cleaved Products—MALDI mass analysis was performed with a surface-enhanced laser desorption/ionization–time-of-flight (SELDI-TOF) mass spectrometer from Ciphergen Biosystems (Fremont, CA) and a Voyager DE/RP MALDI-TOF from Applied Biosystems. Typically, 1 μl of reaction mixture was mixed with 1 μl of a saturated MALDI matrix solution (α-cyanohydroxycinnamic acid in 1:1 water/acetonitrile with 0.1% trifluoroacetic acid) and was then spotted onto Ciphergen NP2 MALDI chips. After drying and crystal formation, sample spots were typically analyzed with 100 laser shots. In all figures containing MALDI mass spectrometric data, the generic notation Aβ X-X refers to the length of the Aβ fragment, such as Aβ 1– 40 or Aβ 1–34. This notation is used to denote the Aβ fragment corresponding to the mass of the singly charged peptide ion (M+H)+. In addition, in cases in which the doubly charged ion (M+2H)2+ is visible, as in Fig. 2, the generic notation (Aβ X-X + 2H)2+, denotes the Aβ species corresponding to the mass of the doubly charged peptide ion. Mass Spectrometric Analysis of APP Processing Products in Media— For analysis of APP processing species secreted into media, the collected media samples were immunoprecipitated with 6E10 antibody and protein G beads (Protein G UltraLink; Pierce Chemical). After incubation at room temperature for 1 h, the supernatants were removed, and the immunoprecipitation beads were first washed three times with a buffer containing 150 mm NaCl, 10 mm Tris, 2 mm EDTA, 0.1% Triton X-100, and 0.1% Igepal CA630, pH 7.8, then washed twice with a buffer consisting of 500 mm NaCl, 10 mm Tris, 2 mm EDTA, 0.1% Triton X-100, and 0.1% Igepal CA630, pH 7.8, and finally washed three times with 100 mm NH4HCO3, pH 7.8. After the final wash, the captured products were eluted from the beads with a minimal volume of a saturated solution of α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile/0.1% trifluoroacetic acid. One microliter of the bead eluate was then spotted on to the an NP-2 SELDI sample target (Ciphergen Biosystems) or a stainless steel target for an ABI Voyager and allowed to air dry. The dried targets were placed into the SELDI-TOF and Voyager mass spectrometers and matrix/analyte coprecipitates were desorbed with N2 laser at 337 nm. The laser power was attenuated and the detector sensitivity manipulated such that spectra, which were the average of 100 laser shots, provided a qualitative assessment of the immunoprecipitation-captured cleavage products. BACE1 or BACE2, but Not Cathepsin D, Cleaves the Aβ-34 Site in Vitro—The activity of purified human full-length BACE1, BACE2, or cathepsin D, a closely related aspartyl protease, was tested for cleavage of a synthetic Aβ peptide 1– 40 in vitro. When BACE1 or BACE2 was incubated with a synthetic Aβ peptide 1-40 in vitro, a cleavage site located between Aβ residues 34 and 35 was clearly identified through mass spectrometric analysis as shown in Fig. 1, B1, B3, and B4. The theoretical mass value for Aβ 1–34 is 3786.9 and the observed mass values for the cleaved 1–34 products were 3785.6 and 3786.6 for BACE1 and BACE2, respectively. However, when Aβ 1– 40 peptide was incubated with cathepsin D, Aβ species cleaved at residues between 19 and 20 were observed by mass spectrometric analysis, but not the Aβ 1–34 product (Fig. 1B, 1 and 2). The absence of Aβ 1–34 species in cathepsin d-treated sample is not the result of a secondary cleavage of Aβ 1–34, because a shorter incubation time and with less enzyme showed similar results (data not shown). In contrast, both purified full-length human BACE1 (Fig. 1B, 3) and BACE2 (Fig. 1B, 4) processed the Aβ-34 site. Thus, the cleavage of Aβ-34 site is specific for BACE1 and BACE2 not for cathepsin D. BACE1 Lacking the Transmembrane and Cytoplasmic Regions Cleaves the Aβ-34 Site in Vitro—One of the major differences between BACE and cathepsin D is that cathepsin D does not contain a transmembrane domain and cytoplasmic tail. To investigate whether the transmembrane and cytoplasmic regions of BACE contribute to its specific activity of processing the Aβ-34 site, we tested the activities of truncated forms of BACE1 lacking both the transmembrane and cytoplasmic domains using the Aβ 1– 40 substrate. Results from mass spectrometric analysis showed that truncated BACE1, both the pro-(amino acids 22– 460) and the mature (amino acids 46–460) forms (19Shi X.-P. Chen E. Yin K.-C. Na S. Garsky V.M. Lai M.-T. Li Y.-M. Platchek M. Register R.B. Sradana M. Tang M.-J. Thiebeau J. Wood T. Shafer J.A. Gardell S.J. J. Biol. Chem. 2001; 276: 10366-10373Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), cleaves the Aβ-34 site in vitro (Fig. 2). Therefore, the differential cleavage of Aβ-34 site between BACE and cathepsin D is probably because of the difference in their enzyme active sites. It is interesting to note that the truncated forms of BACE1 did not cleave the Aβ-20 site as seen with the full-length enzyme. BACE1 or BACE2 Does Not Cleave Aβ-34 Site Using CT99 as Substrate—To further investigate BACE in vitro cleavage specificity, we tested BACE activity using a recombinant CT99 protein substrate. CT99 or CT100 has been described previously as an efficient in vitro substrate for γ-secretase (16Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar, 17Li Y.-M. Xu M. Lai M.-T. Huang Q. Castro J. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M. Shearman M.K. Smith A.L. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (857) Google Scholar). Control mass spectrometric analysis of CT99 alone (Fig. 3A) at mass range 1000–5000 Da did not show any notable species (Fig. 3B). To our surprise, when CT99 was incubated with BACE1, cleavage products of CT99 at either the Aβ-20 site or the Aβ-34 site were not observed (Fig. 3C). This was true for both full-length and truncated forms of BACE1 (data not shown). The absence of BACE1-mediated cleavage at Aβ-20 or Aβ-34 sites in CT99 was not caused by substrate depletion, because the substrate peak was still observed by mass spectrometry at the end of the reaction (data not shown), nor was it caused by enzyme inactivation, because that addition of a small peptide substrate encompassing the β-scissile bond at the end of the CT99 reaction yielded an appropriate cleavage product (data not shown). Likewise, BACE2 also did not cleave CT99 at the Aβ-34 site, although it cleaved at the Aβ-19 and -20 sites (Fig. 3D). Because the CT99 used here is in solution and not embedded in the membrane, the hydrophobic domain of the CT99 might be “abnormally” folded in the buffer. To test whether addition of a limited amount of detergent could expose the Aβ-34 site in CT99 for processing, the in vitro reaction with CT99 was also performed in the presence of 0.05% Triton X-100. Both BACE1 and BACE2 were fully functional in the presence of 0.05% Triton X-100 to cleave the β site of APP (data not shown) (3Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1465) Google Scholar). However, neither of them showed any cleavage of Aβ-34 site in CT99 (data not shown). Therefore, unlike Aβ 1– 40 peptide, CT99 is not a substrate for Aβ-34 site cleavage by either BACE1 or BACE2 in vitro. Overexpression of BACE1 or BACE2 in Cells Increases Aβ 1–34 Production—To evaluate the effect of BACE1 or BACE2 on cellular Aβ 1–34 production, HEK293T cells stably expressing APP695 (HEK293T/APP695) were either mock transfected or transfected with BACE1 or BACE2 cDNA. Aβ 1– 40 level secreted in the media by these cells was determined by a modified ELISA assay using antibodies 6E10 and G2–10 (16Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar) against Aβ 1– 40 (Fig. 4A). Consistent with previous reports, overexpression of BACE1 moderately increased the Aβ 1– 40 production (Fig. 4A, 2) (4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1321) Google Scholar), and overexpression of BACE2 abolished the generation of Aβ 1– 40 (Fig. 4A, 3) (22Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. Chem. 2001; 276: 34019-34027Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). When the conditioned media were subjected to immunoprecipitation and mass spectrometric analysis (see “Materials and Methods”), results showed that HEK293T/APP695 cells produced few Aβ 1–34 species (Fig. 4B, 1); Transfection of BACE1 increased the relative amount of Aβ 1–34 species, in addition to Aβ 1–20 and Aβ 1– 40 (Fig. 4B, 2); transfection of BACE2 similarly increased Aβ 1–34 level, despite abolishing Aβ 1– 40 (Fig. 4B, 3). This suggests that in cells, BACE2 prefers Aβ-19, -20, or -34 sites over the Aβ-1 site. This result not only agrees well with BACE2 activity data in vitro (18Shi X.-P. Wu G.-X. Tugusheva K. Register R.B. Chen-Dodson E. Price E. Sardana M. Lucka A. Bruce J.E. Hazuda D.J. Neurobiol Aging. 2002; 23: S180Google Scholar, 23Farzan M. Schnitzler C. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (340) Google Scholar) but also provides a plausible explanation for the negative effect of BACE2 on cellular Aβ 1– 40 production. In summary, our results indicate that overexpression of BACE1 or BACE2 in cells increases the production of Aβ 1–34. γ-Secretase Inhibitors Block the Production of Aβ 1–34 in Cells—To evaluate the effect of γ-secretase inhibitors on the cellular production of Aβ 1–34 species, HEK293T/APP695 cells, either mock-transfected or transfected with BACE1 or BACE2, were treated with either Me2SO or with 10 μm concentrations of each of the two known γ-secretase inhibitors, compound 1 and compound 2 (24Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith Jr., J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olsen R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 25Beher D. Wrigley J.D.J. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). As expected, both compounds inhibited Aβ 1– 40 production in HEK293TAPP695 cells (Fig. 5A, 1–3) and the cells transfected with BACE1 (Fig. 5A, 4–6). The effect of compounds on the cellular production of Aβ 1– 40 was further confirmed by mass spectrometric analysis of the Aβ species in the conditioned media of these cells (Fig. 5B). Notably, in BACE1 overexpressing cells, these γ-secretase inhibitors blocked the production of not only Aβ 1– 40 species but also Aβ 1–34 species (Fig. 5B, 4–6). Likewise, in BACE2 overexpressing cells, the increased generation of Aβ 1–34 species was also inhibited by these compounds (Fig. 5B, 7–9). In contrast, the generation of other Aβ species such as 1–16, 1–19, or 1–20 were largely unblocked (Fig. 5B). Inhibition of Aβ 1–34 Production by γ-Secretase Inhibitor Is Concentration-dependent and Coincides with the Blockade of Aβ 1– 40 Production—To further evaluate the effect of γ-secretase inhibitors on the cellular production of Aβ 1–34 species, another known γ-secretase inhibitor with a different structural class, compound 3 (17Li Y.-M. Xu M. Lai M.-T. Huang Q. Castro J. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M. Shearman M.K. Smith A.L. Shi X.-P. Yin K.-C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (857) Google Scholar, 26Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (364) Google Scholar), was tested in cells at various concentrations (1 and 10 μm). Results from the modified Aβ ELISA assay revealed a concentration-dependent inhibition of Aβ 1– 40 production in both HEK293T/APP695 cells (Fig. 6A, 1–3) and cells transfected with BACE1 (Fig. 6A, 4–6). Furthermore, the mass spectrometric anal

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.

The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N−Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.

A high-throughput screen at 100 μM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC50 = 0.045 μM, HIV RT RNase H; 13 μM, HIV RT-polymerase; 24 μM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC50 = 0.19 μM) with a modest window with respect to cytotoxicity (CC50 = 3.3 μM).

Host DNA repair enzymes have long been assumed to play a role in HIV replication, and many different DNA repair factors have been associated with HIV. In order to identify DNA repair pathways required for HIV infection, we conducted a targeted siRNA screen using 232 siRNA pools for genes associated with DNA repair. Mapping the genes targeted by effective siRNA pools to well-defined DNA repair pathways revealed that many of the siRNAs targeting enzymes associated with the short patch base excision repair (BER) pathway reduced HIV infection. For six siRNA pools targeting BER enzymes, the negative effect of mRNA knockdown was rescued by expression of the corresponding cDNA, validating the importance of the gene in HIV replication. Additionally, mouse embryo fibroblasts (MEFs) lacking expression of specific BER enzymes had decreased transduction by HIV-based retroviral vectors. Examining the role BER enzymes play in HIV infection suggests a role for the BER pathway in HIV integration.

Raltegravir is the first integrase strand transfer inhibitor approved for the treatment of HIV-1 infection. As the first agent in this new class of antiretroviral therapies, raltegravir has demonstrated safety and efficacy in treatment-naive as well as heavily pretreated HIV-infected patients failing therapy with multidrug-resistant virus. Raltegravir has a favorable drug interaction profile that permits both administration to a wide, demographically diverse patient population and coadministration with many other therapeutic agents, including antiretroviral agents and supportive medications, without restrictions or dose adjustment. Data through 96 weeks of follow-up in three phase III studies, protocol 021 (STARTMRK) in treatment-naive patients, and protocols 018 (BENCHMRK-1) and 019 (BENCHMRK-2) in treatment-experienced patients, demonstrated the potent and durable antiretroviral and immunologic effects and the favorable long-term safety profile of raltegravir in both treatment-naive and treatment-experienced patients. Raltegravir represents an important addition to the current armamentarium for the treatment of HIV infection.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4(+) T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.