Application of ultrasensitive digital ELISA for p24 enables improved evaluation of HIV-1 reservoir diversity and growth kinetics in viral outgrowth assays.

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

Novel Compound Inhibitors of HIV-1NL4-3 Vpu.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

Inhibitors of HIV-1 Nef-Mediated Activation of the Myeloid Src-Family Kinase Hck Block HIV-1 Replication in Macrophages and Disrupt MHC-I Downregulation.

HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730 000 compounds using the Nef·Hck assay identified six unique hit compounds that bound directly to recombinant Nef by surface plasmon resonance (SPR) in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 µM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the µ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size.

BACKGROUND: Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 "next-generation" viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy-suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1-3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6-5.4-fold). Frozen storage did not substantially alter infectious units per million values (-18%; 95% CI, -52% to 39%). CONCLUSIONS: The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.

Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug.

Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.

HBV Core Promoter Inhibition by Tubulin Polymerization Inhibitor (SRI-32007).

Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.

Effector memory differentiation increases detection of replication-competent HIV-l in resting CD4+ T cells from virally suppressed individuals.

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells.

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size.

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.

Investigation of the molecular characteristics of bisindole inhibitors as HIV-1 glycoprotein-41 fusion inhibitors.

In previous work, we described 6-6'-bisindole compounds targeting a hydrophobic pocket on the N-heptad repeat region of viral glycoprotein-41 as effective inhibitors of HIV-1 fusion. Two promising compounds with sub-micromolar IC50's contained a benzoic acid group and a benzoic acid ester attached at the two indole nitrogens. Here we have conducted a thorough structure-activity relationship (SAR) study evaluating the contribution of each of the ring systems and various substituents to compound potency. Hydrophobicity, polarity and charge were varied to produce 35 new compounds that were evaluated in binding, cell-cell fusion and viral infectivity assays. We found that (a) activity based solely on increasing hydrophobic content plateaued at ∼ 200 nM; (b) the bisindole scaffold surpassed other heterocyclic ring systems in efficacy; (c) a polar interaction possibly involving Gln575 in the pocket could supplant less specific hydrophobic interactions; and (d) the benzoic acid ester moiety did not appear to form specific contacts with the pocket. The importance of this hydrophobic group to compound potency suggests a mechanism whereby it might interact with a tertiary component during fusion, such as membrane. A promising small molecule 10b with sub-µM activity was discovered with molecular weight <500 da and reduced logP compared to earlier compounds. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion.

Exploring Modifications of an HIV-1 Capsid Inhibitor: Design, Synthesis, and Mechanism of Action.

Recent efforts by both academic and pharmaceutical researchers have focused on the HIV-1 capsid (CA) protein as a new therapeutic target. An interprotomer pocket within the hexamer configuration of the CA, which is also a binding site for key host dependency factors, is the target of the most widely studied CA inhibitor compound PF-3450074 (PF-74). Despite its popularity, PF-74 suffers from properties that limit its usefulness as a lead, most notably it's extremely poor metabolic stability. To minimize unfavorable qualities, we investigated bioisosteric modification of the PF-74 scaffold as a first step in redeveloping this compound. Using a field-based bioisostere identification method, coupled with biochemical and biological assessment, we have created four new compounds that inhibit HIV-1 infection and that bind to the assembled CA hexamer. Detailed mechanism of action studies indicates that the modifications alter the manner in which these new compounds affect HIV-1 capsid core stability, as compared to the parental compound. Further investigations are underway to redevelop these compounds to optimize potency and drug-like characteristics and to deeply define the mechanism of action.

A single ß-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding.

The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, ß-octyl glucoside (ßOG). X-ray crystallography revealed that a single ßOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-ßOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54-66. Nef dimer formation is stabilized in solution by the addition of ßOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.

Expanding the Antiviral Spectrum of 3-Fluoro-2-(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates: Diamyl Aspartate Amidate Prodrugs.

Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

Enhanced potency of bivalent small molecule gp41 inhibitors.

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40-60times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

Bifunctional aryloxyphosphoramidate prodrugs of 2'-C-Me-uridine: synthesis and anti-HCV activity.

In an attempt to identify novel nucleoside phosphoramidate analogues for improving the anti-HCV activity of 2'-C-Me-uridine, we have synthesized for the first time a series of l-glutamic acid, l-serine, l-threonine and l-tyrosine containing aryloxyphosphoramidate prodrugs of 2'-C-Me-uridine. Evaluation of their activity against HCV revealed that they displayed very potent anti-HCV activity, with EC50 values that are in the same range as of Sofosbuvir.

Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as Dual Inhibitors of HIV and HBV Replication.

The synthesis of four l-2'-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displaying an EC50 of 4.69 µM against HIV-1 and an EC50 value of 0.5 µM against HBV, whereas completely lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led to a boost in antiviral potency. The most potent congeners were a l-aspartic acid diisoamyl ester phenoxy prodrug and a l-phenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low nanomolar range and selectivity indexes of more than 300.

Redoxal, an inhibitor of de novo pyrimidine biosynthesis, augments APOBEC3G antiviral activity against human immunodeficiency virus type 1.

APOBEC3G (A3G) is a cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA; deamination-independent mechanisms are also implicated. HIV-1 Vif protein counteracts A3G by inducing its proteasomal degradation. Thus, the Vif-A3G axis is a potential therapeutic target. To identify compounds that inhibit Vif:A3G interaction, a 307,520 compound library was tested in a TR-FRET screen. Two identified compounds, redoxal and lomofungin, inhibited HIV-1 replication in peripheral blood mononuclear cells. Lomofungin activity was linked to A3G, but not pursued further due to cytotoxicity. Redoxal displayed A3G-dependent restriction, inhibiting viral replication by stabilizing A3G protein levels and increasing A3G in virions. A3G-independent activity was also detected. Treatment with uridine or orotate, intermediates of pyrimidine synthesis, diminished redoxal-induced stabilization of A3G and antiviral activity. These results identify redoxal as an inhibitor of HIV-1 replication and suggest its ability to inhibit pyrimidine biosynthesis suppresses viral replication by augmenting A3G antiviral activity.

Identification of a novel HIV-1 inhibitor targeting Vif-dependent degradation of human APOBEC3G protein.

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-) T cells and had an IC50 as low as 8.4 µM and a TC50 of >100 µM when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 µM). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.

HIV-1, human interaction database: current status and new features.

The 'Human Immunodeficiency Virus Type 1 (HIV-1), Human Interaction Database', available through the National Library of Medicine at http://www.ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/interactions, serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. Each HIV-1 human protein interaction can be retrieved without restriction by web-based downloads and ftp protocols and includes: Reference Sequence (RefSeq) protein accession numbers, National Center for Biotechnology Information Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. In addition to specific HIV-1 protein-human protein interactions, included are interaction effects upon HIV-1 replication resulting when individual human gene expression is blocked using siRNA. A total of 3142 human genes are described participating in 12,786 protein-protein interactions, along with 1316 replication interactions described for each of 1250 human genes identified using small interfering RNA (siRNA). Together the data identifies 4006 human genes involved in 14,102 interactions. With the inclusion of siRNA interactions we introduce a redesigned web interface to enhance viewing, filtering and downloading of the combined data set.

Discovery and optimization of novel small-molecule HIV-1 entry inhibitors using field-based virtual screening and bioisosteric replacement.

With the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The inhibition of HIV-1 entry is an attractive, yet underexploited therapeutic approach with implications for salvage and preexposure prophylactic regimens, as well as topical microbicides. Using the combination of a field-derived bioactive conformation template to perform virtual screening and iterative bioisosteric replacements, coupled with in silico predictions of absorption, distribution, metabolism, and excretion, we have identified new leads for HIV-1 entry inhibitors.