Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Lenacapavir: A Novel, Potent, and Selective First-in-Class Inhibitor of HIV-1 Capsid Function Exhibits Optimal Pharmacokinetic Properties for a Long-Acting Injectable Antiretroviral Agent.

Lenacapavir (LEN) is a picomolar first-in-class capsid inhibitor of human immunodeficiency virus type 1 (HIV-1) with a multistage mechanism of action and no known cross resistance to other existing antiretroviral (ARV) drug classes. LEN exhibits a low aqueous solubility and exceptionally low systemic clearance following intravenous (IV) administration in nonclinical species and humans. LEN formulated in an aqueous suspension or a PEG/water solution formulation showed sustained plasma exposure levels with no unintended rapid drug release following subcutaneous (SC) administration to rats and dogs. A high total fraction dose release was observed with both formulations. The long-acting pharmacokinetics (PK) were recapitulated in humans following SC administration of both formulations. The SC PK profiles displayed two-phase absorption kinetics in both animals and humans with an initial fast-release absorption phase, followed by a slow-release absorption phase. Noncompartmental and compartmental analyses informed the LEN systemic input rate from the SC depot and exit rate from the body. Modeling-enabled deconvolution of the input rates from two processes: absorption of the soluble fraction (minor) from a direct fast-release process leading to the early PK phase and absorption of the precipitated fraction (major) from an indirect slow-release process leading to the later PK phase. LEN SC PK showed flip-flop kinetics due to the input rate being substantially slower than the systemic exit rate. LEN input rates via the slow-release process in humans were slower than those in both rats and dogs. Overall, the combination of high potency, exceptional stability, and optimal release rate from the injection depot make LEN well suited for a parenteral long-acting formulation that can be administered once up to every 6 months in humans for the prevention and treatment of HIV-1.

Forgiveness of INSTI-Containing Regimens at Drug Concentrations Simulating Variable Adherence In Vitro.

The integrase strand transfer inhibitor (INSTI)-based regimens bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF), dolutegravir (DTG)+FTC/TAF, DTG/lamivudine (3TC), and DTG/rilpivirine (RPV) are all approved for treatment of HIV-infected patients, with various limitations. Here, time to in vitro viral breakthrough (VB) and resistance barrier using simulated human drug exposures at either full or suboptimal treatment adherence to each regimen were compared. At drug concentrations corresponding to full adherence and 1 missed dose (Cmin and Cmin-1), no VB occurred with any regimen. At Cmin-2, VB occurred only with DTG+3TC, with emergent resistance to both drugs. At Cmin-3, VB occurred with all regimens: 100% of DTG+3TC cultures had VB by day 12, and <15% of BIC+FTC+TAF, DTG+FTC+TAF, and DTG+RPV cultures had VB. Emergent reverse transcriptase (RT) or integrase (IN) resistance was seen with DTG+RPV and DTG+3TC but not with BIC+FTC+TAF or DTG+FTC+TAF. At Cmin-4, 100% VB occurred with DTG+3TC and DTG+FTC+TAF by day 12, while 94% VB occurred with DTG+RPV by day 25 and only 50% VB occurred with BIC+FTC+TAF by day 35. Emergent Cmin-4 drug resistance was seen with all regimens but at differing frequencies; DTG+RPV had the most cultures with resistance. Emergent resistance was consistent with clinical observations. Overall, under high adherence conditions, no in vitro VB or resistance development occurred with these INSTI-based regimens. However, when multiple missed doses were simulated in vitro, BIC+FTC+TAF had the highest forgiveness and barrier to resistance of all tested regimens. Compared to DTG+3TC and DTG+FTC+TAF, DTG+RPV had higher forgiveness but lower resistance barrier after several simulated missed doses.

Simulating HIV Breakthrough and Resistance Development During Variable Adherence to Antiretroviral Treatment.

BACKGROUND: Barriers to lifelong HIV-1 suppression by antiretrovirals include poor adherence and drug resistance; regimens with higher tolerance to missed doses (forgiveness) would be beneficial to patients. To model short-term nonadherence, in vitro experiments monitoring viral breakthrough (VB) and resistance development were conducted. METHODS: HIV breakthrough experiments simulated drug exposures at full adherence or suboptimal adherence to bictegravir+emtricitabine+tenofovir alafenamide (BIC+FTC+TAF) or dolutegravir + lamivudine (DTG+3TC). MT-2 cells were infected with wild-type or low frequency M184V HIV-1, exposed to drug combinations, monitored for VB, and rebound virus was deep sequenced. Drug concentrations were determined using human plasma-free adjusted clinical trough concentrations (Cmin), at simulated Cmin after missing 1 to 3 consecutive doses (Cmin - 1 or Cmin - 2, and Cmin - 3) based on drug or active metabolite half-lives. RESULTS: Cultures infected with wild-type or low frequency M184V HIV-1 showed no VB with BIC+FTC+TAF at drug concentrations corresponding to Cmin, Cmin - 1, or Cmin - 2 but breakthrough did occur in 26 of 36 cultures at Cmin - 3, where the M184V variant emerged in one culture. Experiments using DTG + 3TC prevented most breakthrough at Cmin concentrations (9/60 had breakthrough) but showed more breakthroughs as drug concentrations decreased (up to 36/36) and variants associated with resistance to both drugs emerged in some cases. CONCLUSIONS: These in vitro VB results suggest that the high potency, long half-lives, and antiviral synergy provided by the BIC/FTC/TAF triple therapy regimen may protect from viral rebound and resistance development after short-term lapses in drug adherence.

Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.

Identification of NK Cell Subpopulations That Differentiate HIV-Infected Subject Cohorts with Diverse Levels of Virus Control.

HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8+ T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection.IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.

TLR7 Agonist GS-9620 Is a Potent Inhibitor of Acute HIV-1 Infection in Human Peripheral Blood Mononuclear Cells.

GS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4+ T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4+ T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal.

Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile.

Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.

Rapid In Vitro Evaluation of Antiretroviral Barrier to Resistance at Therapeutic Drug Levels.

Failure of combination antiretroviral (ARV) therapy in HIV-infected patients is often associated with the emergence of drug resistance-associated mutations (RAMs). To facilitate analysis of the barrier to resistance at therapeutically relevant ARV concentrations, we performed fixed-dose in vitro HIV-1 drug resistance selection assays using the immortalized MT-2 T-cell line and primary human CD4+ T cells with a panel of FDA-approved ARVs, each at their respective cell culture equivalent clinical trough concentration (CCE Cmin). At high multiples of its CCE Cmin, emtricitabine (FTC) selected for the rapid emergence of M184I/V, a result consistent with resistance emergence in vivo. While the rate of viral breakthrough in the presence of rilpivirine or efavirenz was delayed relative to FTC, both inhibitors selected for virus with known clinically relevant RAMs. No viral breakthrough was observed for the protease inhibitor atazanavir even at subtherapeutic drug concentrations, which is consistent with its previously characterized high in vivo barrier to resistance. Depending on assay conditions, treatment with integrase inhibitors elvitegravir and raltegravir resulted in breakthrough of both resistant and wild-type virus. The RAMs observed in drug selections were not detected above a 2% threshold by deep sequencing in the in vitro virus inoculum, and only rarely in isolates from treatment-naive HIV+ patients. These new viral breakthrough assays facilitate the analysis of multiple experimental replicates and conditions in parallel and provide a rapid quantitative means to evaluate drug resistance emergence at therapeutically relevant drug concentrations, which should facilitate the identification of new ARVs with a high barrier to resistance.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

Activation of 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) and other tenofovir phosphonoamidate prodrugs by human proteases.

9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) is an isopropylalaninyl phenyl ester prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV; 9-[(2-phosphonomethoxy)propyl]adenine) exhibiting potent anti-HIV activity and enhanced ability to deliver parent TFV into peripheral blood mononuclear cells (PBMCs) and other lymphatic tissues in vivo. The present study focuses on the intracellular metabolism of GS-7340 and its activation by a variety of cellular hydrolytic enzymes. Incubation of human PBMCs in the presence of GS-7340 indicates that the prodrug is hydrolyzed slightly faster to an intermediate TFV-alanine conjugate (TFV-Ala) in quiescent PBMCs compared with activated cells (0.21 versus 0.16 pmol/min/10(6) cells). In contrast, the conversion of TFV-Ala to TFV and subsequent phosphorylation to TFV-diphosphate occur more rapidly in activated PBMCs. The activity of GS-7340 hydrolase producing TFV-Ala in PBMCs is primarily localized in lysosomes and is sensitive to inhibitors of serine hydrolases. Cathepsin A, a lysosomal serine protease has recently been identified as the primary enzyme activating GS-7340 in human PBMCs. Results from the present study indicate that in addition to cathepsin A, a variety of serine and cysteine proteases cleave GS-7340 and other phosphonoamidate prodrugs of TFV. The substrate preferences displayed by these enzymes toward TFV amidate prodrugs are nearly identical to their preferences displayed against oligopeptide substrates, indicating that GS-7340 and other phosphonoamidate derivatives of TFV should be considered peptidomimetic prodrugs of TFV.

Suppression of HIV-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring.

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.

Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue.

An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective concentration (EC(50)) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 microM compared to an EC(50) of 5 microM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1,000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37 degrees C and a half-life of 28.3 min in an MT-2 cell extract at 37 degrees C. The antiviral activity (>10 x the EC(50)) and the metabolic stability in MT-2 cell extracts (>35 x) and plasma (>2.5 x) were also sensitive to the stereochemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 microg-eq/ml compared to 0.2 microg-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available prodrug tenofovir DF (Viread).