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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. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection.

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. A new compound that inhibits HIV capsid assembly and nuclear transport functions offers potential as a long-acting antiretroviral.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases., Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5′-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC50) values of RDV ranged from 1.7 to >20 μM, resulting in selectivity indices (SI, CC50/EC50) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (50% effective concentration [EC50] of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential for RDV to elicit off-target toxicity, including mitochondria-specific toxicity, consistent with the reported clinical safety profile.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition (Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

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...

With the worldwide number of human immunodeficiency virus positive patients stagnant and the increasing emergence of viral strains resistant to current treatment, the development of novel anti-human immunodeficiency virus drug candidates is a perpetual quest of medicinal chemists. Herein, we report a novel group of diarylpyrimidines, non-nucleoside reverse transcriptase inhibitors, which represents an important class of current anti-human immunodeficiency virus therapy. Series of diarylpyrimidines containing o, o-difluorophenyl (A-arm), 4-cyanophenylamino (B-arm), and a small substituent (e.g. NH2, OMe) at positions 2, 4, and 6 of the pyrimidine ring were prepared. The A-arm was modified in the para position (F or OMe) and linked to the central pyrimidine core with a variable spacer (CO, O, NH). Antiviral activities of 20 compounds were measured against wild type human immunodeficiency virus-1 and mutant reverse transcriptase strains (K103N, Y181C) using a cytoprotection assay. To the most promising structural motives belong the o, o-difluoro- p-methoxy A-arm in position 4, and the amino group in position 6 of pyrimidine. Single digit nanomolar activities with no significant toxicity (CC50 > 17,000 nM) were found for compounds 35 (EC50 = 2 nM), 37 (EC50 = 3 nM), and 13 (EC50 = 4 nM) having O, NH, and CO linkers, respectively.

Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′- C -methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.

Tenofovir alafenamide (TAF) is an investigational oral prodrug of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). Tenofovir disoproxil fumarate (TDF) is another TFV prodrug, widely used for the treatment of HIV-1 infection. TAF is converted mostly intracellularly to TFV and, in comparison to TDF, achieves higher tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells. As a result, TAF has demonstrated potent anti-HIV-1 activity at lower doses than TDF in monotherapy studies. Here, the in vitro virology profile of TAF was evaluated and compared to that of TDF. TAF displayed potent antiviral activity against all HIV-1 groups/subtypes, as well as HIV-2. TAF exhibited minimal changes in the drug concentration needed to inhibit 50% of viral spread (EC 50 ) upon removal of the prodrug, similar to TDF, demonstrating intracellular antiviral persistence. While TAF and TDF exhibited comparable potencies in the absence of serum pretreatment, TAF maintained activity in the presence of human serum, whereas TDF activity was significantly reduced. This result demonstrates TAF's improved plasma stability over TDF, which is driven by the different metabolic pathways of the two prodrugs and is key to TAF's improved in vivo antiviral activity. The activity of TAF is specific for HIV, as TAF lacked activity against a large panel of human viruses, with the exception of herpes simplex virus 2, where weak TAF antiviral activity was observed, as previously observed with TFV. Finally, in vitro combination studies with antiretroviral drugs from different classes showed additive to synergistic interactions with TAF, consistent with ongoing clinical studies with TAF in fixed-dose combinations with multiple other antiretroviral drugs for the treatment of HIV.

Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.

Background GS-CA2, an analog of GS-CA1, is a novel and selective inhibitor of HIV-1 capsid function. Safety and pharmacokinetics (PK) of GS-CA2 is currently being evaluated in healthy human subjects. Herein, we present the anti-HIV activity and nonclinical PK of GS-CA2, demonstrating its potential as a first-in-class long-acting antiretroviral agent. Methods GS-CA2 antiviral activity was evaluated in MT-4 cells and in human peripheral blood mononuclear cells (PBMCs) acutely infected with HIV-1 (IIIb) and clinical HIV-1 isolates, respectively. Standard in vitro methods were used to characterize compound lipophilicity (LogD), solubility and relative binding to cell culture and plasma proteins. Metabolic stability was assessed in cryopreserved hepatocytes. GS-CA2 PK parameters following intravenous and subcutaneous (SC) administration were assessed in rat and dog. GS-CA2 plasma concentrations were determined by HPLC-MS/MS. Results GS-CA2 showed potent and selective anti-HIV activity in MT-4 cells (EC50 = 0.1 nM; CC50 = 26.6 µM). In PBMCs, GS-CA2 displayed a mean EC50 of 0.05 nM (0.02–0.16 nM) against 23 HIV-1 clinical isolates representing all major subtypes. GS-CA2 is highly lipophilic (LogD of 3.7) with low aqueous solubility (3 months. These nonclinical data support clinical development of GS-CA2 as a novel long-acting antiretroviral agent suitable for the treatment of HIV-1 infection. Disclosures J. Zheng, Gilead Sciences, Inc.: Employee, Salary. S. R. Yant, Gilead Sciences, Inc.: Employee, Salary. S. Ahmadyar, Gilead Sciences, Inc.: Employee, Salary. T. Y. Chan, Gilead Sciences, Inc.: Employee, Salary. A. Chiu, Gilead Sciences, Inc.: Employee, Salary. T. Cihlar, Gilead Sciences, Inc.: Employee, Salary. J. O. Link, Gilead Sciences, Inc.: Employee, Salary. B. Lu, Gilead Sciences, Inc.: Employee, Salary. J. Mwangi, Gilead Sciences, Inc.: Employee, Salary. W. Rowe, Gilead Sciences, Inc.: Employee, Salary. S. D. Schroeder, Gilead Sciences, Inc.: Employee, Salary. G. J. Stepan, Gilead Sciences, Inc.: Employee, Salary. K. W. Wang, Gilead Sciences, Inc.: Employee, Salary. R. Subramanian, Gilead Sciences, Inc.: Employee, Salary. W. C. Tse, Gilead Sciences, Inc.: Employee, Salary.

An extract of Humicola fuscoatra (UCSC strain no. 108111A) was shown to reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells. We report the bioassay-guided isolation and structure determination of several resorcyclic acid lactones, including four known compounds, radicicol (1, aka. monorden) and pochonins B (2), C (3), and N (4), and three new analogues, radicicols B–D (5–7). Compounds 1–3 and 5 showed moderate activities in the memory T cell model of HIV-1 latency. Radicicol (1) displayed lower potency in reactivating latent HIV-1 (EC50 = 9.1 μM) relative to the HDAC inhibitors apicidin (EC50 = 0.3 μM), romidepsin (EC50 = 0.003 μM), and SAHA (EC50 = 0.6 μM); however, it achieved equivalent maximum efficacy relative to the positive control compounds (98% of SAHA and romidepsin).

To elucidate the structure-geometry-activity relationship in diarylpyrimidine family (DAPYs) containing carbonyl linker between the central pyrimidine core and phenyl type B-arm, a series of (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones was designed, prepared and tested for their anti-HIV-1 activity. The carbonyl linker bearing B phenyl arm was successfully attached at both C-2 and C-4 positions of the central pyrimidine ring using a new synthetic approach. Further modifications of target compounds are present at C-5 position of the pyrimidine ring. In vitro anti-HIV-1 activity study performed on a series of 22 compounds confirmed the crucial importance of both conformational rigidity between phenyl B arm and the pyrimidine core linked through the carbonyl bridge, as well as presence of fluoro substituents in ortho-positions of phenyl B moiety. The most potent derivative of the series, compound 17, having almost perpendicular angle within the two planes made from the B aromatic arm and the pyrimidine ring, exhibited low nanomolar anti-HIV-1 activity (EC50 = 4 nM) with no significant toxicity (CC50 > 57.1 μM).

The radioligand binding profiles and in vivo pharmacological characteristics of YM-44781, YM-44778 and YM-49598, novel non-peptide tachykinin receptor antagonists, were examined and compared to those of FK-888 and GR-159897. Since no functional NK3 receptors were found in the rat bladder, the emphasis will be on the other two subtypes. YM-44781 and YM-49598 exhibited high binding affinities at NK2 (pKi = 9.94 ± 0.03) and NK1 (pKi = 9.09 ± 0.02) receptors, respectively, whereas YM-44778 exhibited high binding affinities at both NK1 (pKi = 8.08 ± 0.07) and NK2 (pKi = 8.55 + 0.04) receptors stably transfected in CHO-K1 cells (Chinese hamster ovary cells). In an in vivo rat model, a drug-induced bladder contraction model, antagonism of the contractions produced by the selective NK2 receptor agonist, [βAla8]neurokinin A (4–10) (10 µg·kg–1 i.v.) was observed after intravenous administration (dose range 0.001–1 mg·kg–1) of YM-44781 and YM-44778 (IC50 = 27 ± 8 and 100 ± 44 µg· kg–1, respectively). YM-44781 was more potent (about 3-fold) than YM-44778. YM-49598 was almost inactive but produced a potent inhibition (IC50 = 11 ± 7 µg·kg–1) of the contraction of the rat urinary bladder induced by challenge with the NK1-selective receptor agonist [Sar9,Met(O2)11]substance P sulphone (0.3 µg·kg–1). YM-44781 and YM-44778 did not produce major inhibition of [Sar9,Met(O2)11]substance P-induced bladder contraction. These findings indicate that YM-44781 and YM-49598 are potent NK2 and NK1 receptor antagonists, respectively, whereas YM-44778 is a nonselective NK2/NK1 receptor antagonist in the drug-induced bladder contraction model.

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C -nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC 50 ], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC 50 , 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with K i / K m values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′- C -Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro . Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Graphical abstract, New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH2P(O)(OiPr)2 or BrCH2P(O)(OiPr)2 followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using β-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.

Reservoirs of HIV that persist during ART represent barriers to eradication of this virus. One well documented reservoir of latent HIV is found in memory CD4+ T-cells. Identifying means to safely eliminate latently infected memory CD4+ T-cells is an important goal that may contribute to a cure for HIV. One approach toward this end is to activate latent proviruses with the premise that viral particles emanating from these cells will cause a cytopathic effect leading to the demise of the host cell. We have optimized and automated a primary cell-based HIV latency assay that can be used for high throughput screening of small molecule libraries in search of HIV activators. Using this assay, we have identified novel histone deacetylase (HDAC) inhibitors fromGilead’s compound collection that activate latent HIV. Analysis of these inhibitors revealed that the magnitude of HIV expression correlated with the breadth of cellular HDAC inhibition. In addition, we have identified a variety of other compounds that activate latent HIV such as kinase inhibitors which may point to novel mechanisms that govern HIV latency. This screening assay has the potential to identify novel molecular targets for drug discovery and new chemical classes that could be optimized to create new drugs to eliminate reservoirs of latent HIV.

The recombinant alpha1A-adrenoceptor displays a distinct pharmacological profile ('classical alpha1A-adrenoceptor') in homogenate binding assays, but displays the properties of the so-called alpha1L-adrenoceptor in functional studies in whole cells at 37 degrees C. As three splice variants of the human alpha1A-adrenoceptor have been described previously (alpha1A-1, alpha1A-2 and alpha1A-3), we have compared their functional pharmacological profiles, when expressed stably in Chinese hamster ovary (CHO-K1) cells (antagonist inhibition of noradrenaline-stimulated [3H]inositol phosphates accumulation). A fourth, novel isoform (alpha1A-4) has also been studied: alpha1A-4 mRNA predominates in several human tissues including prostate, liver, heart and bladder. In homogenate binding studies, all four isoforms displayed essentially identical affinity profiles, with prazosin (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furoyl)piperazine), tamsulosin (5-[2-[[2-(2-ethoxyphenoxy)ethyl]-amino]propyl]-2-methoxybenzen esulfonamide), RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alphad imethyl-1H-indole-3-ethanamine hydrochloride), WB 4101 ((2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and 5-Me-urapidil (5-methyl-6[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]amino]-1,3-d imethyuracil) all displaying subnanomolar affinities. In functional studies, noradrenaline accelerated [3H]inositol phosphates production with potencies (p[A]50) of between 5.8 and 6.6. The affinities of prazosin, RS-17053, WB 4101 and 5-Me-urapidil, at antagonizing responses to noradrenaline, were reduced by approximately 10-fold (cf. binding data), while those for tamsulosin and indoramin (N-[1-[2-(1H-indol-3-yl)ethyl]-4-piperidinyl]benzamide) remained constant or increased, consistent with the previously described alpha1L-adrenoceptor. Thus, all four human recombinant alpha1A-adrenoceptor isoforms display the pharmacology of the alpha1L-adrenoceptor when studied in functional assays, consistent with the hypothesis that the putative alpha1L-adrenoceptor represents a functional phenotype of the alpha1A-adrenoceptor.