Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition.

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted in vitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

HIV-1 Resistance to Islatravir/Tenofovir Combination Therapy in Wild-Type or NRTI-Resistant Strains of Diverse HIV-1 Subtypes.

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4'-ethynyl-2-fluoro-2'-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies.

Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies.

The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (∼55-fold), with major decrease in WA1 replicon fitness (∼20-fold), but not BA.1 (∼2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and ß-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through "wiggling and jiggling" accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a ß-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.

Drug Interactions in Lenacapavir-Based Long-Acting Antiviral Combinations.

Long-acting (LA) anti-HIV regimens show promise for increasing dosing intervals and consequently, improving the patients' quality of life. The first FDA-approved LA therapy is Cabenuva, which comprises rilpivirine (a non-nucleoside reverse transcriptase inhibitor) and cabotegravir (integrase strand transfer inhibitor). Novel promising LA anti-HIV agents such as lenacapavir (a capsid-targeting antiviral) and islatravir (EFdA, a nucleoside reverse transcriptase translocation inhibitor) need to be explored as combination therapies. Therefore, we sought to determine whether combination of lenacapavir with islatravir, rilpivirine, or cabotegravir displayed synergy, additivity, or antagonism. We performed dose-response matrices of these drug combinations in an HIV-1 reporter cell line and subsequently analyzed the data with SynergyFinder Plus, which employs four major drug interaction models: highest single agent, Bliss independence, Loewe additivity, and zero interaction potency. Most of these models predict additive inhibition by the studied drug combinations This work highlights the importance of effective drug combinations in LA-regimens.

Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env.

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from EnvSF162 into the EnvHXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the EnvHXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312-315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.

Development of Human Immunodeficiency Virus Type 1 Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor-Resistant Strains.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against wild-type (WT) and drug-resistant HIV-1 in phase III clinical trials. EFdA resistance is not well characterized. To study EFdA resistance patterns that may emerge in naive or tenofovir (TFV)-, emtricitabine/lamivudine (FTC/3TC)-, or zidovudine (AZT)-treated patients, we performed viral passaging experiments starting with WT, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless of the starting viral sequence, all selected EFdA-resistant variants included the M184V reverse transcriptase (RT) mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than either M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than was WT HIV-1. These mutants also had significantly lower specific infectivities than did WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (kcat/Km) of WT versus A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data suggest that EFdA is an excellent therapeutic candidate for naive, AZT-, FTC/3TC-, and especially tenofovir-treated patients.

Avoiding Drug Resistance in HIV Reverse Transcriptase.

HIV reverse transcriptase (RT) is an enzyme that plays a major role in the replication cycle of HIV and has been a key target of anti-HIV drug development efforts. Because of the high genetic diversity of the virus, mutations in RT can impart resistance to various RT inhibitors. As the prevalence of drug resistance mutations is on the rise, it is necessary to design strategies that will lead to drugs less susceptible to resistance. Here we provide an in-depth review of HIV reverse transcriptase, current RT inhibitors, novel RT inhibitors, and mechanisms of drug resistance. We also present novel strategies that can be useful to overcome RT's ability to escape therapies through drug resistance. While resistance may not be completely avoidable, designing drugs based on the strategies and principles discussed in this review could decrease the prevalence of drug resistance.

Potency and metabolic stability: a molecular hybrid case in the design of novel PF74-like small molecules targeting HIV-1 capsid protein.

PF74 (1) is a potent and well-characterized prototypical small molecule targeting human immunodeficiency virus type 1 (HIV-1) capsid protein (CA), but not a viable antiviral lead due to the lack of metabolic stability. We report herein our molecular hybridization-based medicinal chemistry efforts toward potent and metabolically stable PF74-like small molecules. The design of the new sub-chemotype 4 rationally combines binding features of two recently reported PF74-like compounds 2 and 3. The subsequent confirmation and structure-activity relationship (SAR) of hit 4a entailed the chemical synthesis of 37 novel analogs, most of which showed modest but meaningful thermal shift, and low µM antiviral activity. The most potent analogs (4a, 4d, 4o, and 4r) all exhibited noticeably improved metabolic stability over PF74. Molecular modeling suggests that these new analogs bind to the PF74 binding site. Overall, our work demonstrated that the molecular hybridization approach is suitable for designing compounds with balanced potency and metabolic stability.