Despite significant progress in hepatitis C virus (HCV) protease inhibitor (PI) drug design, resistance remains a problem causing treatment failure. Double-substitution variants, notably Y56H/D168A, have emerged in patients who fail therapy with a PI-containing regimen. The resistance conferred by Asp168 substitutions has been well characterized and avoided in newer inhibitors. However, an additional mutation at Tyr56 confers resistance to even the most robust inhibitors. Here, we elucidate the molecular mechanisms of resistance for the Y56H/D168A variant against grazoprevir (and four analogs), paritaprevir, and danoprevir through inhibition assays, co-crystal structures, and molecular dynamics simulations. The PIs' susceptibility to Y56H/D168A varies, with those stacking on the catalytic His57 losing the most potency. For such inhibitors, the Y56H substitution disrupts favorable stacking interactions with the neighboring catalytic His57. This indirect mechanism of resistance threatens to cause multi-PI failure as all HCV PIs in clinical development rely on interactions with the catalytic triad.

The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine versus daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).

Cabotegravir long-acting (LA) intramuscular (IM) injection is being investigated for HIV preexposure prophylaxis due to its potent antiretroviral activity and infrequent dosing requirement. A subset of healthy adult volunteers participating in a Phase I study assessing cabotegravir tissue pharmacokinetics underwent serial magnetic resonance imaging (MRI) to assess drug depot localization and kinetics following a single cabotegravir LA IM targeted injection.

Cabotegravir is an integrase strand transfer inhibitor in clinical development as long-acting (LA) injectable HIV preexposure prophylaxis.

A long-acting injectable formulation of the HIV integrase inhibitor cabotegravir (CAB-LA) is currently in clinical development for PrEP. Although the long plasma half-life of CAB-LA is an important attribute for PrEP, it also raises concerns about drug resistance emergence if someone becomes infected with HIV, or if PrEP is initiated during undiagnosed acute infection. Here we use a macaque model of SHIV infection to model risks of drug resistance to CAB-LA PrEP. Six macaques infected with SHIV received CAB-LA before seroconversion. We show integrase mutations G118R, E92G/Q, or G140R in plasma from 3/6 macaques as early as day 57, and identify G118R and E92Q in viruses from vaginal and rectal fluids. G118R and G140R confer > 800-fold resistance to CAB and cross-resistance to all licensed integrase inhibitors. Our results emphasize the need for appropriate HIV testing strategies before and possibly shortly after initiating CAB LA PrEP to exclude acute infection.

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

Cabotegravir is an integrase inhibitor in clinical development for the treatment and prevention of HIV infection using oral tablets for short-term, lead-in use before subsequent administration of a long-acting injectable formulation. This phase 1, single-center, randomized, 2 × 2 crossover study evaluated the effect of a high-fat meal on the pharmacokinetics (PK) of oral cabotegravir. Healthy adults received oral cabotegravir 30 mg as a single dose on 2 separate occasions, either after fasting or following a high-fat meal (∼53% fat, ∼870 kcal). Safety evaluations and serial PK samples were collected, and a mixed-effects model was used to determine within-participant treatment comparison of noncompartmental PK parameters. Twenty-four patients were enrolled and had a mean body mass index of 25.6 kg/m2 ; 67% were male. Compared with the fasting state, coadministration of cabotegravir with a high-fat meal increased plasma cabotegravir area under the concentration-time curve and maximal drug concentration, each by 14%. The slight 14% to 17% increase in exposure associated with a high-fat, high-calorie meal was not considered clinically significant. No grade 3/4 adverse events (AEs), drug-related AEs, or AEs leading to discontinuation were reported.