Background: Doravirine (DOR) is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with improved potency against common NNRTI-associated resistance mutants as compared to the other approved NNRTIs. MK-8591 is a novel and potent nucleoside reverse transcriptase translocation inhibitor (NRTTI). The combination of DOR and MK-8591 is currently being studied in Phase 2. It has previously been reported that the NNRTI-associated substitution Y181C confers hypersusceptibility to MK-8591. This study aimed to characterize the effect of DOR-selected NNRTI resistant mutants from Phase 3 trials on MK-8591 as well as evaluate the resistance profile of DOR in combination with MK8591 in vitro.
Methods: HIV-1 mutant viruses were generated via a site-directed mutagenesis (SDM) method with gene synthesis and subcloning into plasmid RT112 (R8). These mutants were tested for susceptibility to MK-8591 and 3TC in MT4-GFP cells. In vitro resistance selection studies were performed with MT4-GFP cells in a 96-well plate format. The following combinations were evaluated: DOR/MK8591, DOR/3TC and DTG/3TC. For every passage, the plates were scanned to monitor viral replication by analyzing the intensity of green fluorescence. When evidence of virus breakthrough was observed, the supernatant was removed and the virus subjected to genotyping analysis of the HIV-1 RT region.
Results: In Phase 3 trials of DOR, seven of 747 (0.9%) patient developed NNRTI mutations, predominately F227C with additional substitutions. SDM viruses were generated with RT Y188L, A98G/F227C, V106I/F227C, V106I/H221Y/F227C, A98G/V106I/H221Y/F227C, V106A/P225H/Y318F, and V106M/F227C. Most of these mutants exhibited a fold change (FC) >100 (mutant_EC50 versus WT_EC50) to DOR. In contrast, viruses with F227C alone or in combination (A98G/F227C, V106I/F227C, V106M/F227C, and V106I/H221Y/F227C) were 2X or greater more susceptible to MK-8591 whereas no change in susceptibility to 3TC. The combination of DOR/MK8591 was less susceptible to breakthrough compared to either DOR/3TC or DTG/3TC. Mutations identified in DOR/MK-8591 combination were mostly DOR-associated mutations whereas M184V/I were identified with the DTG/3TC.
Conclusions: DOR-associated clinical mutants containing F227C, alone or in combination, are hypersusceptible to the NRTTI MK-8591. This hypersensitivity may, in part, explain the observation that the combination of DOR with MK-8591 has a higher barrier to resistance development than the combination of DOR/3TC or DTG/3TC in vitro.