No antagonism or cross-resistance and a high barrier to the emergence of resistance in vitro for the combination of islatravir and lenacapavir.

Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits HIV-1 at multiple stages throughout the viral life cycle. ISL and LEN are being investigated as once-weekly combination oral therapy for the treatment of HIV-1. Here, we characterized ISL and LEN in vitro to assess combinatorial antiviral activity, cytotoxicity, and the potential for interactions between the two compounds. Bliss analysis revealed ISL with LEN demonstrated additive inhibition of HIV-1 replication, with no evidence of antagonism across the range of concentrations tested. ISL exhibited potent antiviral activity against variants encoding known LEN resistance-associated mutations (RAMs) with or without the presence of M184V, an ISL RAM in reverse transcriptase (RT) . Static resistance selection experiments were conducted with ISL and LEN alone and in combination, initiating with either wild-type virus or virus containing the M184I RAM in RT to further assess their barrier to the emergence of resistance. The combination of ISL with LEN more effectively suppressed viral breakthrough at lower multiples of the compounds' IC50 (half-maximal inhibitory concentration) values and fewer mutations emerged with the combination compared to either compound on its own. The known pathways for development of resistance with ISL and LEN were not altered, and no novel single mutations emerged that substantially reduced susceptibility to either compound. The lack of antagonism and cross-resistance between ISL and LEN support the ongoing evaluation of the combination for treatment of HIV-1.

Doravirine and Islatravir Have Complementary Resistance Profiles and Create a Combination with a High Barrier to Resistance.

Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor (NNRTI), was approved for treatment of HIV-1 infection in 2018. In the pivotal phase 3 trials, DRIVE-FORWARD and DRIVE-AHEAD, 7 out of 747 (0.9%) treatment-naive participants treated with DOR plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) met protocol-defined virologic failure criteria and showed phenotypic resistance to DOR at week 48. The most common DOR resistance-associated mutation (RAM) detected in 5 of the 7 resistant isolates was F227C. Six isolates bearing NRTI RAMs (M184V and/or K65R) were resistant to lamivudine (3TC) and emtricitabine (FTC) but not to other approved NRTIs. All DOR-resistant isolates were susceptible or hypersusceptible (fold change of <0.25) to islatravir (ISL), a nucleoside reverse transcriptase translocation inhibitor (NRTTI). Isolate hypersusceptibility to ISL required F227C, in contrast to zidovudine, an NRTI, which required M184V. Based on the frequent emergence of F227C, we hypothesized that DOR and ISL would create a combination (DOR/ISL) with a high barrier to resistance. In de novo resistance selection studies in MT4-GFP cells (MT4 cells engineered to express green fluorescent protein), DOR/ISL synergistically prevented viral breakthrough at a threshold of 2× the half-maximal inhibitory concentration (IC50). DOR/ISL exhibited a higher barrier to resistance than DOR/3TC and dolutegravir (DTG)/3TC. Resistance analysis showed no emergence of substitutions at F227, an observation consistent with its ability to confer hypersusceptibility to ISL. Overall, the data demonstrate that DOR/ISL creates a 2-drug combination with a higher barrier to resistance, consistent with the reported clinical activity.

Islatravir Has a High Barrier to Resistance and Exhibits a Differentiated Resistance Profile from Approved Nucleoside Reverse Transcriptase Inhibitors (NRTIs).

Islatravir (ISL) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that inhibits human immunodeficiency virus (HIV) reverse transcription by blocking reverse transcriptase (RT) translocation on the primer:template. ISL is being developed for the treatment of HIV-1 infection. To expand our knowledge of viral variants that may confer reduced susceptibility to ISL, resistance selection studies were conducted with wild-type (WT) subtype A, B, and C viruses. RT mutations encoding M184I and M184V were the most frequently observed changes. Selection studies were also initiated with virus containing a single known resistance-associated mutation in RT (K65R, L74I, V90I, M184I, or M184V), and no additional mutations were observed. Antiviral activity assays were performed on variants that emerged in selection studies to determine their impact. M184I and M184V were the only single-codon substitutions that reduced susceptibility >2-fold compared to WT. A114S was an emergent substitution that when combined with other substitutions further reduced susceptibility >2-fold. Viruses containing A114S in combination with M184V did not replicate in primary blood mononuclear cells (PBMCs), consistent with the rare occurrence of the combination in clinical samples. While A114S conferred reduced susceptibility to ISL, it increased susceptibility to approved nucleoside reverse transcriptase inhibitors (NRTIs). This differential impact of A114S on ISL, an NRTTI, compared to NRTIs likely results from the different mechanisms of action. Altogether, the results demonstrate that ISL has a high barrier to resistance and a differentiated mechanism compared to approved NRTIs.

Purification of untagged HIV-1 reverse transcriptase by affinity chromatography.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Review of Doravirine Resistance Patterns Identified in Participants During Clinical Development.

BACKGROUND: Doravirine (DOR) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) approved for the treatment of HIV-1 infection in patients with no known DOR resistance-associated mutations. DOR was rationally designed to address limitations associated with other approved NNRTIs, particularly resistance from common NNRTI resistance-associated mutants containing K103N, Y181C, or G190A reverse transcriptase substitutions. SETTING: Data to date from both in vitro studies and clinical trials have been compiled to summarize the resistance profile of DOR. METHODS: We analyzed data from in vitro studies and phase 2 and 3 trials to assess the emergence of resistance-associated mutations and their impact on efficacy among participants treated with DOR. RESULTS: DOR exhibited a distinct resistance profile compared with efavirenz and rilpivirine in vitro and in vivo; mutant viruses that were resistant to DOR showed limited cross-resistance to efavirenz and rilpivirine. In clinical trials, the development of DOR resistance-associated substitutions in reverse transcriptase was uncommon. CONCLUSION: Overall, minimal cross-resistance across NNRTIs was observed for DOR and limited development of DOR-related resistance. These data should assist clinicians in further understanding the resistance profile of DOR, so appropriate treatment decisions can be made for their patients.