Using EFdA to target HIV from various subtypes:Inhibition mechanism and efficacy against clinical resistant mutants

项目摘要

PROJECT ABSTRACT HIV is a major public health challenge that currently affects 38 million people worldwide. There are several subtypes of HIV that have reported differences in drug susceptibility and resistance profiles. Understanding such differences are important to inform prevention and treatment strategies of HIV. Patient adherence to current antivirals is a major concern, as non-adherence can lead to viral resistance mutations, which result in failure of current therapies. This work will contribute to the development of long-acting HIV regimens that are expected to improve patient adherence. This proposal uses a novel nucleoside reverse transcriptase translocation inhibitor (NRTTI), known as 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, or Islatravir). If EFdA is implemented as a therapeutic, it could contribute to long-acting regimens for the prevention and treatment of HIV on a global scale, especially in low-income countries. There have been several EFdA inhibition and resistance studies, however, these studies primarily use subtype B of HIV (HIV-B: primarily found in North America, Europe, and Japan) and HIV-B only accounts for 11% of HIV infections. In order to implement EFdA on a global scale, HIV- nonB subtypes that account for the vast majority of global HIV infections need to be studied. Other subtypes that will be studied in this proposal include CRF_AE and CRF_AG (West Africa and SE Asia), and C (sub-Saharan Africa, India, Brazil), which account for about 60% of the global prevalence of HIV. Therefore, it is critical to understand EFdA in the context of these various subtypes. The overarching hypothesis is that subtype-specific sequence differences dictate the inhibition mechanism(s) and thus virus susceptibility to EFdA. This hypothesis will be addressed in two specific aims. Aim 1 will determine the effect of clinical resistance mutations to existing drugs on the susceptibility of HIV-nonB viruses to EFdA and on viral fitness. This will be determined by dose- response curves, viral replication, competition, and viral passaging assays. In addition, the mechanism of EFdA inhibition and resistance has been primarily studied in vitro using only HIV subtype B RT. Therefore, Aim 2 will determine the in vitro and in cellulo mechanism(s) of EFdA inhibition and resistance in various HIV subtypes. These in vitro gel-based biochemical assays will utilize purified RT enzymes. In addition, a novel technique will be used to determine the in cellulo inhibition mechanism of EFdA that has single-nucleotide resolution at the 3’- termini of the reverse transcriptase products. These studies will be conducted with a variety of cell lines that enable facile development of drug resistance. This work will reveal the inhibition mechanism of EFdA in the cellular environment and EFdA efficacy across various subtypes, leading to better EFdA-based therapies that can help prevent and treat HIV worldwide.