Project Summary The overarching obstacle in obtaining a functional cure for the 37.9 millions of people living with HIV globally is eliminating the HIV-1 reservoirs. Viral integrase (IN) establishes lifelong infection in transcriptionally inactive but replication competent cells by IN integrating proviral DNA into the host chromosome. The localization of integration is directed by host factor LEDGF/p75 binding IN tethering to active transcriptional gene dense regions of DNA. HIV-1 IN is one of the major antiviral targets, and multiple IN inhibitors (INSTs) have been developed as a key component of current antiretroviral therapy (ART) but are subjected to viral resistance, cross-resistance and toxicity. Rapid development of IN antivirals with high genetic barrier, new modes of action (MOA), with safe DMPK profiles are in immediate need. Allosteric IN inhibitors (ALLINIs) are a new class of IN inhibitors targeting non-catalytic sites of HIV-1 IN. STP0404 is a pyrrolopyridine-based ALLINI with outstanding efficacy, PK and safety profiles observed in both preclinical in vitro and animal investigations. STP0404 became the first-in-human (FIH) ALLINI compound demonstrating its outstanding safety and human PK profile for once-a-day oral formulations, which further supports advanced clinical evaluations for its antiviral efficacy in HIV-1 patients. To this effect, I propose to explore a new 2nd-generation ALLINI compound our laboratory designed for potentially improved genetic barrier and possible utility as an HIV-1 cure agent. I have identified A128T IN mutant rendering viral resistance to STP0404, and our X-ray structural study confirmed the longer side chain of A128T clashes against 3-methyl group at the pyrrole moiety of STP0404, contributing to the reduced binding affinity to STP0404 and viral resistance to STP0404. To avoid contact between the A128T mutant and the methyl group of STP0404, our group recently synthesized a new STP0404 derivative, EKC110, with missing the methyl group contacting the A128T side chain. Here, I propose to test the hypothesis that EKC110 shows improved genetic barrier with altered resistance mutation profiles, compared to the parental STP0404. Our preliminary data has supported this hypothesis, additionally emerging evidence has reported ALLINIs blocking LEDGF/p75 binding to IN, called LEDGINs, can drive the integration site specificity of HIV-1 toward non-actively transcribed regions of the chromosomes, reducing HIV-1 reactivation from latently infected T cell reservoirs. In Aim 1, I will investigate a new STP0404 derivative, EKC110 using molecular, virological and cell biological techniques for its genetic barrier, viral resistance, antiviral MOA to determine the impact of structural modifications on HIV-1 antiviral activity. Aim 2, I will investigate the effect of STP0404 and EKC110 on HIV-1 integration site selection and reactivation suppression from HIV-1 latently infected T cells using biochemical, molecular, and c...