ABSTRACT The HIV-1 capsid protein (CA) is an attractive target for the development of novel antiretrovirals due to its essential and multifaceted roles in the virus biology. Lenacapavir (LEN) is a recently discovered first-in-class, long-acting capsid-targeting inhibitor with picomolar potency against HIV-1. Phase 2/3 clinical trials have revealed that subcutaneous administration of LEN with a six-month dosing interval enables high rates of HIV-1 suppression in heavily treatment-experienced patients infected with multi-drug resistant viral phenotypes. These findings have highlighted LEN as a promising agent that could complement current antiretroviral compounds to treat people living with HIV-1. However, cell culture-based viral breakthrough assays and clinical trials have identified a number of CA substitutions that confer substantial resistance to LEN. Specifically, Q67H/N74D and M66I substitutions emerged as major drug-resistance associated variants. Therefore, there is a need to develop improved LEN analogs with a higher barrier to resistance. We have established synthesis of LEN in academic setting by utilizing a modular approach of independently preparing three unique LEN subunits (A, B, and C) from commercially available reagents. Such modular approach allows for straightforward modifications of each subunit which can then be combined in any order to prepare LEN and its analogs. Thus, our medicinal chemistry strategy is highly advantageous for analog development. Our complementary virology, biochemistry and structural biology experiments have allowed us to characterize a multi-modal mechanism of action of LEN. Furthermore, we have recently determined high-resolution X-ray crystal structures of CA hexamers containing major drug-resistance associated Q67H/N74D and M66I changes. We propose to exploit these findings to rationally develop improved LEN analogs with a higher barrier to resistance. In Aim 1, our efforts will focus on modifications to LEN subunits A and C to overcome steric hindrance and electrostatic repulsions created by the drug resistant Q67H/N74D CA variant. In aim 2, we will use our recent promising findings from MiniFrags screening studies, which identified fragments that bind to the CA hydrophobic pocket in close vicinity to LEN. We will synthesize new chemotypes by connecting these fragments to LEN to generate second-generation analogs to target the major drug-resistant M66I variant. Newly synthesized compounds from both Aims 1 and 2 will be evaluated by a stepwise approach using antiviral activity, cytotoxicity, surface plasmon resonance and X- ray crystallography to identify and characterize the lead compound(s). Taken together, the proposed research is expected to generate novel chemotypes with improved antiviral activities against major drug resistant CA variants that confer substantial resistance to parental LEN.