ABSTRACT Despite continuous progress in anti-HIV therapy, drug toxicity and emergence of drug-resistant isolates during long term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel anti-viral agents. A number of reports demonstrated that both HIV entry into and exit from the target cells involves cholesterol-rich micro domains called lipid rafts. The HIV protein Nef evolved to regulate the abundance of lipid rafts by targeting the main cellular cholesterol transporter, ABCA1. Nef in HIV-infected cells interacts with the endoplasmic reticulum chaperone, calnexin, to disrupt calnexin interaction with ABCA1, thus impairing ABCA1 maturation and sending it to a degradation pathway. As a result, cholesterol efflux is inhibited, cells accumulate cholesterol and the abundance of lipid rafts is increased promoting virus assembly and release. Similarly, Nef delivered to uninfected cells by extracellular vesicles (EVs) released from HIV-infected cells suppresses ABCA1 activity and increases the abundance of lipid rafts, increasing susceptibility to HIV infection. In addition, widespread suppression of ABCA1 and increased abundance of lipid rafts in myeloid cells exacerbate inflammatory responses that underlie HIV- associated co-morbidities. Our recent studies identified a small molecule, NSC13987, that thwarted the interaction of Nef and calnexin and reversed the deleterious effects of Nef on cholesterol metabolism. However, the anti-HIV activity of this compound has not been tested, and solubility issues prevented its testing in the in vivo models. This proposal is focused on improving the biological activity and aqueous solubility of the lead molecule via computationally-guided chemical changes. We predict that the proposed research will improve drug likeness of NSC13987. Aim 1 described in this proposal is focused on the design, synthesis and chemical characterization of NSC13987 analogues. We also propose to study the pharmacokinetic properties of the most promising analogue. Aim 2 will involve in vitro and in vivo testing of the novel analogues using human CD4+ T cells, monocyte-derived macrophages and HIV-infected humanized mice. The proposed studies are highly significant since they will provide new therapeutic options to minimize HIV replication and HIV-associated co-morbidities. The proposal incorporates expertise in the area of synthetic medicinal chemistry (Dr. Kulkarni), biological screening (Dr. Bukrinsky), and computer-assisted drug design (Dr. Adzhubei). It is fully consistent with the goals of this PA and is expected to define anti-HIV compounds working through a novel mechanism different from that of any other currently used drug.