PROJECT SUMMARY: While currently available antiretrovirals block viral replication and thus control HIV-1 infection, they do not cure the disease; latent reservoirs of replication-competent virus persist. To eradicate HIV-1 infection, novel an- tiretrovirals must be developed. These drugs would ideally induce the killing of infected cells once latency is reversed. An attractive direction in developing such antiretrovirals is the inhibition of the HIV-1 Nef protein. By modulating surface-levels of immune receptors, Nef enables infected cells to evade host defense mechanisms. Among the many functions of Nef, surface downregulations of CD4 and major histocompatibility complex class I (MHC-I) are the most prominent and presumably most relevant in antiretroviral drug discovery. By downregu- lating CD4 from the cell surface, Nef enables CD4-induced epitopes of the viral Env protein to remain con- cealed, which renders infected cells less sensitive to antibody-dependent cellular cytotoxicity (ADCC). By downregulating MHC-I, Nef disrupts host antigen presentation so that infected cells are protected from killing by cytotoxic T lymphocytes (CTLs). Conceivably, therapeutic inhibition of these Nef functions may restore the activities of ADCC and CTLs, thus facilitating the detection and clearance of infected cells. Crystal structures solved by us showed that Nef-mediated downregulations of CD4 and MHC-I involve a common site on Nef. In each case, however, this site is remodeled by Nef’s association with target-specific, hijacked clathrin adaptor proteins (APs) to uniquely accommodate the intended substrate. Furthermore, when bound to Nef, both the CD4 cytosolic tail and the MHC-I cytosolic tail adopt curved, near-circular postures, which suggests that this multifunctional site of Nef is structurally poised to bind curved peptide sequences. We therefore hypothesize that cyclic peptides mimicking these cytosolic tails may function efficiently as inhibitors to block the correspond- ing cellular activities of Nef. Cyclic peptides (or cyclopeptides) are a promising, novel class of therapeutics, which are uniquely capable of disrupting protein-protein interactions. Importantly, high-affinity cyclopeptide in- hibitors can be developed efficiently using recently established strategies. In this project, we will develop such cyclopeptide-based Nef inhibitors. Our specific aims are: 1) use our established MOrPH-PhD platform to screen and select CD4-mimetic cyclopeptide inhibitors that bind to the Nef/AP2 complex in high affinity, and solve high-resolution crystal structures of the cyclopeptide-bound Nef/AP2 complexes to enable structure- based derivatization of the inhibitors; 2) use the same work flow to develop and optimize MHC-I-mimetic cyclo- peptides into potent inhibitors, which block recruitment of MHC-I into the Nef/AP1 complex; 3) use cell-based assays to characterize the identified Nef inhibitors for their efficacy, cell permeability, and cellular toxicity, ...