Developing Cyclopeptide Nef Inhibitors to Facilitate HIV-1 Eradication

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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 antiretro- virals 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 downregulating CD4 from the cell surface, Nef enables CD4-induced epitopes of the viral Env protein to remain concealed, 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 can be targeted by cyclic peptides, a promising new class of therapeutics well-suited to disrupt protein- protein interactions. Supported by promising preliminary data, this project aims to develop small-sized macrocy- clic peptides capable of mimicking the cytosolic tails of CD4 and MHC-I and thus blocking the cellular activities of Nef through inhibition of Nef-mediated protein-protein interactions. High-affinity cyclopeptide Nef inhibitors will be developed through enabling-strategies recently established in our laboratories. Specifically, a powerful phage display platform will be applied to optimize CD4-mimetic cyclopeptide inhibitors that can bind to the Nef/AP2 complex with high affinity. In parallel, a similar workflow will be applied to develop and optimize MHC-I-mimetic cyclopeptides into potent inhibitors that can block recruitment of MHC-I into the Nef/AP1 complex. High-resolu- tion structures of the cyclopeptide-Nef complexes will be obtained to enable structure-based optimization of the Nef inhibitors. Using a panel of cell-based assays, these compounds will be characterized for their efficacy in blocking Nef functions in cells, cell permeability, and cellular toxicity, and this knowledge will be leveraged to guide further derivatization for improved cellular activity. Successful completion of this work should yield cyclic peptide-based Nef inhibitors with high affinity in vitro and significant efficacy in cells, which could ideally be developed into novel antiretrovirals with unique therapeutic potentials.