Gregory Alan Weiss, UC Irvine

This project proposes the development of inhibitors targeting HIV Nef. Nef weakens the immune system, enhances the pathogenicity of HIV, and contributes to the transition from HIV infection to AIDS, which suggests Nef inhibitors could provide a useful adjunct to other anti-HIV therapies. The experiments proposed here apply combinatorial chemistry and medium throughput screening to identify a new class of anti-HIV candidate drugs. Next biophysical techniques, including combinatorial mutagenesis and NMR structure determination, will be used to characterize the molecular basis for Nef inhibition. In turn, detailed structural information will guide the design and synthesis of improved inhibitors.

The PI's laboratory has recently reported the first small molecule inhibitors effective against Nef.
Versatile assays have been developed to identify inhibitors capable of disrupting Nef binding to CD4, Fyn, p56lck, actin, p53, or MHC-1. In published preliminary results, we (in collaboration with the Robinson laboratory) have already demonstrated four sensitive, cell-based assays for quantifying the affects of perturbing Nef function at different stages of the HIV lifecycle. The initial leads identified from these assays, guanidine alkaloids of the batzellidine and crambescidin class, proved too cytotoxic for cell culture studies. This likely resulted from the anti-cancer pedigree of the initial lead comopunds. More recently, the PI's laboratory has screened thousands of additional compounds to uncover new Nef inhibitors. This strategy of using an initial lead as a toe-hold to improve anti-Nef inhibition forms the basis for the proposed Specific Aim 1.

In Specific Aim 2, anti-Nef inhibitors will be characterized structurally to elucidate the molecular basis for inhibition. This specific aim leverages the PI's extensive experience with phage-displayed combinatorial mutagenesis. Here, alanine scanning will be used to identify specific sidechains responsible for the interactions of Nef inhibitors with phage-displayed Nef. Such structure-function analysis will be combined with NMR structural analysis to characterize specific functional groups mediating small molecule binding to Nef. This information, in turn, will inform design of the next generation of anti-Nef compounds.

Thus, the proposed pilot studies could guide development of a new class of anti-HIV pharmaceuticals and potentially uncover a useful new tool for better characterization of Nef contributions to HIV.