Emerging drug resistance of HIV-1 to current antiretroviral therapies requires the development of new drugs. By targeting the interaction of HIV-1 proteins with host cell proteins, it may be possible to minimize the drug resistance problems associated with current therapies targeted to viral enzymes, since viral proteins that interact with human proteins cannot freely mutate in order to conserve binding affinity. The HIV-1 accessory protein Nef makes critical interactions with Src family tyrosine kinases that have been implicated in the HIV lifecycle and may represent an important target for the disruption of HIV-1 infection. By making single point mutations in the SH3 domains we have identified a small pocket located in Nef in which small molecules could bind. Compounds were screened by isothermal titration calorimetry to identify non-peptidic small molecule scaffolds that inhibit Nef/SH3 binding. In addition, NMR chemical shift perturbation experiments were used to identify residues from Nef that are involved in binding the small molecule inhibitors and confirmed that the residues that we expected were affected upon binding the small molecule.

In addition, NMR experiments identified an allosteric path in Nef between the SH3 and CD4 binding sites. This pathway was affected by the addition of Lck-SH3 or that of the small compound, EPF-0037. This makes sense considering the connection between Nef, CD4, and Lck-SH3 in the HIV life cycle and the increase of affinity for CD4 in the presence of SH3 domains This thesis also addressed whether the binding affinity of Nef for CD4 was increased in the presence of the small molecular weight inhibitor. Since a cooperative path between the SH3 and CD4 binding sites are observed by NMR, the importance of the N-terminal arm in Nef binding Hck-SH3 was examined. Isothermal titration experiments were performed to shed light on which residues could be important in propagating this affect and possibly structuring the SH3 binding site.