The present proposal will extend ongoing efforts in developing HIV1 inhibitors to block the interaction of the HIV1 envelope protein with its cellular receptor, the human CD4 surface glycoprotein, to prevent the subsequent virus-cell fusion. In prior funding periods, we have produced, crystallized and determined the structure of the amino terminal two domains (D1 and D2) of CD4 (sCD4/1-183) and HIV-gp41 to high resolution. Mutational analysis has defined a 900 A/2 surface on the CD4 D1 C'C"""""""" ridge where HIV-gp120 binds and a additional area on D and D2 were various class II MHC molecules interact. Class II MHC binding, unlike gp120 binding, is dependent on oligomerization of CD4 through its membrane proximal D3-D4 module. Here three groups of investigators will utilize their talents in immunology, crystallography and NMR spectroscopy to pursue structure-based design efforts. The Reinherz group will characterize CD4-based class II MHC binding, both in vitro and in vivo, using transgenic mice bearing hCD4 point mutants in the murine CD4 -/- background. They will engineer and purify Lec3.2.8.1-produced gp120 (gp100) derived from SF2 and ADA, T- and macrophage-tropic HIV-1 strains, respectively. In complex with sCD4/1-183 and mAbs derived from mouse and human sources, these structures will be resolved crystallographically by the Harrison group. Analysis of such complexes will provide detailed information about the CD4 binding site as well as neutralizing antibody responses to HIV. Structure analysis of the corresponding uncomplexed gp120 trimers will also be attempted to ascertain information about the pre-fusion gp41 state and of the native gp120 component. Subsequently, generation of gp120 and gp160 mini-proteins amenable to NMR analysis for drug screening will be attempted. The Wagner group will perform structure activity relationship by NMR (SAR by NMR) on sCD4/1-183, whose solution structure they have already completed and on gp41 whose structure who underway. For this purpose, a library of small, drug-like organic compounds is being screened. The binding mode of two compounds which interact at adjacent sites of the /15N-labeled proteins will be determined and compounds chemically linked to yield high affinity binders (Kd approximately 10-/9 M). Successfully engineered mini-proteins will be screened by this method. The anti-viral and/or immunosuppressive nature of potential lead compounds will be assessed.
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