Carole Bewley LBC, NIDDK, NIH Annual Report, Fiscal Year 2000 Current research focuses on several projects, all of which involve either the identification of small-molecule inhibitors, studies of the mechanism of action of such inhibitors, or both. Systems we are studying include human-immunodeficiency virus 1 (HIV-1) envelope (Env)-mediated fusion, and mycothiol biosynthesis and mycothiol-conjugate-dependent detoxification. During the past 8 months (my lab was set up during January, 2000) we have established a screening effort where we are testing biodiverse marine plant and invertebrate extracts for inhibtion in 1) a vaccinia virus-based reporter gene assay that measures HIV 1 Env-mediated fusion; and 2) a fluorescence assay that measures the enzymatic activity of mycothiol-conjugate amidase (MCA), an enzyme that is critical for redox control and detoxification in mycobacteria. To date we have tested approximately 1500 extracts in each assay. Active extracts are prioritized for further isolation and structure elucidation based on ancillary testing data, including cytotoxicity and cytolytic activity, provided to us by the Natural Products Branch of NCI. We have several leads for both fusion-blocking and MCA ihibitory compounds: In the case of inhbitors of MCA, we have so far identified two unrelated classes of compounds, specifically bromo-tyrosine derivatives and cyclic alkyl-pyridinium compounds, that inhibit MCA at micromolar concentrations. Chemistry on one other still unrelated group of inhibitors is ongoing. Results from the fusion assay have yielded several unreported sesterterpenes with cytotoxic activity, and several other leads are under way. A related area of research includes determining the structural requirements and more detailed mechnism of action of HIV 1 fusion-blocking molecules, where we are studying in some depth the potent HIV-inactivating protein cyanovirin-N (CV-N). CV-N exerts its potent fusion blocking activity by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Carbohydrates on gp120 were suspected to play a role in CV-N/gp120 interactions, but experimental evidence has been lacking. By using the fusion assay, we have determined that CV-N recognizes and binds with high affinity to two specific oligomannose structures, and that recognition requires a tri-antennary branch where the 1,3 terminal mannosyl units must be present. Moreover, these specific branched oligomannose structures act as divalent ligands to CV-N. Given the terms that the surface of gp120 comprises mainly unordered loops and N-linked carbohydrates, our results strongly suggest that CV-N/gp120 interactions are mediated by these high affinity interactions with oligomannose (JBC, submitted). Despite a complete lack of sequence and structural homology with any other proteins, including mannose-binding proteins, a carbohydrate-binding motif comprising four key residues is present in CV-N. Further studies including mutagenesis and co-crystal complex formation are underway. Last, by making use of dipolar couplings and NMR spectroscopsy, and rigid-body docking and minimization, we determined the solution structure of the domain-swapped dimer of CV-N. (Bewley, CA and Clore, GM. JACS, 2000).
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