Preliminary analysis of a defined combinatorial library of inhibitors targeted at aspartic proteases with similarity of the HIV-1 protease has identified lead structures with potent in vitro activity against Giardia lamblia. Additionally, the active compounds are strong inhibitors of a protease that have been partially purified from cell extracts. This represents a new target for therapeutic intervention against this important parasite. The ease of achieving success with this technique demonstrates the utility of coupling combinatorial chemistry approaches to synthesis with high- throughput screening with defined assays. The long range goal of this method is to develop a systematic, coordinated process by which either thematic or targeted libraries of compounds can be rapidly analyzed and utilized as leads for new drug development in any biological system. The first attempt has resulted in identifying a new therapeutic target for G. lamblia and lead structural motifs from the original combinatorial library. The next step is to refine the target and hence allow the refinement of the inhibitors. This will be accomplished in this phase by molecular cloning of the gene to the target protease enzyme form G. lamblia, overproducing its product, and utilizing this target for more detailed biochemical analysis. Success at the Phase I stage will lead the way to subsequent development efforts of a new treatment for giardiasis, namely further refinement of the inhibitor, initial clinical evaluation of the compound, including specificity, toxicity, metabolism, pharmacokinetic, and distribution studies of the lead compound. Specifically, the aims of the Phase I STTR proposal are: 1. Clone the gene encoding the aspartic protease inhibited by the novel inhibitors form Giardia lamblia. 2. Perform DNA sequence analysis of the gene so that the sequence can be compared with other consensus aspartic porteases that have been crystallized such as the HIV-1 protease, pepsin, and cathepsin D. 3. Engineer the gene to allow the overproduction and purification of large amount of this recombinant product. 4. Begin detailed biochemical analysis of the lead inhibitor against the recombinant product. 5. Design of second generation compounds. 6. Synthesis of second generation compounds.
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| Zwick, Michael B; Parren, Paul W H I; Saphire, Erica O et al. (2003) Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120. J Virol 77:5863-76 |
