Our molecular target is matriptase. Matriptase is an epithelial cell derived, integral membrane serine protease. This enzyme was originally isolated by our biologist collaborators at the Lombardi Cancer Center (Dr. Robert Dickson), from human breast cancer cells in culture, and has been implicated in breast cancer invasion and metastasis. Dickson et al demonstrated that matriptase can convert hepatocyte growth factor /scatter factor to its active form and it can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells, and thus function in extracellular matrix degradation and epithelial cell migration. Our specific objective is to develop selective inhibitors of matriptase, as antimetastatic agents. We have identified a small disulfide bridged bicyclic-peptide natural product, termed sunflower derived trypsin inhibitor (SFTI-1), that we then found to inhibit matriptase with high potency (Ki of 0.92 nM). We developed a facile synthesis of this cage-like molecule that appears to have considerable proteolytic stability. SFTI-1 serves as a lead compound for further design. An important parameter for success is to develop effective protease inhibitors to matriptase and possibly to related type II transmembrane serine proteases, but not to serine proteases, such as trypsin, thrombin, or Factor Xa, that are required for normal physiological function. Using homology modeling based design, we synthesized 12 additional analogs. These new agents were evaluated for inhibitory potency to matriptase, and the promising analogs will be assayed for selectivity to the various serine proteases. Plans have been made to evaluate the effect of selected SFTI-1 analogs in xenograph models of human ovarian malignant ascites in the nude mouse model. In related efforts, we developed peptidomimetic agents that inhibit cellular signaling, initiated by the erbB2 growth factor receptor mediated oncogenic ras activation pathway. Our lead compounds, typified by G1TE, are 10 amino acid long cyclic peptides, originally discovered by phage library methodologies. These agents selectively inhibit the association of ErbB2 and EGFr with the adaptor protein Grb2. The newly developed analogs are unique in that binding affinity to the Grb2 SH2 domain is not dependent on the phosphotyrosine motif, and our peptides are conformationally restricted by virtue of being cyclized. Our newly designed analogs are non-phosphorylated and do not contain a pTyr mimic, yet they are inhibitory at sub-micromolar concentrations, as demonstrated by Biacore and by ELISA binding assays. One such example is typified by the G1TE(Gla1, -tBuAla8)-linker sulfoxide analog, IC50 = 300 nM, which contains three major modifications compared to the original prototype agent. The carrier conjugated analog of G1TE(Gla1) is active in cell cultures of MDA-453 breast cancer cell lines at low micromolar concentrations. Our SAR studies, hand in hand with molecular modeling efforts and NMR solution studies on G1TE and analogs, allowed the identification of the structural motif that is critical for high Grb2 binding affinity, and is different from the prototypical pTyr binding motif. In the past year we also synthesized a number of phosphatase-stable phosphonotyrosine containing cyclic peptides, such as G1TE(A1, Pmp3, Ach4, NPG8)S=O-I) that have shown high Grb2 antagonist activities in ELISA assays (IC50 = 8 nM). These new agents and their membrane translocating carrier conjugates will be evaluated for their effect in ErbB2 overexpressing MDA-453 breast cancer cells in culture. Peptidomimetic structural design was successfully applied to the development of a new family of integrase inhibitors. Integrase enzyme plays a pivotal role in the infectious process of the HIV virus. It allows for the integration of the viral genome into its targeted host genome, and thus for the replication of the virus. The complex mechanism of integrase function poses a challenging opportunity for enzyme inhibitory design. An attractive structural model presented itself by the discovery of a 6 amino acid long Trp rich peptide, His-Cys-Lys-Phe-Trp-Trp, by Plasterk et al, using combinatorial peptide library methodologies. However, we discovered that disulfide homo-dimerization through the cysteines improved inhibitory activity approx. 10 fold (IC50 = 3.7 uM, 3'-processing). Subsequently, we prepared several dimeric analogs that indicated that the side-chain linker length was a crucial determinant for inhibitory activity. In the past year we embarked on a synthetic efforts to prepare redox stable covalently linked dimeric analogs, incorporating various lanthionine building blocks, and to evaluate these for integrase inhibitory activity. Results indicate that the all-D configuration analogs are also good inhibitors, and these promise to be proteolytically stable under physiological conditions. Our results support the structural model based on the recent X-ray structure of integrase complexed with small molecule inhibitors (Goldgur et al). It appears that in the complex of three integrase protein subunits that are complexed with two small-molecule inhibitors, the inhibitors are in near vicinity to each other. Our novel dimeric inhibitors could conceivably occupy two inhibitory sites simultaneously within the trimeric or tetrameric integrase complex. X-ray structural studies are planned to test this hypothesis.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Sun, Haiying; Nikolovska-Coleska, Zaneta; Chen, Jianyong et al. (2005) Structure-based design, synthesis and biochemical testing of novel and potent Smac peptido-mimetics. Bioorg Med Chem Lett 15:793-7
Koehler, Niklas K U; Yang, Chao-Yie; Varady, Judith et al. (2004) Structure-based discovery of nonpeptidic small organic compounds to block the T cell response to myelin basic protein. J Med Chem 47:4989-97
Sun, Haiying; Nikolovska-Coleska, Zaneta; Yang, Chao-Yie et al. (2004) Structure-based design of potent, conformationally constrained Smac mimetics. J Am Chem Soc 126:16686-7
Nikolovska-Coleska, Zaneta; Wang, Renxiao; Fang, Xueliang et al. (2004) Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem 332:261-73
Krajewski, Krzysztof; Marchand, Christophe; Long, Ya-Qiu et al. (2004) Synthesis and HIV-1 integrase inhibitory activity of dimeric and tetrameric analogs of indolicidin. Bioorg Med Chem Lett 14:5595-8
Sun, Haiying; Nikolovska-Coleska, Zaneta; Yang, Chao-Yie et al. (2004) Structure-based design, synthesis, and evaluation of conformationally constrained mimetics of the second mitochondria-derived activator of caspase that target the X-linked inhibitor of apoptosis protein/caspase-9 interaction site. J Med Chem 47:4147-50
Nikolovska-Coleska, Zaneta; Xu, Liang; Hu, Zengjian et al. (2004) Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database. J Med Chem 47:2430-40
Song, Yan-Li; Roller, Peter P; Long, Ya-Qiu (2004) Development of l-3-aminotyrosine suitably protected for the synthesis of a novel nonphosphorylated hexapeptide with low-nanomolar Grb2-SH2 domain-binding affinity. Bioorg Med Chem Lett 14:3205-8
Li, Peng; Peach, Megan L; Zhang, Manchao et al. (2003) Structure-based design of thioether-bridged cyclic phosphopeptides binding to Grb2-SH2 domain. Bioorg Med Chem Lett 13:895-9
Krajewski, Krzysztof; Long, Ya-Qiu; Marchand, Christophe et al. (2003) Design and synthesis of dimeric HIV-1 integrase inhibitory peptides. Bioorg Med Chem Lett 13:3203-5

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