Matriptase is a large transmembrane serine protease, originally isolated from the extracellular domain of human mammary epithelial cells. Overexpression of this membrane bound protease on cancer cells functions in the degradation of basement membrane extracellular matrix. Matriptase is also known to activate hepatocyte growth factor by a specific proteolytic cleavage of the inactive pre-pro/growth factor. Matriptase also activates urokinase-type plasminogen activator, and the protease activated receptor-2 (Par-2). Based on these biological functions of Matriptase, developing inhibitors to matriptase may provide therapeutic applications for prevention of cancer cell invasion and metastasis.
Our aim i n this project is to develop selective inhibitors to Matriptase, as a way of inhibiting the metastatic and oncogenic cell proliferation We discovered that a bicyclic cage-like conformationally restricted, and proteolytically stable cyclopeptide, termed SFTI-1, inhibited the matriptase enzyme, with a very impressive Ki of 0.92 nM. Our current goals are two-fold. 1. to modify the current therapeutic candidate, SFTI-1, such that it selectively inhibits matriptase. And 2. To evaluate the prototype cyclopeptide, SFTI-1 in in vivo systems. We peptidomimetically modified SFTI-1 in order to improve its proteolytic stability. The 12 new analogs will be evaluated with various serine proteases, in order to achieve better matriptase selective enzyme inhibition. Our collaborators at the Lombardi Cancer Center evaluated SFTI-1 in matriptase expressing tumor bearing mice. Results demonstrate the significant survival promoting effect of as little as 10 ug SFTI-1 per day treatment of mice with implanted ovarian tumor cells. Overexpression of Her-2 (neu/erbB2) is found in 25-30% of human breast cancers and correlates with more aggressive tumors and a poorer prognosis. Our molecular target is the Grb2 protein that links the Tyr-phosphorylated segments of the GF-receptor to the protein complex of the oncogenic ras activation pathway. Our lead compound, typified by G1TE, is a 10 amino acid long cyclic peptide, originally discovered by phage library methodologies. This agents inhibits the association of ErbB2 and EGFr with the adaptor protein Grb2, with specificity. Our specific focus is the development of conformationally compact cyclopeptides that do not require a phosphotyrosine motif or its phosphonate analog. In order to achieve high affinity, we considered both intramolecular hydrophobic and hydrophilic interactions within the inhibitor, including the oxidation state of the cyclization linker, the sulfur atom. Two recently discovered agents merit mentioning that we recently developed: 1. RRK-132, G1TE(Gla1,Ach4,NPG8)S=O-(R) (IC50 = 75 nM); and RRK-168, G1TE(Gla1,Phe(4-Me)2, Ach4, NPG8, Phe(4-NH2)9, S=O)-(R)) (IC50 = 28 nM). These new analogs are unique in that binding affinity to the Grb2 SH2 domain is not dependent on the phosphotyrosine motif, and these agents are conformationally restricted by virtue of being cyclized. Several of these newly designed agents are inhibitory at low nanomolar concentrations as demonstrated by Biacore and by ELISA binding assays. More significantly, one of the extensively modified analog, termed RRK-169, was effective in inhibiting Grb2-SH2 domain association with the erbB2 receptor in intact MDA-453 breast cancer cells, at 10 uM concentration, demonstrating at the same time that this agent is also able to cross the cell membrane. These agents merit a more thorough biological evaluation, especially in various ErbB2 overexpressing cell lines. 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. Integrase does not have an obvious cellular counterpart, therefore inhibitors developed to it may not be toxic to mammalian cells. The complex mechanism of integrase function poses a challenging opportunity for enzyme inhibitory design. We have focused for some time to develop anti-HIV therapeutics by designing inhibitors to the HIV specific enzyme, integrase. Our lead compound for this project was a 6-amino acid long tryptophan rich peptide, discovered by Plasterk using combinatorial chemistry methodology. Carrying out systematic SAR studies we discovered that all D-amino acid containing peptides were favored for better inhibitory activity. Further studies demonstrated that oligomerization of selected peptides provided a 200-fold improvement in inhibitory activity (IC50 = 0.50 microM, 3'-processing). The mechanistic implications of inhibitory action suggest that the tetrameric inhibitory peptide may act by simultaneously occupying several neighboring catalytic sites in tandem, with entropic advantage, within the integrase oligomeric complex. Based on this new observation, we are synthesizing a variety of covalently linked metabolically stable multimeric structures for evaluation. We also embarked on developing reverse transcriptase inhibitors, by further development of an agent discovered by Vinay Pathak's group, on screening the NCI chemical repository. This very rigid chair shaped molecule was re-synthesized in our laboratory, its enantiomers separated by chiral HPLC, and the X-ray structure of one of the analogs determined. This very rigid RT inhibitory agent provides a new pharmacophore model for developing new types of reverse transcriptase inhibitors.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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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
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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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