RESUMO
Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.
Assuntos
Ciclotídeos/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cães , Impedância Elétrica , Células HEK293 , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Células Madin Darby de Rim Canino , Masculino , Terapia de Alvo Molecular , Invasividade Neoplásica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Precursores de Proteínas/metabolismo , Serina Endopeptidases/genética , Especificidade por Substrato , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/enzimologia , Fatores de Tempo , TransfecçãoRESUMO
Stereospecific deprotonation of the epoxy proton at the beta-position of the alpha,beta-epoxy esters 5 and 6 yielded oxiranyl "remote" anions 7 and 8, which could then be used for alkylation. The anions 7 and 8 underwent a consecutive aldol lactonization to give, respectively, epoxy lactones 11 and 13 with high stereoselectivity. Generation of the remote anions as well as their stereoselective reactions served as a new synthetic route to the naturally occurring alpha-methylenebis-gamma-butyrolactones, 1.