Characterization of new matrix metalloproteinases inhibitors from westerdykella multispora F50733

Abstract

Matrix metalloproteinases (MMPs) have been implicated in a variety of disease states, including rheumatoid arthritis, periodontal disease, tumor invasion, and metastasis. In the MMP family, MMP-2 (gelatinase A) is unique in its ability to cleave type Ⅳ collagen, a principal structural components of basement membrane, and has been focused on as a pharmacological target. Consequently, specific inhibitors of MMP-2 may be of value in the therapy of cancers as well as other disease states involving tissue remodeling. ProMMP-2 was purified from a conditioned media of T98G human glioblastoma cells. Tissue inhibitor of metalloproteinase-2 (TIMP-2) complexed proMMP-2 and TIMP-2 free proMMP-2 were separated by heparin affinity HPLC. The proMMP-2 was homogeneous on SDS-PAGE, with a molecular weight of 64-kDa without reduction. In addition, TIMP-2 and proMMP-2 were separated from the TIMP-2 complexed proMMP-2 by reverse phase HPLC in the presence of trifluoroacetic acid. Two high-throughput screening assay methods were developed with the purified proMMP-2. As results, a fluorescence detection method using 96-well microplate was used as a primary screening system and a direct reaction injection method was used for the confirmation of inhibitory activities of screening samples. In the course of screening for MMP-2 inhibitors from fungi, a fungal strain F50733 was selected. The strain F50733 was identified as Westerdykella multispora on the basis of its cultural and morphological characteristics. The inhibitors of MMP-2, designated gelastatins A and B, and their precursor, dykellic acid were isolated from the culture broth of W. multispora F507343 by successive chromatographies and preparative HPLC. The structures of gelastatins A and B, and dykellic acid were determined by spectroscopic analysis of UV, IR, FAB-MS, $^1H-$, $^{13}C-NMR$, DEPT, HMQC, HMBC, and NOESY. Gelastatins A and B were determined to be 3-(5E-hexa-2E,4E-dienylidene-2-oxo-5,6-dihydro-2H-pyran-3-yl)-pr...