Characterization of the interaction between human alpha-thrombin and methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carboxylate (LY806303) using electrospray mass spectrometry and tandem mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) have been used for the first time to study the interaction of human alpha-thrombin with methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carbox ylate (LY806303; 1), a potent and selective inhibitor whose mechanism of action was never fully defined. Using ESI-MS, it is shown that inhibitor 1 covalently modifies human alpha-thrombin as evidenced by a shift in the molecular weight of the native protein by 72 Da, which is consistent with isobutyrylation (C4H7O; 71 Da) of the enzyme at a single site. Tryptic digestion of the modified protein and tandem mass spectral analysis of isolated peptide fragments indicate that compound 1 acylates Ser-205 of the heavy chain of alpha-thrombin. Ser-205, along with His-43 and Asp-99 make up the catalytic triad within the active site of thrombin.