X-ray crystallographic analyses of human alpha-thrombin complexed to peptidy aminophosphonates: Evidence of a binding mechanism

We sought to study O,O-Diphenyl dipeptidylaminophosphonates and their inter action with the coagulation protease, human a-thrombin. The 'fibrinogen-lik e' recognition sequence D-Phe-Pro-Arg, is a template of high affinity for t hrombin. This was modified by replacing the 'P1' Arg by a neutral side chai n, and the unnatural amino acid diphenylalanine was used in place of the 'P 3' Phe. The tripeptides of general formula D-Dpa-Pro-NHCHRP(OPh)(2) are hig hly selective for thrombin amongst other serine proteases; and, more import antly fbr the design of an efficacious anti-thrombotic agent, show particul arly low activity toward other coagulation serine proteases of the cascade. However the kinetics of thrombin inhibition were seen to be dependent on t he compounds' structure. To explain this we have studied compounds (1), whe re R is pentyl and compound (2) where ii is (3-methoxy)propyl, for which th e kinetics of inhibition were analysed as competitive (2 mu M) and slow, ti ght-binding (final K-i 20nM) respectively. Analysis of the X-Ray crystal st ructure of these compounds complexed to human a-thrombin, unusually shows n o covalent bond formed between the phosphorus nucleus and the serine 195 in the catalytic site of the enzyme. These observations are at variance to th e proposed mechanism of action of other phosphorus based serine protease in hibitors.