Design of Weakly Basic Thrombin Inhibitors Incorporating Novel P1 Binding Functions:  Molecular and X-ray Crystallographic Studies

Abstract

To prepare weakly basic thrombin inhibitors with modified S1 anchoring groups, two series of compounds were synthesized by reaction of guanidine or aminoguanidine with acyl halides and N,N-disubstituted carbamoyl chlorides. pK(a) measurements of these acylated guanidines/aminoguanidines showed a reduced basicity, with pK(a) values in the range of 8.4-8.7. These molecules typically showed inhibition constants in the range of 150-425 nM against thrombin and 360-965 nM against trypsin, even though some bulky derivatives, such as N,N-diphenylcarbamoylguanidine/aminoguanidine and their congeners, showed much stronger thrombin inhibitory activity, with inhibition constants in the range of 24-42 nM. Unexpectedly, very long incubation times with both proteases revealed that aminoguanidine derivatives behaved as irreversible inhibitors. To assess the molecular basis responsible for the high affinity observed for these molecules toward thrombin, the crystal structure of the thrombin-hirugen-N,N-diphenylcarbamoylaminoguanidine complex has been solved at 1.90 A resolution. The structural analysis of the complex revealed an unexpected interaction mode with the protease, resulting in an N,N-diphenylcarbamoyl intermediate covalently bound to the catalytic serine as a consequence of its hydrolysis together with the release of the aminoguanidine moiety. Surprisingly, in this covalent adduct a phenyl group was found in the S1 specificity pocket, which usually recognizes positively charged residues. These findings provide new insights in the design of low basicity serine protease inhibitors.

Recent developments in guanylating agents

“…3e Isolation of guanidine compounds as metabolites provides leads for the prevention of metabolic disorders and helps the prognosis of cancer, cardiovascular diseases, diabetes etc. 3f Recent examples of synthetic, biologically-active guanidines include antimicrobial activity, 4a,b thrombin inhibitors, 5 Na + /H + exchanger (NHE) inhibitors, 6a,b transport for the delivery of anti-cancer agents, 7a,b anti-influenza agents.…”

A Simple Yet Multifaceted Enzyme

“…The activator field, still in its infancy from the pharmacologic viewpoint, may lead to a better understanding of cognition but may as well lead to applications in therapeutic areas, such as phobias, obsessive-compulsive disorder, generalized anxiety, and post-traumatic stress disorders, for which few efficient therapies are available. Thus, my involvement for more than 30 years in the research field of just a single, yet highly multifaceted enzyme, may be considered of some relevance, although I was and am working on various other drug targets, such as metalloproteases [131][132][133][134][135], serine proteases [136,137] or viral proteases [138][139][140]. However, the work on CAs remain the most well-known one.…”

A Statistical Survey on the Binding Constants of Covalently Bound Protein–Ligand Complexes

“…Case 4: a two‐step mechanism in which a reversible noncovalent complex ( EI ) forms first, and then it transforms irreversibly into a covalent complex ( EI* ) 27 Case 5: another two‐step mechanism, in which a reversible covalent complex ( EI 1 * ) forms first, and then it transforms irreversibly into another covalent complex ( EI 2 * ) 28 …”