Oxyanion-Mediated Inhibition of Serine Proteases^,

Abstract

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].

Structural binding evidence of the trypanocidal drugs Berenil® and Pentacarinate® active principles to a serine protease model

“…In ␤-tryp/dmz-B, the electron density around Gln192 side chain is continuous and well defined, suggesting the occurrence of a single conformation. This latter Gln192 side chain conformation (␤-tryp/dmz-B) is particularly different from those observed in ␤-tryp/pnt and ␤-tryp/dmz-A, as well as in a number of previously reported ␤-trypsin/ligand complex structures (PDB IDs: 1BJU and 1BJV [1], 1MTS, 1MTU, 1MTV and 1MTW [19], 1QB1, 1QB6 and 1QBN [12], 1C2F, 1C1T and 1C1Q [11], 1PPH [20], 1F0U [21], 1OYQ [22], 1K1I, 1K1J, 1K1M, 1K1N and 1K1P [13], 2ZDK, 2ZDL, 2ZDM and 2ZDN and 2ZFS; 1Y3U, 1Y3V and 1Y3W [23]). However, Katz et al [15] described an almost identical conformation for this amino acid residue in the crystallographic structure of bovine ␤-trypsin in complex with the ligand 124 (2-(2-hydroxy-phenyl)-1H-indole-5-carboxamidine) (PDB ID 1GI6 [15], rmsd of 0.36 Å for all Gln192 side chain atoms).…”

“…Trypsin-like serine proteases play critical roles in many physiological processes, such as blood coagulation, complement activation, digestion, fibrinolysis, kinin formation, reproduction and phagocytosis [1]. This class of enzymes is an important target for contemporary medicinal chemistry, as many diseases are related to a malfunctioning in the regulation mediated by these enzymes [2].…”

“…In particular, benzamidine, a structural mimic of arginine and itself a potent inhibitor [8], is the primary component of many larger inhibitors [9]. In fact, potency and specificity for different serine proteases can be attained by incorporating the small inhibitor benzamidine into larger and more complex synthetic inhibitors [1].…”

“…They maintain the benzamidine scaffold essential for binding into the specificity pocket of the enzyme but present a number of variations either in their terminal chemical groups (benzimidazole-, amidinobenzimidazole-, pirrolydine-, naphthyl-, cyclopentyloxy-) or in the linker moieties (urea-, amido-, methylene-, saccharides-, peptidyl-, pyrrolizin-, benzoimidazol-, benzoyl-, piperidynil-, acetimidoyl) which connect the benzamidine and the terminal groups. Some of these compounds have been tested for their activities against serine proteases (particularly trypsin) with binding affinities varying between the sub-nanomolar [11][12][13][14] and the micromolar range [1,11,12,15]. Nevertheless, only few studies concerning the binding of bis-benzamidine compounds to serine proteases [9,12,16] have been reported.…”

“…Additionally, isothermal titration calorimetry (ITC) assays performed on the binding of pentamidine to the enzyme allowed a complete thermodynamic description of the interaction. In the light of previously described thermodynamic data [2,5] and crystallographic structures of trypsin in complex with benzamidine-based compounds (PDB IDs: 1BJU and 1BJV [1]; 1MTS, 1MTU, 1MTV and 1MTW [19]; 1GI4, 1GI5 and 1GI6 [15]; 1QB1, 1QB6 and 1QBN [12]; 1C2F, 1C1T and 1C1Q [11]; 1PPH [20]; 1F0U [21]; 1OYQ [22]; 1K1I, 1K1J, 1K1M, 1K1N and 1K1P [13] and 1Y3U, 1Y3V and 1Y3W [23]), a comparative analysis with related complexes is presented in order to establish direct correlations between bindings mode and binding affinity in benzamidine-based platforms.…”