1. The substrate specificity of proteinase B from baker's yeast
E Kominami, H Hoffschulte, L Leuschel, K Maier, H Holzer Biochim Biophys Acta. 1981 Sep 15;661(1):136-41. doi: 10.1016/0005-2744(81)90092-9.
The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The Km value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1/20 that of chymotrypsin A. Km and kcat for Bz-Arg-OEt are 1/50 and 1/7 as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.
2. Serine protease specificity for peptide chromogenic substrates
L E Mattler, N U Bang Thromb Haemost. 1977 Dec 15;38(4):776-92.
Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
3. Discovery of transition state factor Xa inhibitors as potential anticoagulant agents
B Y Zhu, W Huang, T Su, C Marlowe, U Sinha, S Hollenbach, R M Scarborough Curr Top Med Chem. 2001 Jun;1(2):101-19. doi: 10.2174/1568026013395425.
Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].
