On active duty: The available drugs for cardiovascular diseases can promote blood clotting but can also lead to life‐threatening bleeding episodes. A promising target for the development of safer alternatives is the transglutaminase Factor XIII (FXIII), the active structure of which is presented. The binding and coordination of three calcium ions induce major domain movements in the enzyme upon activation.

New peptidic Michael acceptor based cysteine protease inhibitors displaying antiparasitic activity were identified by testing a broad series of 45 compounds in total, containing Asn, Gln, or Phe. As target enzymes, falcipain-2 and -3 from P. falciparum and rhodesain from T. b. rhodesiense were used. In the case of the Asn/Gln containing compounds, the trityl-protected, diastereomeric E-configured vinylogous dipeptide esters 16 (Boc-(S)-Phg-(R/S)-vGln(Trt)-OEt) were discovered as most active inhibitors concerning both protease inhibition and antiparasitic acitivity, with inhibition constants in the submicromolar range. The compounds were shown to display time-dependent and competitive inhibition. In the case of the Phe containing compounds, the maleic acid derivatives 42 and 43 (BnO-Phe<--Mal-Phe-OBn, BnO-Phe<--Mal-Phe-Ala-OBn, Mal = maleic acid) displayed good inhibition of rhodesain as well as good antitrypanosomal activity, while the fumaric acid derived E-analogue 14 (BnO-Phe<--Fum-Phe-OBn) only displayed inhibition of the target enzymes but no antiparasitic activity. Inhibition by these Phe derivatives was shown to be time-independent and competitive.

A series of cis-configured epoxides and aziridines containing hydrophobic moieties and amino acid esters were synthesized as new potential inhibitors of the secreted aspartic protease 2 (SAP2) of Candida albicans. Enzyme assays revealed the N-benzyl-3-phenyl-substituted aziridines 11 and 17 as the most potent inhibitors, with second-order inhibition rate constants (k(2)) between 56,000 and 121,000 M(-1) min(-1). The compounds were shown to be pseudo-irreversible dual-mode inhibitors: the intermediate esterified enzyme resulting from nucleophilic ring opening was hydrolyzed and yielded amino alcohols as transition-state-mimetic reversible inhibitors. The results of docking studies with the ring-closed aziridine forms of the inhibitors suggest binding modes mainly dominated by hydrophobic interactions with the S1, S1', S2, and S2' subsites of the protease, and docking studies with the processed amino alcohol forms predict additional hydrogen bonds of the new hydroxy group to the active site Asp residues. C. albicans growth assays showed the compounds to decrease SAP2-dependent growth while not affecting SAP2-independent growth.

Background: Factor XIII (FXIII) is the final enzyme of the coagulation cascade. While the other enzymatic coagulation factors are proteases, FXIII belongs to the transglutaminase family. FXIIIa covalently crosslinks the fibrin clot and represents a promising target for drug development to facilitate fibrinolysis. However, no FXIII-inhibiting compound has entered clinical trials. Here, we introduce the features of a peptidomimetic inhibitor of FXIIIa (ZED3197) as a potential drug candidate. Methods:The potency of ZED3197 against FXIIIa and the selectivity against other human transglutaminases were characterized using transamidation and isopeptidase assays. The inhibition of fibrin crosslinking was evaluated by biochemical methods and thromboelastometry. Further, the pharmacology of the compound was explored in a rabbit model of venous stasis and reperfusion.Results: ZED3197 proved to be a potent and selective inhibitor of human FXIIIa. Further, the compound showed broad inhibitory activity against cellular FXIIIA from various animal species. Rotational thromboelastometry in whole human blood indicated that the inhibitor, in a dose-dependent manner, prolonged clot formation, reduced clot firmness, and facilitated clot lysis without affecting the clotting time, indicating minimal impact on hemostasis. In vivo, the novel FXIIIa inhibitor effectively decreased the weight of clots and facilitated flow restoration without prolongation of the bleeding time.Conclusions: ZED3197 is the first drug-like potent compound targeting FXIIIa, a yet untapped target in anticoagulation. Due to the function of FXIII downstream of thrombin the approach provides minimal impact on hemostasis. In vivo data imply that the inhibitor dissociates an antithrombotic effect from increased bleeding tendency.

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K(i) values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.