RESUMO
Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.
Assuntos
Compostos Aza/farmacologia , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Leishmania mexicana/efeitos dos fármacos , Tripanossomicidas/farmacologia , Compostos Aza/síntese química , Compostos Aza/química , Catepsina B/metabolismo , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Leishmania mexicana/enzimologia , Simulação de Dinâmica Molecular , Estrutura Molecular , Nitrilas/síntese química , Nitrilas/química , Nitrilas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a Kiapp of 1.3 × 10-9 M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 µM and 15.4 µM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.