RESUMEN
Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.
Asunto(s)
Carboxipeptidasa B2/antagonistas & inhibidores , Fibrinólisis/efectos de los fármacos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Carboxipeptidasa B/antagonistas & inhibidores , Carboxipeptidasa B/química , Carboxipeptidasa B2/química , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cianobacterias/química , Humanos , Modelos Moleculares , Péptidos Cíclicos/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Otamixaban is a potent (Ki=0.5 nM) fXa inhibitor currently in late-stage clinical development at Sanofi for the management of acute coronary syndrome. Being unproductive in obtaining a suitable crystal of Otamixaban, the required enantiomeric characterization has been accomplished using vibrational circular dichroism (VCD) spectroscopy. Selected by a spectrum similarity index, the calculated spectra of several higher energy conformers were found to match well with the observed spectra. The characteristic IR bands of these conformers were also identified and attributed to the solvation effect. Combined with both the single crystal x-ray diffraction results for an intermediate and the proton NMR study, the absolute configuration of Otamixaban is unambiguously determined to be (R,R).