RESUMEN
The enzymes dimethylargininase [dimethylarginine dimethylaminohydrolase (DDAH); EC 3.5.3.18] and peptidylarginine deiminase (PAD; EC 3.5.3.15) catalyze hydrolysis of substituted arginines. Due to their role in normal physiology and pathophysiology, both enzymes have been identified as potential drug targets, but few useful inhibitors have been reported. Here, we find that 2-chloroacetamidine irreversibly inhibits both DDAH from Pseudomonas aeruginosa and human PAD4 in a time- and concentration-dependent manner, despite the nonoverlapping substrate specificities and low levels of amino acid identity of their catalytic domains. Substrate protection experiments indicate that inactivation occurs by modification at the active site, albeit with modest affinity. Mass spectral analysis demonstrates that irreversible inactivation of DDAH occurs through selective formation of a covalent thioether bond with the active-site Cys249 residue. The mechanism of inactivation by 2-chloroacetamidine is analogous to that of chloromethyl ketones, a set of inhibitors that have found wide application because of their specific covalent modification of active-site residues in serine and cysteine proteases. Likewise, 2-chloroacetamidine may potentially find wide applicability as a general pharmacophore useful in delineating characteristics of the amidinotransferase superfamily.
Asunto(s)
Amidinas/farmacología , Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Clonación Molecular , Cartilla de ADN , Hidrolasas/genética , Hidrolasas/metabolismo , Espectrometría de Masas , Desiminasas de la Arginina ProteicaRESUMEN
Avaliou-se a atividade inibitória de diferentes materiais restauradores e selantes de fóssulas e fissuras sobre microrganismos cariogênicos através do método de difusão em ágar, empregado-se o ágar Mueller-Hinton acrescido de 5 por cento de sangue desfribrinado de carneiro. Verificou-se que a maioria dos materiais testados apresentou alguma atividade inibitória frente aos microrganismos isolados, particularmente os cimentos ionoméricos. As resinas compostas, à exceção do Dyract, não evidenciaram atividade antimicrobiana