RESUMO
(3S)-(Naphthalene-2-sulfonylamino)-1-[2R-(4-amidinophenyl)-1- piperidinocarbonylethyl]-2-pyrrolidinone (1a) is a potent inhibitor of thrombin with an IC50 value by 112 times lower than that of NAPAP (racemate). The selectivity versus trypsin can be improved by incorporation of substituents on the naphthyl ring. The mode of binding of the compound was determined by X-ray crystallography.
Assuntos
Antitrombinas/farmacologia , Benzamidinas/farmacologia , Pirrolidinonas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Trombina/antagonistas & inibidores , Animais , Antitrombinas/síntese química , Antitrombinas/química , Antitrombinas/metabolismo , Benzamidinas/síntese química , Benzamidinas/química , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Dipeptídeos/química , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Desenho de Fármacos , Ligação de Hidrogênio , Lactamas/síntese química , Lactamas/farmacologia , Estrutura Molecular , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Pirrolidinonas/síntese química , Pirrolidinonas/química , Ratos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Trombina/metabolismo , Tripsina/metabolismoRESUMO
A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA. The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket. From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor. Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.