Inhibition of thermolysin by bifunctional N-carboxyalkyl dipeptides

A series of N-carboxyalkyl derivatives of L-leucyl-L-alanine was synthesized and tested as inhibitors of the zinc endoproteinase thermolysin. The purpose of the study was to determine whether bifunctional N-carboxyalkyl compounds with secondary metal coordinating groups are more potent inhibitors than analogs lacking such an additional binding function. Reductive condensation of L-leucyl-L-alanine (LA) with pyruvic, oxalacetic, alpha-ketoglutaric, 2-oxopentanoic, 4-ethyloxalacetic, or imidazoylpyruvic acids gave N-[1(R, S)-carboxyethyl]-LA (I), N-[1(R, S)-carboxy-2-carboxyethyl]-LA (II), N-[1(R, S)-carboxy-3-carboxypropyl]-LA (III), N-[1(R, S)-carboxy-n-butyl]-LA (IV), N-[1(R, S)-2-ethylcarboxyethyl]-LA (V), and N-[1(R, S)-carboxy-2-(4-imidazoyl-ethyl]-LA (VI), respectively. Values of KI determined with furylacryloyl-Gly-Leu-NH2 as substrate were 116 +/- 21, 7.4 +/- 1.8, 6.3 +/- 0.5, 19.7 +/- 1.5, 17.0 +/- 1.0, and 3.3 +/- 0.1 microM for compounds I-VI, respectively. Although bifunctional inhibitors II, III, and VI were indeed more potent than I, they were not much more effective than analogs IV and V that contained noncoordinating functionalities of comparable size. The results do not provide strong evidence for chelation of the active site zinc ion as proposed, although such interactions do not appear to be ruled out altogether.