Various N-sulfonylamino acid derivatives were synthesized and evaluated for their in vitro and in vivo activities to inhibit type IV collagenase (MMP-9 and MMP-2). When the amino acid residue and the sulfonamide moiety were modified, their inhibitory activities were greatly affected by the structure of the sulfonamide moiety. A series of aryl sulfonamide derivatives containing biaryl, tetrazole, amide, and triple bond were found to be potent and highly selective inhibitors of MMP-9 and MMP-2. In addition, these compounds were orally active in animal models of tumor growth and metastasis. These results revealed the potential of the N-sulfonylamino acid derivatives as a new type of candidate drug for the treatment of cancer.

Thermolysin is a well-known protease which exhibits its specificity against hydrophobic amino acid residues such as L-leucine, L-phenylalanine, etc. whose amino groups donate the susceptible peptide bonds (amino-endopeptidase). The present study was undertaken to investigate the effects of neighboring residues surrounding the sensitive amino acid residues at the amino-side in peptide substrates. For the purpose, a kinetic study was made using various synthetic oligopeptides such as Z-A-(Gly),-i;Leu-Ala or Z-Gly+eu-(Gly),-B (A or B = various I)-or L-amino acid residues; n = 0, 1 and/or 2; the arrow shows the bond split) as substrates. Other kinetic or inhibition studies were also made. These studies indicated that the specificity is affected by a t least three amino acid residues on the N-terminal side and by two amino acid residues on C-terminal side from the sensitive amino acid residue (at amino-side) in peptide substrates. The effect of each of the five neighboring amino acid residues for appearance of the specifiicity was similar with that of the corresponding one which had been observed in a neutral protease of Bacillus subtilis, but that was not completely the same.Thermolysin is a thermo-stable protease produced by Bacillus thermopotwlyticus. It was discovered by Endo El], and its enzymatic specificity was first studied by Matsubara et al.[2], using beef cytochrome c as substrate, who assumed that the enzyme is specific for peptide bonds containing the amino group of hydrophobic amino acid residues such as L-leucine, L-isoleucine, L-phenylalanine, etc. This assumption was supported by a further study by the same authors using insulin using Z-Gly-X-NH, (X = various amino acid residues of D-or L-configuration; the arrow shows the bond split) as substrates indicated that the highest activity was obtained when X was L-leucine, or L-phenylalanine, well reflecting the specificity against large moleular peptides or proteins. These characteristics of specificity have since been found to be Unueuul Abbreviations. DFP, diisopropyl phosphofluoridate ; Z, benzyloxyoarbonyl.A 181. The specificities of various neutral proteases from bacterial origin, including thermolysin, were shown [15] to be affected by at least the next five residues adjacent to the sensitive amino acid residue in peptide substrates. A further study of B. subtilis neutral protease [16,17] with various synthetic peptides somewhat clarified the contribution to specificity of each of these five amino acid residues in peptide substrates. The present kinetic study of thermolysin with synthetic peptides was undertaken to compare the influences of neighboring residues on the specificity against the amino acid residue donating the amino group to be hydrolyzed with those found with the B. subtilis enzyme.

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Three-dimensional models for the catalytic domain of gelatinases (MMP-9 and -2) have been constructed based on the X-ray crystal structure of MMP-3. Conformations of the loop segment which forms the bottom half of the S1' subsite but shows conformational diversity among the crystal structures of other MMPs have been explored by simulated annealing of each gelatinase model complexed with two highly potent "probe" inhibitors. Representative catalytic domain models have been selected for each gelatinase from the set of generated conformations based on shape complementarity of the loop to the probe inhibitors. The single model selected for MMP-9 was utilized to explain the structure-activity relationship of our novel sulfonamide inhibitors. Molecular dynamics (MD) simulations of the complex models revealed important features of the binding mechanism of our inhibitors: (i) the ligand carboxylate group coordinating to the catalytic zinc ion and hydrogen bonding to the Glu219 side chain, (ii) one of the sulfonyl oxygens forming hydrogen bonds with the main chain NHs (Leu181 and Ala182), (iii) the sulfonyl substituent making extensive hydrophobic contact with the S1' subsite. The gauche conformation exclusively adopted by the sulfonamide C-N-S-C torsion plays an important role in achieving the third binding feature by properly directing the substituent into the S1' subsite. Improvement of the inhibitory activity according to straight elongation of the sulfonyl substituent was attributed to an increase of the hydrophobic contact between the substituent and the S1' subsite. Structural modifications which alter the straight shape of the substituent lead to deterioration of the activity. On the other hand, the two candidate models selected for MMP-2 differ in the bottom shape of the S1' subsite: one with a channel-like subsite and the other with a pocket-like subsite resembling that of the MMP-9 model. The bottom shape was experimentally probed by chemical synthesis of inhibitors having elongated sulfonyl substituents whose terminal alkyl groups were shown by MD simulations to protrude from the S1' subsite bottom into the solvent. Gelatinase assays of these inhibitors showed that elongation of the substituent significantly reduces activity against MMP-9 while retaining activity against MMP-2, consequently increasing the selectivity between MMP-2 and -9. The results confirm that MMP-9 has a pocket-like S1' subsite with a floorboard and MMP-2 has a channel-like S1' subsite.