The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and Glu, respectively, as well as a triple mutant (Val133Ala/Val157Gly/Ser205Glu), where Val157 is also replaced by Gly. Three synthetic substrates were used for the kinetic characterization of the mutants, as well as wild-type papain and cathepsin B: CBZ-Phe-Arg-MCA, CBZ-Arg-Arg-MCA, and CBZ-Cit-Arg-MCA. The ratio of kcat/KM obtained by using CBZ-Phe-Arg-MCA as substrate over that obtained with CBZ-Arg-Arg-MCA is 8.0 for the Val133Ala/Ser205Glu variant, while the equivalent values for wild-type papain and cathepsin B are 904 and 3.6, respectively. This change in specificity has been achieved by replacing only two amino acids out of a total of 212 in papain and with little loss in overall enzyme activity. However, further replacement of Val157 by Gly as in Val133Ala/Val157Gly/Ser205Glu causes an important decrease in activity, although the enzyme still displays a cathepsin B like substrate specificity. In addition, the pH dependence of activity for the Val133Ala/Ser205Glu variant compares well with that of cathepsin B. In particular, the activity toward CBZ-Arg-Arg-MCA is modulated by a group with a pKa of 5.51, a behavior that is also encountered in the case of cathepsin B but is absent with papain.(ABSTRACT TRUNCATED AT 250 WORDS)

Epoxysuccinyl dipeptide analogs of E-64 (R-EpsLeuPro-R') (Figure 1) have been synthesized with the carboxylate group on the epoxide ring either free (R = OH) or converted to an ester or an amide (R = EtO or i-BuNH) and with the C-terminal amino acid proline either blocked (R' = OBzl) or free (R' = OH). These compounds were used to investigate the recently reported selectivity of this type of inhibitor for the lysosomal cysteine protease cathepsin B. It was shown that derivatization of the carboxylate on the epoxide ring confers selectivity for cathepsin B over papain only when it is combined to a dipeptidyl moiety with a free negatively charged C-terminal residue. It is proposed that this selectivity reflects interactions with histidine residues on a loop located in the primed subsites of cathepsin B which provides a positively charged anchor for the C-terminal carboxylate group of the inhibitor. The primed subsite loop of cathepsin B is not found in other cysteine proteases of the papain family and offers a unique template for designing selectivity in cysteine protease inhibitors.

The structure-activity relationships of two series of novel retinoids (2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates) have been investigated by evaluating their ability to induce differentiation in both human promyelocytic leukemia (HL60) cells and mouse embryonal carcinoma (P19) cells. The most active compound (ED50 = 8.3 x 10(-9) M) of the 2-pyrazinylcarboxamidobenzoates is 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethylquinoxalyl)carboxamido]benzoic acid (9u), while the most active analogue of the beta-ionylideneacetamidobenzoates is 4-[3-methyl-5-(2',6',6'-trimethyl-1'-cyclohexen-1'-yl)-(2E, 4E)-pentadienamido]benzoic acid (10a, ED50 = 3.2 x 10(-8) M). Our studies identify an absolute requirement for the carboxylic acid moiety on the aromatic ring to be para relative to the amide linkage for activity. Benzoate substitutions in the ortho position relative to the terminal carboxylate (9d,k,r) are well-tolerated; however, a methoxy substituent meta relative to the terminal carboxylate gives rise to only weakly active analogues (9x). Conformational studies (NMR, X-ray crystallography) of the 2-pyrazinylcarboxamidobenzoates indicate that the preferred conformation exhibits a trans-amide bond and an internal hydrogen bond between the quinoxaline N1 and HN amide which locks the torsional angle between C2 and CO in the s-trans conformation. N-Methylation (9y) results in loss of activity. Studies indicate that there is now a cis-amide bond present which redirects the carboxylate toward the pharmacophoric gem-dimethyl groups. The distance between the gem-dimethyl group and the terminal carboxylate appears to be too short to activate the retinoid receptor. N-Methylation in the beta-ionylideneacetamidobenzoate series (10c) also results in the formation of a cis-amide bond and loss of activity.

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A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) and benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) were synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if selectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter kinac./Ki and that the specificity of these analogues does not parallel that observed for substrates. This is particularly true in the case of the benzyl ester derivatives where the deviation from substrate-like behaviour is more important than with the benzyl amide derivatives. The results suggest that the amide proton of the benzyl amide group in HO-Eps-AA-NHBzl interacts in the S2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors. The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S2 subsite of the enzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the order of inactivation rates with chloromethane inhibitors (which are believed to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin substrates. In conclusion, the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E64 analogues in a rational manner.