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In order to elucidate the substrate specificity of the Sn subsites (n=1-3) of cathepsin B, its crystal structure inhibited by E64c [(+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]-leucylcarbonyl)oxirane-2-carboxylic acid] was analyzed by the X-ray diffraction method. Iterative manual rebuilding and convenient conjugate refinement of structure decreased R- and free R-factors to 19.7% and to 23.9%, respectively, where 130 water molecules were included for the refinement using 14,759 independent reflections from 10 to 2.3 A resolution. The epoxy carbonyl carbon of E64c was covalently bonded to the Cys(29) S(gamma) atom and the remaining parts were located at Sn subsites (n=1-3). The substrate specificity of these subsites was characterized based on their interactions with the inhibitor. Base on these structural data, we developed a novel cathepsin B-specific noncovalent-type inhibitor, which may bind to S2'-S3. The molecular design of possessing structural elements of both CA074 and E64c, assisted by energy minimization and molecular dynamics (MD) simulation, may lead to a new lead noncovalent-type inhibitor.

The binding mode of CA074 [N-(L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-pr oline], a specific irreversible inhibitor, to bovine spleen cathepsin B was elucidated by X-ray crystal structure analysis of the complex at 2.2 A resolution (conventional R=0.185). Inconsistently with our model used for the development of CA074, the L-isoleucyl-L-proline and propylcarbamoyl moieties are located at the S' and S subsites, respectively. This unexpected binding is primarily due to (i) similar extended chain conformations (due to the same S configurations) at the oxirane C2 and C3 atoms of CA074 and (ii) the just fit formation of double hydrogen bonds between the carboxyl oxygens of L-proline and the imidazole nitrogens of His-110 and His-111 residues (these residues are missing in papain, the tertiary structure of which was used for the design of CA074). The oxirane C3 atom possessing the P' substituent is covalently bound to the Cys-29 Sgamma atom (C3-Sgamma=1.79 A) and the S configuration is maintained. The present result will provide useful information for characterizing the substrate-specificity of cathepsin B.

In order to investigate the stereo specificity of papain Sn subsites (n = 1-4) at the atomic level, two kinds of covalent-type inhibitors were designed based on the previous results on papain-E-64 and papain-E-64-c interactions, and their complex crystals with papain were analyzed by X-ray diffraction. The results show that the hydrophobic regions consisting of Val-133, Val-157 and Asp-158 and of Tyr-61, Gly-66 and Tyr-67 residues interact with the hydrophobic P2 and P3 side chains of inhibitors, thus indicating the function of the former and latter binding pockets as S2 and S3 subsites, respectively. Furthermore, the X-ray analysis suggests that the papain has no definite Sn subsite of nor = 4, and the S3-P3 hydrophobic interaction is significantly affected by the Pn side chain (nor = 4) of both the substrate and the inhibitor.

The specificity of compound CA074 [N-( l -3-trans-propylcarbamoyloxirane-2-carbonyl)- l -isoleucyl- l -proline] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 m −1 · s−1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 μ m , 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

To design a potent inhibitor specific for cathepsin B (rat liver), the tertiary structure was predicted based on the crystal structure of the papain complexed with (+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]leucylcarbonyl)oxirane-2- carbolylic acid (E-64-c), a thiol protease inhibitor. Taking advantage of the structural characteristics of the predicted active site, seventeen inhibitors were chemically synthesized by molecular modeling, and one of them, N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-p rol ine (CA-074) was shown to be the first potent inhibitor specific for cathepsin B. The relationship between the structure and inhibitory activity is discussed based on the model structure of the cathepsin B-inhibitor complex.

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-isoleucyl-L-proline (compound CA-030) and N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline (compound CA-074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n-propylamide group in the oxirane ring were necessary, the ethyl ester group or the n-propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.

The crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R = 0.152 using X-ray diffraction data up to 2.18 A resolution. BSCB is characterized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop structures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 S(gamma) atom (C3-S(gamma)=1.81 A), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n = 1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex.

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.

The synthesis, antibacterial activity and oral absorption in rats of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)ace tamido] cephalosporins (1) having various substituted-alkylthio groups at the C-3 position of the cephem nucleus are described. Of these, the cephalosporins with a cyanomethylthio group (1d) and fluoroethylthio group (1p) at the C-3 position showed a potent in vitro antibacterial activity against Gram-positive and Gram-negative bacteria as well as good oral absorption in rats. When administered orally to mice infected with Klebsiella pneumoniae, 1d had stronger protective effect than 1p. The structure-activity relationships of 1 are also presented.

The synthesis, antibacterial activity and oral absorption of new 7 beta-[D-alpha-amino-alpha-(4-hydroxyphenyl)acetamido]cephalosporins (1) with various O-substituents at the C-3 position of a cephalosporin nucleus are described. Of these, the cephalosporins (1b-1e) having an alkoxycarbonylmethoxy group at the C-3 position showed good oral absorption in rats as well as potent activity against Gram-positive bacteria. The structure-activity relationships of 1 are also presented.

Ethly (+)-(2S, 3S) -3- [(S) -3-methyl-1- (3-methylbutylcarbamoyl) butylcarbamoyl] -2- oxirane-carboxylate (EST ; la) is expected to be useful as an oral therapeutic agent for muscular dystrophy on the basis of its potent inhibitory activities against the cysteine proteinases involved in the myofibrillar protein degradation that occurs in the disease. Through extensive investigations aimed at developing a new synthetic method for la that would be suitable for industrial application, it has been found that L-arginine can be used as a new, efficient resolving agent to obtain optically pure L-trans-epoxysuccinic acid (3a), and the active ester method using p-nitrophenol is very effective in the coupling reaction of ethyl L-trans-epoxysuccinate (7a) and L-leucine isoamylamide (8a) because of the extremely low formation of by-products.To examine the contribution of the stereochemistry of the trans-epoxysuccinic acid and leucine moieties to the inhibitory activity against cysteine proteinases, the diastereomers (lb-d) of la were synthesized by a similar method and the rate constants of inactivation of papain by la-d were measured. Compound la, having L-trans-epoxysuccinic acid and L-leucine moieties, showed the most potent activity among them.

Syntheses a partir de phenol-6 6H-isoselenazolo- ou -isothiazolo [4,3-d] pyrimidones-7 ou de dimethyl-4,6 tetrahydro-4,5,6,7 isoselenazolo- ou -isothiazolo [4,3-d] pyrimidinediones-5,7 via l'acylation par des bromures d'α-bromoalcanoyle d'amides d'acides amino-4 isoselenazole- ou -isothiazolecarboxyliques-3

The three-dimensional structure of plastocyanin, a ‘blue’ or ‘Type 1’ copper-protein, has been determined at a resolution of 2.7 A. The copper atom has a highly distorted tetrahedral coordination geometry. It is coordinated by a cysteine thiol group, a methionine thioether group, and two histidine imidazole groups.

The synthesis, antibacterial activity and oral absorption in rats of 3-alkoxycarbonyl-methoxy-7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(O-substituted oxyimino)acetamido]cephalosporins (1) are described. In this cephalosporin series, 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxy-methoxyimino)acetamid o] cephalosporins (1b, 1i and 1j) with a lower alkoxycarbonylmethoxy group at the C-3 position of a cephem nucleus exhibited not only potent activity against Gram-negative bacteria but also good oral absorption in rats. Structure-activity relationships of 1 are also presented.

Ethly (+)-(2S, 3S) -3- [(S) -3-methyl-1- (3-methylbutylcarbamoyl) butylcarbamoyl] -2- oxirane-carboxylate (EST ; la) is expected to be useful as an oral therapeutic agent for muscular dystrophy on the basis of its potent inhibitory activities against the cysteine proteinases involved in the myofibrillar protein degradation that occurs in the disease. Through extensive investigations aimed at developing a new synthetic method for la that would be suitable for industrial application, it has been found that L-arginine can be used as a new, efficient resolving agent to obtain optically pure L-trans-epoxysuccinic acid (3a), and the active ester method using p-nitrophenol is very effective in the coupling reaction of ethyl L-trans-epoxysuccinate (7a) and L-leucine isoamylamide (8a) because of the extremely low formation of by-products.To examine the contribution of the stereochemistry of the trans-epoxysuccinic acid and leucine moieties to the inhibitory activity against cysteine proteinases, the diastereomers (lb-d) of la were synthesized by a similar method and the rate constants of inactivation of papain by la-d were measured. Compound la, having L-trans-epoxysuccinic acid and L-leucine moieties, showed the most potent activity among them.

The reactions of poplar plastocyanin, PCu(I), with [Fe(CN)6]3– and [Co(phen)3]3+(phen = 1,10-phenanthroline) as oxidants, and of PCu(II) with [Co(phen)3]2+ as reductant, have been studied at 25 °C, I= 0.10 M (NaCl). At pH 7.5, 69% of the reaction of [Co(phen)3]3+ is at the Tyr 83 site, which incorporates the acidic patch, in this case at 42–44. With [Fe(CN)6]3– as oxidant the behaviour observed is consistent with reaction at the His 87 (north) site. From the effects of pH (4.5–7.5) on rate constants, active site (pKa 4.7) and (acidic patch) binding site (pKa′ 5.25) deprotonation/protonation equilibria are identified. For PCu(II) no active site pKa is observed, and the binding site pKa′ is 5.1. Comparisons are made with plastocyanin from parsley, spinach, and French bean. With [Co(phen)3]3+ as oxidant no limiting kinetic behaviour is observed, and association of PCu(I) with [Co(phen)3]3+ is at a lower level (K < 50 M–1). From competitive inhibition studies association with redox inactive [(NH3)5Co(NH2)Co(NH3)5]5+ is also at a lower level (K= 5 650 M–1). The origin of differences in pKa′ for PCu(I) and PCu (II) states is discussed.