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Various 2-arylideneindanones 1, 2-arylidenetetralones 2, and 2-arylidenebenzosuberones 3 were synthesized with the aim of determining the relative orientations of the two aryl rings which favored cytotoxicity. Molecular modeling of the unsubstituted compound in each series revealed differences in the spatial arrangements of the two aryl rings, and evaluation of these compounds against P388, L1210, Molt 4/C8, and CEM cells as well as a panel of human tumor cell lines indicated that in general the order of cytotoxicity was 3 > 2 > 1. In particular 2-(4-methoxyphenylmethylene)-1-benzosuberone (3k) had the greatest cytotoxicity, possessing 11 times the potency of the reference drug melphalan when all five screens were considered. Series 3 was considered in further detail. First, excision of the aryl ring fused to the cycloheptanone moiety in series 3 led to some 2-arylidene-1-cycloheptanones 4 which had approximately one-third of the bioactivity of the analogues 3. Second, in some screens cytotoxicity was correlated negatively with the sigma values and positively with the MR constants of the substituents in the arylidene aryl ring of 3. Third, X-ray crystallography of five representative compounds (3i,k-n) revealed differences in the locations of the aryl rings which may have contributed to the variations in cytotoxicity. Finally three members of series 3 inhibited RNA and protein syntheses and induced apoptosis in human Jurkat T cells. This study has revealed that 2-arylidene-1-benzosuberones are a group of useful cytotoxic agents, and in particular 3k serves as a prototypic molecule for subsequent structural modifications.

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA 29 Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr 232 proximal to Ser 237 , permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.Staphylococcus aureus can colonize and infect virtually every tissue and organ system of the body (1, 2). A key factor in these defining traits is its ability to sustain bacteremia and adhere to tissues. Consequently it has adapted to growth in blood by subversion of plasma proteins (3) and the tissue extracellular matrix (4). Members of the MSCRAMM (microbial extracellular matrix-binding protein) family of adhesion proteins bind extracellular matrix ligands such as collagen, fibronectin, and fibrinogen (4) of which the latter two are also abundant in plasma. Manipulation of other plasma proteins such as IgG and von Willebrand factor is facilitated by staphylococcal Protein A (5), whereas coagulase promotes fibrin clot formation by activation of prothrombin (6), and staphylokinase activates plasminogen (7) to facilitate fibrin clot dissolution. Our studies on secreted proteases are also supportive of S. aureus as a paradigm for the manipulation of plasma and coagulation proteins.The SspA 4 glutamyl endopeptidase, also known as V8 protease, moderates adhesion of S. aureus to fibronectin by degrading cell surface fibronectin-binding proteins in which there is a high glutamic acid content, including a conserved motif that is essential for ligand binding (8, 9). SspA is expressed in an operon with a cysteine protease, SspB, and activates the zSspB zymogen by removing its N-terminal propeptide (10). Mature 20-kDa SspB mimics plasma serine proteases as noted by its ability to (i) convert high molecular weight kininogen into heavy and light chains, (ii) hydrolyze the Bz-Pro-Phe-Arg substrate of kallikrein, a serine protease that processes single chain kininogen by excision of the vasoactive peptide bradykinin (RPPGFSPFR), (iii) process the N terminus of the fibrinogen ␤-chain at the same site as plasmin, and (iv) remove the N-terminal domain of fibronectin with a specificity equivalent to plasminogen activator (10). We have also found that antibodies to SspB are produced when mice are challenged with hypervirulent strains of community-acquired methicillin-resistant S. aureus and that SspA and SspB are r...

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

. Can. J. Chem. 68, 1116 (1990).The lectin IV of Griffonia simplicifolia (GS4) specifically binds the terminal tetrasaccharide unit of the Lewis b human blood group determinant (~e~) .The single crystal X-ray analysis of the complex with L~~-o M~ has demonstrated that the binding site on the lectin is a shallow depression with a negatively charged aspartate side chain at the bottom of the cavity. In addition to this aspartate, a serine and an asparagine side chain provide the polar groups that hydrogen bond to the three hydroxyl groups of Leb, which has been termed the key polar grouping for complex formation. A notable characteristic of the binding site is that five aromatic amino acid side chains (one Phe, two Tyr, and two Trp residues) surround these polar interactions and make van der Waals contacts with the tetrasaccharide. Thus, as predicted from previous solution binding studies, extensive nonpolar interactions are involved, which contribute importantly both to the specificity of the reaction and the stability of the noncovalent complex that is formed. These results represent the first structural example of the molecular recognition of a human blood group determinant by the receptor site of a protein. Extensive sequence homology exists between GS4 and the concanavalin A (Con A), pea, and favin lectins. The main hydrophilic groups of the carbohydrate-binding site of GS4 and Con A are aspartate, asparagine, and serine residues; the homology suggests that the serine is replaced by asparagine in the case of the pea and favin lectins. It appears probable that these two latter lectins possess very similar, if not identical, specificities.Key words: lectin, carbohydrate, molecular recognition, binding.Lours T. J. DELBAERE, MARGARET VANDONSELAAR, LATA PRASAD, J. WILSON QUAIL, JOYCE R. PEARLSTONE, MICHAEL R. CARPENTER, LAWRENCE B. SMILLIE, PANDURANG V. NIKRAD, ULRIKE SPOHR et RAYMOND U. LEMIEUX. Can. J. Chem. 68, 1116 (1990).La lectine IV du Grrffonia simplicifolia (GS4) se lie sptcifiquement a l'unitt tktrasaccharide terminale du determinant du groupe sanguin humain Lewis b (Leb). Une analyse par diffraction des rayons-X effectute sur un cristal unique de son complexe avec le Leb-OM^ a perrnis de dtmontrer que le site de liaison de la lectine est une ltgtre dkpression au fond de laquelle on retrouve une chaine lattrale d'aspartate chargke nkgativement. En plus de cet aspartate, des chaines lattrales de strine et d'asparagine fournissent des groupements polaires qui foment des points hydrogenes avec les trois groupements hydroxyles du ~e~; il s'agit du groupement polaire clt pour la formation de complexes. Le fait que cinq chaines lattrales formtes d'acides amints arornatiques entourent ces interactions dipolaires et qu'elles ttablissent des contacts de van der Waals avec le tktrasaccharide est une caracteristique notable de ce site de liaison. Tel que prkdit 2 partir d'ttudes anttrieures de liaisons en solution, il existe donc de nornbreuses interactions non-polaires qui apportent une contribution importante tant...

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