Peptidyl Arginine Deiminases and Neurodegenerative Diseases.

Peptidyl arginine deiminases (PADs) are a small group of isozymes that convert Arg residues on the surface of proteins into citrulline residues, typically as a part of posttranslational processing. PADs are present in most of the tissues, and the isozyme distribution is tissue-specific. In the past 15 years, it is becoming apparent that PADs are either upregulated or their catalytic activity is enhanced in certain disease conditions, including neurological diseases. In particular, hypercitrullinated proteins and elevated PAD activities are discovered in neurodegenerative conditions such as multiple sclerosis, Alzheimer's disease etc. This review article reviews the status of PAD enzymes as targets in neurodegenerative conditions, and briefly outlines the efforts in medicinal chemistry to identify PAD inhibitors for the treatment of various neurodegenerative conditions.

Inhibition of orotidine 5'-monophosphate decarboxylase and its therapeutic potential.

Orotidine 5'-monophosphate decarboxylase (ODCase) is among the most proficient enzymes, and catalyzes the decarboxylation of OMP to UMP. An overview of ODCase and various proposals for its catalytic mechanism of decarboxylation are briefly presented here. A number of inhibitors of ODCase and new developments in the X-ray structures of ODCases from different species are discussed in the context of their therapeutic potential against cancer and infectious diseases. Latest discoveries in the inhibition of ODCase, for example using the novel C6 substitutions on the uridine, open new doors for drug discovery targeting parasitic diseases such as malaria.

RU-79115 (Aventis Pharma).

Aventis Pharma AG is investigating a series of novel coumarin DNA gyrase B inhibitors, including RU-79115, as potential treatments for bacterial infections. These inhibitors have displayed potent inhibitory activity in vitro against DNA supercoiling by DNA gyrase B and have antibacterial activity. Good antibacterial activity against vancomycin- and teicoplanin-resistant enterococci was observed with most analogs (where the 3-acylamino residue was replaced with reversed isosteres). Amide derivatives were also active against novobiocin-resistant strains [335149]. RU-79115 has shown activity against Gram-positive bacteria, particularly staphylococci and enterococci, and its activity compares favorably to vancomycin. Against oxacillin- and ofloxacin-resistant staphylococci, RU-79115 had MIC50 values 7.5-fold lower than eperezolid and significantly lower than vancomycin. In vivo data suggest that the compound is safe and carries a satisfactory pharmacokinetic profile [216256], [340969], [376199]. The compound is the most potent in the series which also includes RU-78535 and RU-64135 [340969], [341039].

Insight into the complex and dynamic process of activation of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are important hydrolytic enzymes with profound physiological and pathological functions in living organisms. MMPs are produced in their inactive zymogenic forms, which are subsequently proteolytically activated in an elaborate set of events. The propeptide in the zymogen blocks the active site, with a cysteine side-chain thiolate from this propeptide achieving coordination with the catalytically important zinc ion in the active site. Molecular dynamics simulations, ab initio calculations, and wet chemistry experiments presented herein argue for the critical importance of a protonation event at the coordinated thiolate as a prerequisite for the departure of the propeptide from the active site. Furthermore, a catalytically important glutamate is shown to coordinate transiently to the active-site zinc ion to "mask" the positive potential of the zinc ion and lower the energy barrier for dissociation of the protonated cysteine side chain from the zinc ion. In addition, a subtle conformational change by the propeptide is needed in the course of zymogen activation. These elaborate processes take place in concert in the activation process of MMPs, and the insight into these processes presented herein sheds light on a highly regulated physiological process with profound consequences for eukaryotic organisms.

X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme.

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.

Substrate hydrolysis by matrix metalloproteinase-9.

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.

'Infectious web'.

Exhaustive information on the Epstein-Barr virus, a member of the herpes family, is described at the International Herpes Management Forum web-site. Cervical cancer associations, AIDS treatment projects, and the Los Alamos National Laboratories provide useful information on papillomavirus infections, as well as hyperlinks to recent international papillomavirus conferences. A private pharmaceutical company, in collaboration with the National Institutes of Health, has launched a lively web-site covering different aspects of microbial infections for the general public.

Tethered bisubstrate derivatives as probes for mechanism and as inhibitors of aminoglycoside 3'-phosphotransferases.

Aminoglycoside 3'-phosphotransferases [APH(3')s] phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to aminoglycosides in bacteria. APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism. Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms. Four bisubstrate analogues, compounds 1-4, were designed and synthesized as inhibitors for APH(3')s. These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons. The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.

'Infectious web'.

Infections by Helicobacter pylori are responsible for duodenal and gastric ulcers and are a significant risk factor for the development of gastric adenocarcinoma. H. pylori was discovered in 1983, but many institutes in Canada, Europe, and the United States are already involved in programs to understand and treat the infections, as reflected by the growing number of internet sites devoted to this bacterium. Most AIDS patients and about 20% of children with acute lymphoblastic leukemia develop Pneumocystis carinii pneumoniae. Information on clinical symptoms and treatment, as well as the P. carinii genome sequencing project, are described at several web sites. Students and researchers wishing to understand the correlation between telomere length and AIDS may turn to web sites of the University of Colorado and Washington University School of Medicine for the latest on telomeres and telomerase, and their function in aging and cancer.

Stereoselective reduction of alpha-bromopenicillanates by tributylphosphine.

[reaction: see text] Diastereoselective reduction of 6-bromo-6-substituted penicillanate esters has been achieved by treatment with tributylphosphine to give 6-substituted penicillanate esters. This reaction would appear to proceed through a phosphonium beta-lactam enolate species, followed by a diastereoselective protonation. This method has the advantage of being simple to carry out and it is mild, gives high diastereoselectivity, and should tolerate a number of functional groups in the substrates. Implications of these observations are discussed.

From genes to sequences to antibiotics: prospects for future developments from microbial genomics.

The entire genome sequences for a number of microbial organisms have become available over the past few years. This knowledge is the beginning point for understanding the fundamental principles of bacterial structure and function. The prospects for gain in knowledge from genomics are discussed in this report.

'Infectious web'.

This issue of 'Infectious Web' includes web-sites related to AIDS/HIV, pathogenic characteristics and resistance to Staphylococcus spp., diagnostic and clinical aspects of arthritis, and comprehensive information resources on malaria, cystic fibrosis and biological weapons.

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors.

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.

Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1.

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.

'Infectious web'.

A comprehensive list of all known bacterial pathogens of humans is now available at various web-sites on the internet. The sites contain hyperlinks to original scientific literature, along with general information on laboratory testing, antibiotic resistance and clinical treatment. More specific sites highlight the fungus Pneumocystic carinii, arguably the main cause of pneumonia in immunosuppressed individuals.

Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa.

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.

Mechanistic and clinical aspects of beta-lactam antibiotics and beta-lactamases.

Bacterial infections have been a major cause of concern in the recent years due to the emergence of drug resistance strains and inability of the current therapeutic regimens to treat these infections in certain cases. Beta-Lactam antibiotics have been drugs of choice since the introduction of penicillin. These drugs inhibit bacterial cell-wall-synthesizing enzymes, the so-called penicillin-binding proteins (PBPs) selectively, thus providing an effective strategy for treatment of the bacterial infections. Significantly, bacteria have developed resistance mechanisms to neutralize the antibiotic action of beta-lactam drugs. Beta-Lactamases are enzymes that hydrolyze the beta-lactam moiety of these drugs, rendering them inactive. This is the primary mechanism of resistance to this class of antibiotics. There are 255 known beta-lactamases to date and the continued use of beta-lactams may select for newer variants yet. A discussion of the roles of these enzymes in the manifestation of the drug-resistant phenotype and their implications for pathogenecity of clinical strains of bacteria is presented.

Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams.

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.

Structure determination of 4-azido-2-pyrimidinone nucleoside analogs using mass spectrometry.

The nucleoside prodrugs 4-azido-ara-C and 2'-fluoro-2', 3'-dideoxy-4-azido-ara-C and their base-catalyzed reaction products were thoroughly characterized by mass spectrometry. The structures of the base-catalyzed reaction products were determined and confirmed using a combination of high-resolution and tandem mass spectrometry with deuterium exchange. An intra-molecular rearrangement reaction occurred in 4-azido-ara-C at physiological pH leading to the formation of a 2',6-anhydro product. A nucleoside of similar structure, 2'-fluoro-2'3'-dideoxy-4-azido-ara-C was studied to determine if the formation of the 2',6-anhydro ring was due to the presence of the 4-azido group or the arabinose 2'-OH group. The 6-position of 2'-fluoro-2',3'-dideoxy-4-azido-ara-C was found to be unreactive at physiological pH, but could add ammonia under strongly basic conditions (pH 11.0, ammonia solution). Finally, the formation of an intriguing tetrazole ring by the 4-azido moiety was observed.