A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.     

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.     

Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene

Mechanism-based inactivators of β-lactamases are used to overcome the resistance of clinical pathogens to β-lactam antibiotics. This strategy can itself be overcome by mutations of the β-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 β-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with β-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp β-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM β-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.     

The pharmacokinetics and pharmacodynamics of adinazolam: multi-ethnic comparisons

The pharmacokinetics and pharmacodynamics of adinazolam and N-demethyladinazolam (NDMAD), its major active metabolite, were compared in 39 healthy male volunteers (13 Asian, 12 Caucasian and 14 African-American). In a four-way, double-blind crossover design, subjects were administered (1) 30 mg oral adinazolam mesylate SR tablets, (2) 10 mg parenteral (IV) adinazolam mesylate, (3) 30 mg IV NDMAD and (4) placebo. Venous blood samples were collected at specific time intervals after drug administration and assayed for adinazolam and NDMAD concentrations. Sedation was rated at the time of each blood draw according to the Nurse-Rated Sedation Scale, and the digit-symbol substitution test was administered to evaluate psychomotor performance. After IV administration of adinazolam, Asians manifested significantly higher C_max, larger AUC and lower CL of both adinazolam and NDMAD than their Caucasian and African-American counterparts. Likewise, after IV NDMAD Asians had significantly higher NDMAD C_max and AUC than Caucasians and African-Americans. Most of these differences remained statistically significant after controlling for body surface area. With PO adinazolam, Asians also manifested substantially higher C_max, larger AUC and lower CL for both adinazolam and NDMAD; however, with the exception of C_max, these differences did not reach statistical significance. These results are in accordance with previous observations for ethnic-related differences in drug pharmacokinetics. In contrast, pharmacodynamic differences were not noted among the three study groups. Received: 19 June 1996/Final version: 17 September 1996     

De novo design of nonpeptidic compounds targeting the interactions between interferon-alpha and its cognate cell surface receptor

Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus erythematosus, respectively. Specific sequences on the surface of IFN, IFN receptor recognition peptides (IRRPs) mediate the binding and signal transduction when IFN interacts with IFNAR. Structural features of two such IRRPs, IRRP-1 and IRRP-3, were used as templates to design small molecule mimetics. In silico screening was used to identify the molecular structural features mimicking their surface characteristics. A set of 26 compounds were synthesized and their ability to interfere with IFN-IFNAR interactions was investigated. Two compounds exhibited antagonist activity, specifically, blocking IFN-inducible Stat phosphorylation Stat complex-DNA binding. Design principles revealed here pave the way toward a novel series of small molecules as antagonists for IFN-IFNAR interactions.     

Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.     

Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes

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Acinar choroid plexus adenoma of the fourth ventricle

Mucus-secreting adenomas of the choroid plexus are very rare. We report the case of a 72-year-old woman who was first seen because of difficulty in walking. Her CT scan showed a lesion compatible with the appearance of a cystic mass in the fourth ventricle. A posterior fossa exploration was performed and an acinar choroid plexus adenoma of the fourth ventricle was removed.     

A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of beta-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-A resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type D-alanyl-D-alanine carboxypeptidase/transpeptidase (EC ) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.