High priority preparedness research and its support.

The US Federal Government has considerable interest in supporting research into preparedness. Because of the diverse nature of possible threats and the responsibilities of different agencies, a number of different programs have been developed. Perspectives from representatives from 3 of the leading agencies; the Department of Homeland Security, the Centers from Disease Control and Prevention, and the National Institutes of Health, are described herein.

Accelerating global innovation to address antibacterial resistance: introducing CARB-X.

A global response to the chronic shortfall in antibiotic innovation is urgently needed to combat antimicrobial resistance. Here, we introduce CARB-X, a new global public-private partnership that will invest more than US$350 million in the next 5 years to accelerate the progression of a diverse portfolio of innovative antibacterial products into clinical trials.

PolC-type polymerase III of Streptococcus pyogenes and its use in screening for chemical inhibitors.

The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag. The purified recombinant enzyme showed an apparent molecular mass of 160 kDa on SDS-PAGE and a specific activity of 3.5 nmol/min/mg when assayed in the presence of calf thymus DNA and the four deoxyribonucleoside triphosphates. This activity was inhibited by TMAU, a specific inhibitor of PolC. To facilitate kinetic studies, and high-throughput assays, a double-stranded oligo DNA primer/template was used as a substrate. The minimum requirement for the length of the substrate was a 20-base oligo primer annealed to a 35-base template. PolC activity was detected either by a filter-binding format or by a novel homogeneous scintillation proximity assay (SPA). Sensitivity to inhibition by anilinouracil analogs was improved by incorporating three deoxycytidines in the template strand as the first 3 bases to be copied by the polymerase. Inhibition of PolC activity by trimethyleneanilinouracil by the filtration and SPA methods gave comparable results, but the SPA assay uses less radioactive label, is less time-consuming, and is amenable to high-throughput formatting.

Development of a whole-cell assay for peptidoglycan biosynthesis inhibitors.

Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of (14)C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 microM. Flavomycin, bacitracin, vancomycin, D-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the E. coli cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors.

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.

Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.

A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan biosynthesis.

Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG. Conditions have been optimized for the production of large amounts of membranes with increased levels of MurG, allowing the development of an assay suitable for high-throughput screening of large compound libraries. The quality of the purified membranes was assessed by electron microscopy and also by testing cross-linked peptidoglycan production. Moreover, kinetic studies allowed the determination of optimal concentrations of the substrates and membranes to be utilized for maximum sensitivity of the assay. Using a 96-well assay format, the IC50 values for vancomycin, tunicamycin, flavomycin, and bacitracin were 1.1 microM, 0.01 microg/ml, 0.03 microg/ml, and 0.7 microg/ml, respectively.

Regulated expression of the Escherichia coli lepB gene as a tool for cellular testing of antimicrobial compounds that inhibit signal peptidase I in vitro.

Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on L-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of L-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 micro M was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 micro M. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation.