Potential Antibacterial Target Research Articles

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 μM with IC 50 value of 2 μM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 μM with IC 50 value of 6 μM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU).

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X-ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active-site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substrate-binding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo-like conformation until N-acetylglucosamine-1-phosphate (GlcNAc-1-P) binds and induces a conformational change at the GlcNAc-binding subsite. Following the binding of GlcNAc-1-P to the UTP-charged uridyltransferase active site, the non-esterified oxygen of GlcNAc-1-P performs a nucleophilic attack on the alpha-phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure-guided design of novel antibacterial agents targeting GlmU activity.

Structural and functional investigations of Ureaplasma parvum UMP kinase - a potential antibacterial drug target

The crystal structure of uridine monophosphate kinase (UMP kinase, UMPK) from the opportunistic pathogen Ureaplasma parvum was determined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family. Recombinant UpUMPK exhibited Michaelis-Menten kinetics with UMP, with K(m) and V(max) values of 214 +/- 4 microm and 262 +/- 24 micromol.min(-1).mg(-1), respectively, but with ATP as variable substrate the kinetic analysis showed positive cooperativity, with an n value of 1.5 +/- 0.1. The end-product UTP was a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP. Unlike UMPKs from other bacteria, which are activated by GTP, GTP had no detectable effect on UpUMPK activity. An attempt to create a GTP-activated enzyme was made using site-directed mutagenesis. The mutant enzyme F133N (F133 corresponds to the residue in Escherichia coli that is involved in GTP activation), with F133A as a control, were expressed, purified and characterized. Both enzymes exhibited negative cooperativity with UMP, and GTP had no effect on enzyme activity, demonstrating that F133 is involved in subunit interactions but apparently not in GTP activation. The physiological role of UpUMPK in bacterial nucleic acid synthesis and its potential as target for development of antimicrobial agents are discussed.

The Structural Basis of Signal Transduction for the Response Regulator PrrA from Mycobacterium tuberculosis

The structure of the two-domain response regulator PrrA from Mycobacterium tuberculosis shows a compact structure in the crystal with a well defined interdomain interface. The interface, which does not include the interdomain linker, makes the recognition helix and the trans-activation loop of the effector domain inaccessible for interaction with DNA. Part of the interface involves hydrogen-bonding interactions of a tyrosine residue in the receiver domain that is believed to be involved in signal transduction, which, if disrupted, would destabilize the interdomain interface, allowing a more extended conformation of the molecule, which would in turn allow access to the recognition helix. In solution, there is evidence for an equilibrium between compact and extended forms of the protein that is far toward the compact form when the protein is inactivated but moves toward a more extended form when activated by the cognate sensor kinase PrrB.

Discovery of Bacterial Fatty Acid Synthase Inhibitors from aPhomaSpecies as Antimicrobial Agents Using a New Antisense-Based Strategy

Fatty acids are essential for bacterial growth and viability, with the type II fatty acid synthesis (FAS II) pathway being a potential antibacterial target. A new, selective, and highly sensitive whole cell-based antisense strategy has been designed to screen for natural product inhibitors of FabH/F of the FAS II pathway using a high-throughput two-plate agar-based differential sensitivity assay (FabF(2)p). An antisense assay along with the FASII enzyme prepared from Staphylococcus aureus was used for bioactivity-guided fractionation, leading to the isolation of phomallenic acids A-C (1-3) from a leaf litter fungus identified as Phoma sp. Compounds 1-3 exhibited minimum detection concentrations (MDC) of 0.63, 0.31, and 0.15 microg/mL in the FabF(2P) assay, IC(50) values of 22, 3.4, and 0.77 microg/mL in the FASII enzyme assay, and minimum inhibitory concentrations (MIC) of 250, 7.8, and 3.9 microg/mL, respectively, against wild-type S. aureus. Phomallenic acid C (3), the analogue with the longest chain, exhibited the best overall activity within the phomallenic acids obtained and was superior to cerulenin and thiolactomycin, the two most studied and commonly used FabF inhibitors.

Overview of Antibacterial Target Selection

AbstractResearch in microbial genomics has yielded a vast amount of DNA sequence data and a number of new or improved tools for defining the bacterial genome. These findings have dramatically increased the number of potential antibacterial targets, particularly enzymatic sites, and have greatly enhanced their characterization.Potential targets are prioritized according to necessity for survival (essentiality), spectrum, and selectivity using a combination of bioinformatic analyses and experimental results. Experimental methods such as mutagenesis and conditional expression techniques are used to assess essentiality in vitro or virulence in vivo in animal hosts.

Use of Structure-Based Drug Design Approaches to Obtain Novel Anthranilic Acid Acyl Carrier Protein Synthase Inhibitors

Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.

Unique GMP‐binding site in Mycobacterium tuberculosis guanosine monophosphate kinase

Bacterial nucleoside monophosphate (NMP) kinases, which convert NMPs to nucleoside diphosphates (NDP), are investigated as potential antibacterial targets against pathogenic bacteria. Herein, we report the biochemical and structural characterization of GMP kinase from Mycobacterium tuberculosis (GMPKMt). GMPKMt is a monomer with an unusual specificity for ATP as a phosphate donor, a lower catalytic efficiency compared with eukaryotic GMPKs, and it carries two redox-sensitive cysteines in the central CORE domain. These properties were analyzed in the light of the high-resolution crystal structures of unbound, GMP-bound, and GDP-bound GMPKMt. The latter structure was obtained in both an oxidized form, in which the cysteines form a disulfide bridge, and a reduced form which is expected to correspond to the physiological enzyme. GMPKMt has a modular domain structure as most NMP kinases. However, it departs from eukaryotic GMPKs by the unusual conformation of its CORE domain, and by its partially open LID and GMP-binding domains which are the same in the apo-, GMP-bound, and GDP-bound forms. GMPKMt also features a unique GMP binding site which is less close-packed than that of mammalian GMPKs, and in which the replacement of a critical tyrosine by a serine removes a catalytic interaction. In contrast, the specificity of GMPKMt for ATP may be a general feature of GMPKs because of an invariant structural motif that recognizes the adenine base. Altogether, differences in domain dynamics and GMP binding between GMPKMt and mammalian GMPKs should reveal clues for the design of GMPKMt-specific inhibitors.

Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0 Å Resolution

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.

Inteins as targets for potential antimycobacterial drugs.

Protein splicing is a self-catalyzed process mediated by inteins. The observation that inteins occur only in microorganisms and that they often interrupt genes that play an essential role in nucleic acid metabolism makes them attractive as potential antibacterial targets. Because mycobacteria are the only intein-containing bacteria associated with human hosts, inteins would represent highly specific antimycobacterial targets. In Mycobacterium tuberculosis, two important proteins of DNA repair and replication, RecA and DnaB, respectively, are interrupted by inteins that must be excised by protein splicing before these proteins can function. This review describes the screening systems for the detection of mutations or inhibitors that interfere with proteins splicing that have been developed and published to date. In three of these experimental system, inteins have been inserted into proteins that are toxic under certain conditions. Protein splicing therefore leads to conditional growth inhibition or cell death and its inhibition can be monitored in terms of bacterial growth. A fourth assay for protein splicing and its inhibition is based on purified proteins and measures the formation of Green Fluorescent Protein or its inhibition. The advantages of inteins as antimycobacterial targets are discussed.

Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 Å resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a gram-positive pathogen Streptococcus pneumoniae.

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.

Antifungal properties and target evaluation of three putative bacterial histidine kinase inhibitors.

Histidine protein kinases have been explored as potential antibacterial drug targets. The recent identification of two-component histidine kinases in fungi has led us to investigate the antifungal properties of three bacterial histidine kinase inhibitors (RWJ-49815, RWJ-49968, and RWJ-61907). All three compounds were found to inhibit growth of the Saccharomyces cerevisiae and Candida albicans strains, with MICs ranging from 1 to 20 microg/ml. However, deletion of SLN1, the only histidine kinase in S. cerevisiae, did not alter drug efficacy. In vitro kinase assays were performed by using the Sln1 histidine kinase purified from bacteria as a fusion protein to glutathione S-transferase. RWJ-49815 and RWJ-49968 inhibited kinase a 50% inhibitory concentration of 10 microM, whereas RWJ-61907 failed to inhibit at concentrations up to 100 microM. Based on these results, we conclude that these compounds have antifungal properties; however, their mode of action appears to be independent of histidine kinase inhibition.

Biochemical characterization of tissue and recombinant mammalian dihydroorotate dehydrogenase

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.