sep413

Related Articles

Chemical tools to characterize peptidoglycan synthases.

Curr Opin Chem Biol. 2019 12;53:44-50

Authors: Taguchi A, Kahne D, Walker S

Abstract
The peptidoglycan cell wall is a unique macromolecular structure in bacteria that defines their shape and confers protection from the surrounding environment. Decades of research has focused on understanding the peptidoglycan synthesis pathway and exploiting its essentiality for antibiotic development. Recently, a new class of peptidoglycan polymerases known as the SEDS (shape, elongation, division and sporulation) proteins were identified; these polytopic membrane proteins function together with the better-known penicillin-binding proteins (PBPs) to build the cell wall. In this review, we will highlight recent developments in chemical tools and methods to label the bacterial cell wall and discuss how these developments are leading to a better understanding of peptidoglycan synthases and their cellular roles.

PMID: 31466035 [PubMed - indexed for MEDLINE]

Related Articles

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis.

ACS Chem Biol. 2019 12 20;14(12):2745-2756

Authors: Kuru E, Radkov A, Meng X, Egan A, Alvarez L, Dowson A, Booher G, Breukink E, Roper DI, Cava F, Vollmer W, Brun Y, VanNieuwenhze MS

Abstract
Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

PMID: 31743648 [PubMed - indexed for MEDLINE]

Related Articles

d-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis.

Acc Chem Res. 2019 09 17;52(9):2713-2722

Authors: Hsu YP, Booher G, Egan A, Vollmer W, VanNieuwenhze MS

Abstract
The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.

PMID: 31419110 [PubMed - indexed for MEDLINE]

Related Articles

The role of MurT C-terminal domain in the amidation of Staphylococcus aureus peptidoglycan.

Antimicrob Agents Chemother. 2019 Jul 29;:

Authors: Gonçalves BV, Portela R, Lobo R, Figueiredo TA, Grilo IR, Ludovice AM, de Lencastre H, Dias JS, Sobral RG

Abstract
Glutamate amidation, a secondary modification of the peptidoglycan was first identified in Staphylococcus aureus It is catalyzed by the protein products of the murT and gatD genes that are conserved and co-localized in the genome of most sequenced gram-positive species. The MurT-GatD complex is required for cell viability, full resistance to beta-lactam antibiotics and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. A great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex.In this work we demonstrate, through non-structural approaches, the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, on MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Co-purification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence on the effect of specific residue substitutions in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

PMID: 31358586 [PubMed - as supplied by publisher]

Related Articles

Minimal exposure of lipid II cycle intermediates triggers cell wall antibiotic resistance.

Nat Commun. 2019 Jun 21;10(1):2733

Authors: Piepenbreier H, Diehl A, Fritz G

Abstract
Cell wall antibiotics are crucial for combatting the emerging wave of resistant bacteria. Yet, our understanding of antibiotic action is limited, as many strains devoid of all resistance determinants display far higher antibiotic tolerance in vivo than suggested by the antibiotic-target binding affinity in vitro. To resolve this conflict, here we develop a comprehensive theory for the bacterial cell wall biosynthetic pathway and study its perturbation by antibiotics. We find that the closed-loop architecture of the lipid II cycle of wall biosynthesis features a highly asymmetric distribution of pathway intermediates, and show that antibiotic tolerance scales inversely with the abundance of the targeted pathway intermediate. We formalize this principle of minimal target exposure as intrinsic resistance mechanism and predict how cooperative drug-target interactions can mitigate resistance. The theory accurately predicts the in vivo efficacy for various cell wall antibiotics in different Gram-positive bacteria and contributes to a systems-level understanding of antibiotic action.

PMID: 31227716 [PubMed - in process]

Related Articles

Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles' heel for the TB-causing pathogen.

FEMS Microbiol Rev. 2019 Jun 10;:

Authors: Maitra A, Munshi T, Healy J, Martin LT, Vollmer W, Keep NH, Bhakta S

Abstract
Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. M. tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors against ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

PMID: 31183501 [PubMed - as supplied by publisher]

Related Articles

Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays.

Nat Chem. 2019 04;11(4):335-341

Authors: Hsu YP, Hall E, Booher G, Murphy B, Radkov AD, Yablonowski J, Mulcahey C, Alvarez L, Cava F, Brun YV, Kuru E, VanNieuwenhze MS

Abstract
Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.

PMID: 30804500 [PubMed - indexed for MEDLINE]

Related Articles

FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein.

Nat Microbiol. 2019 04;4(4):587-594

Authors: Taguchi A, Welsh MA, Marmont LS, Lee W, Sjodt M, Kruse AC, Kahne D, Bernhardt TG, Walker S

Abstract
The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria1. For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA-a member of the unrelated SEDS protein family-also acts as a peptidoglycan polymerase2-4. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly5,6. FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane7,8. Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division.

PMID: 30692671 [PubMed - indexed for MEDLINE]

Related Articles

Kinetic mechanism of Enterococcus faecium D-aspartate ligase.

Biochimie. 2019 Jan 22;:

Authors: Škedelj V, Fonović UP, Molek P, Magnet S, Mainardi JL, Blanot D, Gobec S, Stojan J, Zega A

Abstract
Enterococcus faecium D-aspartate ligase (Aslfm) is a peptide bond-forming enzyme that is involved in the peptidoglycan assembly pathway. It catalyzes the ATP-dependent ligation of the β-carboxylate of D-Asp to the ε-amino group of L-Lys in the nucleotide precursor UDP- MurNAc-pentapeptide. The enzyme is of interest as a target of new, potential, narrow-spectrum antibiotics directed against multiresistant E. faecium. The kinetic mechanism of Aslfm has not been fully characterized. To determine it, a progress curve analysis of Aslfm catalytic process using pyruvate kinase/lactate dehydrogenase ATPase detection assay was performed. With an inspection of the shape of measured progress curves and the results of specific qualitative experiments, the Aslfm reaction mechanism was singled out. The proposed Aslfm kinetics reaction scheme was evaluated by fitting the parameters of the corresponding differential equations to progress curves using the computer program ENZO. The complete kinetic analysis result is consistent with the substrate binding order 1) ATP, 2) D-Asp, and 3) UDP-MurNAc-pentapeptide. The analysis suggests that slowly establishing non-productive equilibria between the free and ATP-bound enzyme with the participating pentapeptide are responsible for initial reaction burst followed by a steady-state period before the complete depletion of the reactant added in the lowest concentration.

PMID: 30682389 [PubMed - as supplied by publisher]

Related Articles

Chemoselective, Postpolymerization Modification of Bioactive, Degradable Polymers.

Biomacromolecules. 2019 02 11;20(2):1018-1027

Authors: Fishman JM, Zwick DB, Kruger AG, Kiessling LL

Abstract
Degradable polymers promote sustainability, mitigate environmental impact, and facilitate biological applications. Tailoring degradable polymers is challenging because installing functional group-rich side chains is difficult when the backbone itself is susceptible to degradation. A convenient means of side chain installation is through postpolymerization modification (PPM). In functionalizing polyoxazinones, a class of degradable polymers generated by the ring-opening metathesis polymerization (ROMP), we predictably found PPM challenging. Even the versatile azide-alkyne cycloaddition click reaction was ineffective. To solve this problem, we screened PPM reactions whose efficiencies could be assessed using photochemistry (excimer formation). The mildest, pH-neutral process was functionalization of a ketone-containing polymer to yield either oxime (acid labile)- or alkyoxylamine (stable)-substituted polymers. Using this approach, we equipped polymers with fluorophores, reporter groups, and bioactive epitopes. These modifications imbued the polymers with distinctive spectral properties and biological activities. Thus, polyoxazinones are now tunable through a modular method to diversify these macromolecules' function.

PMID: 30608163 [PubMed - indexed for MEDLINE]

Related Articles

Structural basis for the interaction and processing of β-lactam antibiotics by L,D- transpeptidase 3 (LdtMt3) from Mycobacterium tuberculosis.

ACS Infect Dis. 2018 Dec 17;:

Authors: Libreros-Z Uacute Ntilde Iga GAES, Dos Santos Silva C, Salgado Ferreira R, Dias MVB

Abstract
Targeting Mycobacterium tuberculosis peptidoglycans with β-lactam antibiotics represents a strategy to address increasing resistance to anti-tubercular drugs. β-Lactams inhibit peptidoglycan synthases such as L,D-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3→3 cross-links. M. tuberculosis encodes five L,D-transpeptidases (LdtMt1-5) of which LdtMt3 is one of the less understood. Herein, we structurally characterized the apo and faropenem-acylated forms of LdtMt3 at 1.3 and 1.8 Å resolution, respectively. These structures revealed a fold and catalytic diad similar to those of other LdtsMt enzymes, supporting its involvement in transpeptidation reactions despite divergences in active site size and charges. The LdtMt3-faropenem structure indicated that faropenem is degraded after Cys-246 acylation and possibly only a β-OH-butyrate or an acetyl group (C2H3O) covalently attached to the enzyme remains, an observation that strongly supports the notion that LdtMt3 is inactivated by β-lactams. Docking simulations with intact β-lactams predicted key LdtMt3 residues that interact with these antibiotics. We also characterized the heat of acylation involved in the binding and reaction of LdtMt3 for ten β-lactams belonging to four different classes and imipenem had the highest inactivation constant. This work provides key insights into the structure, binding mechanisms, and degradation of β-lactams by LdtMt3 which may be useful for the development of additional β-lactams with potential anti-tubercular activity.

PMID: 30556998 [PubMed - as supplied by publisher]

Related Articles

Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division.

Nat Commun. 2018 11 30;9(1):5090

Authors: Pazos M, Peters K, Casanova M, Palacios P, VanNieuwenhze M, Breukink E, Vicente M, Vollmer W

Abstract
During the transition from elongation to septation, Escherichia coli establishes a ring-like peptidoglycan growth zone at the future division site. This preseptal peptidoglycan synthesis does not require the cell division-specific peptidoglycan transpeptidase PBP3 or most of the other cell division proteins, but it does require FtsZ, its membrane-anchor ZipA and at least one of the bi-functional transglycosylase-transpeptidases, PBP1A or PBP1B. Here we show that PBP1A and PBP1B interact with ZipA and localise to preseptal sites in cells with inhibited PBP3. ZipA stimulates the glycosyltransferase activity of PBP1A. The membrane-anchored cell division protein FtsN localises at preseptal sites and stimulates both activities of PBP1B. Genes zipA and ftsN can be individually deleted in ftsA* mutant cells, but the simultaneous depletion of both proteins is lethal and cells do not establish preseptal sites. Our data support a model according to which ZipA and FtsN-FtsA have semi-redundant roles in connecting the cytosolic FtsZ ring with the membrane-anchored peptidoglycan synthases during the preseptal phase of envelope growth.

PMID: 30504892 [PubMed - indexed for MEDLINE]

Related Articles

The bacterial lipid II flippase MurJ functions by an alternating-access mechanism.

J Biol Chem. 2018 Nov 27;:

Authors: Kumar S, Rubino FA, Mendoza AG, Ruiz N

Abstract
The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. Bacteria use multi-protein machineries for the synthesis of the PG cell wall during cell division and elongation that can be targeted by antibiotics such as the β-lactams. Lipid II, the lipid-linked precursor for PG biogenesis, is synthesized in the inner leaflet of the cytoplasmic membrane and then translocated across the bilayer, where it is ultimately polymerized into PG. In Escherichia coli, MurJ, a member of the MOP exporter superfamily, has been recently shown to have lipid II flippase activity that is dependent on membrane potential. Because of its essentiality, MurJ could potentially be targeted by much needed novel antibiotics. Recent structural information suggests that a central cavity in MurJ alternates between inward- and outward-open conformations to flip lipid II, but how these conformational changes occur are unknown. Here, we utilized structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe the conformational changes of MurJ in E. coli cells. We found that paired cysteine substitutions in transmembrane domains 2 and 8, and periplasmic loops of MurJ could be cross-linked with homobifunctional cysteine cross-linkers, indicating that MurJ can adopt both inward- and outward-facing conformations in vivo. Furthermore, we show that dissipating the membrane potential with an ionophore decreases the prevalence of the inward-facing, but not the outward-facing state. Our study provides in vivo evidence that MurJ uses an alternating-access mechanism during the lipid II transport cycle.

PMID: 30482840 [PubMed - as supplied by publisher]

Related Articles

Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus.

BMC Microbiol. 2018 Nov 29;18(1):201

Authors: Coyle JF, Pagliai FA, Zhang D, Lorca GL, Gonzalez CF

Abstract
BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP.
RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS.
CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.

PMID: 30497377 [PubMed - in process]

With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5'-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB₂FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in Escherichia coli.

Preliminary finding turns heads at neuroscience meeting

Structural insights into tunicamycin’s toxic interactions with the human N-linked glycosylation pathway allows the identification of non-toxic antibiotics effective against tuberculosis in mice

Antibiotic resistance is often the result of mutations that block drug activity; however, bacteria also evade antibiotics by transiently expressing genes such as multidrug efflux pumps. A crucial question is whether transient resistance can promote permanent genetic changes. Previous studies have established that antibiotic treatment can select tolerant cells that then mutate to achieve permanent resistance. Whether these mutations result from antibiotic stress or preexist within the population is unclear. To address this question, we focused on the multidrug pump AcrAB-TolC. Using time-lapse microscopy, we found that cells with higher acrAB expression have lower expression of the DNA mismatch repair gene mutS, lower growth rates, and higher mutation frequencies. Thus, transient antibiotic resistance from elevated acrAB expression can promote spontaneous mutations within single cells.

Related Articles

Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.

Sci Rep. 2017 01 23;7:41147

Authors: Ganesan L, Shieh P, Bertozzi CR, Levental I

Abstract
Palmitoylation is a widespread, reversible lipid modification that has been implicated in regulating a variety of cellular processes. Approximately one thousand proteins are annotated as being palmitoylated, and for some of these, including several oncogenes of the Ras and Src families, palmitoylation is indispensable for protein function. Despite this wealth of disease-relevant targets, there are currently few effective pharmacological tools to interfere with protein palmitoylation. One reason for this lack of development is the dearth of assays to efficiently screen for small molecular inhibitors of palmitoylation. To address this shortcoming, we have developed a robust, high-throughput compatible, click chemistry-based approach to identify small molecules that interfere with the palmitoylation of Ras, a high value therapeutic target that is mutated in up to a third of human cancers. This assay design shows excellent performance in 384-well format and is sensitive to known, non-specific palmitoylation inhibitors. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. The screening approach described here provides an integrated platform to identify specific modulators of palmitoylated proteins, demonstrated here for Ras and Fyn, but potentially applicable to pharmaceutical targets involved in a variety of human diseases.

PMID: 28112226 [PubMed - indexed for MEDLINE]

Related Articles

Use of a Fluorescent Analogue of a HBV Core Protein-Directed Drug To Interrogate an Antiviral Mechanism.

J Am Chem Soc. 2018 11 14;140(45):15261-15269

Authors: Nair S, Li L, Francis S, Turner WW, VanNieuwenhze M, Zlotnick A

Abstract
Heteroaryldihydropyrimidines (HAPs) are antiviral small molecules that enhance assembly of HBV core protein (Cp), lead to assembly of empty and defective particles, and suppress viral replication. These core protein allosteric modulators (CpAMs) bind to the pocket at the interface between two Cp dimers and strengthen interdimer interactions. To investigate the CpAM mechanism, we wanted to examine the cellular distributions of Cp and the CpAM itself. For this reason, we developed a fluorescently labeled CpAM, HAP-ALEX. In vitro, HAP-ALEX modulated assembly of purified Cp and at saturating concentrations induced formation of large structures. HAP-ALEX bound capsids and not dimers, making it a capsid-specific molecular tag. HAP-ALEX labeled HBV in transfected cells, with no detectable background with a HAP-insensitive Cp mutant. HAP-ALEX caused redistribution of Cp in a dose-dependent manner consistent with its 0.7 μM EC50, leading to formation of large puncta and an exclusively cytoplasmic distribution. HAP-ALEX colocalized with the redistributed Cp, but large puncta accumulated long before they appeared saturated with the fluorescent CpAM. CpAMs affect HBV assembly and localization; with a fluorescent CpAM both drug and target can be identified.

PMID: 30375863 [PubMed - indexed for MEDLINE]

Related Articles

Synthesis of Lipid-Carbohydrate-Peptidyl-RNA Conjugates to Explore the Limits Imposed by the Substrate Specificity of Cell Wall Enzymes on the Acquisition of Drug Resistance.

Chemistry. 2018 Oct 09;24(56):14911-14915

Authors: Fonvielle M, Bouhss A, Hoareau C, Patin D, Mengin-Lecreulx D, Iannazzo L, Sakkas N, El Sagheer A, Brown T, Ethève-Quelquejeu M, Arthur M

Abstract
Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNAGly and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 =56 nm). The synthetic route was exploited to generate substrates and inhibitors containing d-lactate residue (d-Lac) instead of d-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.

PMID: 30020544 [PubMed - indexed for MEDLINE]

Related Articles

A pathway-directed screen for inhibitors of the bacterial cell elongation machinery.

Antimicrob Agents Chemother. 2018 Oct 15;:

Authors: Buss JA, Baidin V, Welsh MA, Flores-Kim J, Cho H, Wood BM, Uehara T, Walker S, Kahne D, Bernhardt TG

Abstract
New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue towards the discovery of such next generation therapies is to identify novel inhibitors of clinically validated targets like cell wall biogenesis. We have therefore developed a pathway-directed, whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli This conserved multi-protein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod- cells. A screen of ∼690,000 identified 1,300 compounds active against E. coli Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB-antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

PMID: 30323039 [PubMed - as supplied by publisher]

Related Articles

Design, synthesis and biological evaluations of quaternization harman analogues as potential antibacterial agents.

Eur J Med Chem. 2018 Oct 06;160:23-36

Authors: Dai J, Dan W, Ren S, Shang C, Wang J

Abstract
Thirty-three new quaternization harman analogues were synthesized and their antibacterial activity against four Gram-positive and two Gram-negative bacteria were evaluated. The structure-activity relationships were summarized and compounds 4f, 4i, 4l, 4u, 4w, 4x and 5c showed excellent antibacterial activity, low cytotoxicity, good thermal stability and "drug-like" properties. In particular, compound 4x exhibited better bactericidal effect (4-fold superiority against methicillin-resistant Staphylococcus aureus) than standard drugs fosfomycin sodium and ampicillin sodium (minimum inhibitory concentration = 50 nmol/mL). Scanning electron microscopy revealed morphological changes of the bacterial cell surface and the docking evaluation provided a good total score (6.4952) for 4x which is close to the score of ciprofloxacin (6.9723). The results indicated that the quaternization harman analogues might exert their bactericidal effect by damaging bacterial cell membrane and wall, and disrupting the function of type II topoisomerase. In addition, the in vivo antibacterial assay with a protective efficacy of 81.3% further demonstrated the potential of these derivatives as new bactericides and antibiotics.

PMID: 30317023 [PubMed - as supplied by publisher]

Related Articles

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

J Biol Chem. 2018 12 07;293(49):19113-19126

Authors: McDonald ND, DeMeester KE, Lewis AL, Grimes CL, Boyd EF

Abstract
Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

PMID: 30315110 [PubMed - indexed for MEDLINE]

Related Articles

Drug resistance in Mycobacterium tuberculosis and targeting the l,d-transpeptidase enzyme.

Drug Dev Res. 2018 Oct 12;:

Authors: Gokulan K, Varughese KI

Abstract
Hit, Lead & Candidate Discovery Tuberculosis (TB) is a disease that has afflicted mankind for thousands of years, but in the last seven decades, much progress has been made in anti-TB therapy. Early drugs, such as para-aminosalicylic acid, streptomycin, isoniazid, and rifamycins were very effective in combatting the disease, giving rise to the hope that TB would be eradicated from the face of the earth by 2010. Despite that optimism, TB continues to kill more than a million people annually worldwide. A major reason for our inability to contain TB is the emergence drug resistance in Mycobacterium tuberculosis. This commentary is based on our recent publication on the structure of l,d-transpeptidase enzyme, relevant to drug resistance. As a background, we briefly outline the history and development of anti-TB therapy. Based on the crystal structure, we suggest a potential direction for designing more potent drugs against TB.

PMID: 30312987 [PubMed - as supplied by publisher]