RESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.
Asunto(s)
Antibacterianos , Lipopolisacáridos , Proteínas de Transporte de Membrana , Animales , Humanos , Ratones , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/clasificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Desarrollo de MedicamentosRESUMEN
Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
Asunto(s)
Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Lipopolisacáridos , Proteínas de Transporte de Membrana , Acinetobacter/química , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipidâ II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S.â aureus FtsW in vitro. Using a non-polymerizable Lipidâ II derivative, we showed that this compound competes with Lipidâ II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.
Asunto(s)
Proteínas Bacterianas , Peptidoglicano Glicosiltransferasa , Proteínas Bacterianas/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Peptidoglicano/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de la Membrana/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Pared Celular/metabolismoRESUMEN
Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of â¼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Glucolípidos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Mutaciones Letales SintéticasRESUMEN
Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type Escherichia coli and Acinetobacter baumannii but not the growth of the Gram-positive organism Bacillus subtilis. Resistance mutations in E. coli and A. baumannii mapped exclusively to the aminoacyl-tRNA synthetase PheRS. We confirmed biochemically that the compound inhibited PheRS from these organisms and showed that it did not inhibit PheRS from B. subtilis or humans. To understand the basis for the compound's high selectivity for only some PheRS enzymes, we solved crystal structures of E. coli and A. baumannii PheRS complexed with the inhibitor. The structures showed that the compound's benzyl group mimics the benzyl of phenylalanine. The other amine substituent, a 2-(cyclohexen-1-yl)ethyl group, induces a hydrophobic pocket in which it binds. Through bioinformatic analysis and mutagenesis, we show that the ability to induce a complementary hydrophobic pocket that can accommodate the second substituent explains the high selectivity of this remarkably simple molecular scaffold for Gram-negative PheRS. Because this secondary amine scaffold is active against wild-type Gram-negative pathogens but is not cytotoxic to mammalian cells, we suggest that it may be possible to develop it for use in combination antibiotic therapy to treat Gram-negative infections.
Asunto(s)
Aminas/farmacología , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Aminas/química , Antibacterianos/química , Bacillus subtilis/enzimología , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fenilalanina-ARNt Ligasa/metabolismoRESUMEN
New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward 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 multiprotein 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 (also known as amdinocillin) 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 â¼690,000 compounds identified 1,300 compounds that were 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 those from 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.
Asunto(s)
Antibacterianos/farmacología , Pared Celular/metabolismo , Escherichia coli/efectos de los fármacos , Peptidoglicano/metabolismo , Amdinocilina/farmacología , Amdinocilina/toxicidad , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas del Citoesqueleto/antagonistas & inhibidores , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Proteínas de Unión a las Penicilinas/metabolismoRESUMEN
New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Acinetobacter baumannii/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Lipopolisacáridos/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lipopolisacáridos/genéticaRESUMEN
Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.