RESUMEN
Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, ß-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and ß-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 µg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring ß-thujaplicin and the α- and ß-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 µM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Tropolona/farmacología , Antibacterianos/química , Calorimetría , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/enzimología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Tropolona/químicaRESUMEN
We report the first Raman spectroscopic study of propargyl-linked dihydrofolate reductase (DHFR) inhibitors being taken up by wild type Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus cells. A novel protocol is developed where cells are exposed to the fermentation medium containing a known amount of an inhibitor. At a chosen time point, the cells are centrifuged and washed to remove the extracellular compound, then frozen and freeze-dried. Raman difference spectra of the freeze-dried cells (cells exposed to the drug minus cells alone) provide spectra of the compounds inside the cells, where peak intensities allow us to quantify the number of inhibitors within each cell. A time course for the propargyl-linked DHFR inhibitor UCP 1038 soaking into E. coli cells showed that penetration occurs very quickly and reaches a plateau after 10 min exposure to the inhibitor. After 10 min drug exposure, the populations of two inhibitors, UCP 1038 and UCP 1089, were ~1.5 × 10(6) molecules in each E. coli cell, ~4.7 × 10(5) molecules in each K. pneumonia cell, and ~2.7 × 10(6) in each S. aureus cell. This is the first in situ comparison of inhibitor population in Gram-negative and Gram-positive bacterial cells. The positions of the Raman peaks also reveal the protonation of diaminopyrimidine ring upon binding to DHFR inside cells. The spectroscopic signature of protonation was characterized by binding an inhibitor to a single crystal of DHFR.
Asunto(s)
Escherichia coli/metabolismo , Antagonistas del Ácido Fólico/farmacocinética , Klebsiella pneumoniae/metabolismo , Microscopía/métodos , Espectrometría Raman/métodos , Staphylococcus aureus/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Cristalografía por Rayos X , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Resistance to the antibacterial antifolate trimethoprim (TMP) is increasing in members of the family Enterobacteriaceae, driving the design of next-generation antifolates effective against these Gram-negative pathogens. The propargyl-linked antifolates are potent inhibitors of dihydrofolate reductases (DHFR) from several TMP-sensitive and -resistant species, including Klebsiella pneumoniae. Recently, we have determined that these antifolates inhibit the growth of strains of K. pneumoniae, some with MIC values of 1 µg/ml. In order to further the design of potent and selective antifolates against members of the Enterobacteriaceae, we determined the first crystal structures of K. pneumoniae DHFR bound to two of the propargyl-linked antifolates. These structures highlight that interactions with Leu 28, Ile 50, Ile 94, and Leu 54 are necessary for potency; comparison with structures of human DHFR bound to the same inhibitors reveal differences in residues (N64E, P61G, F31L, and V115I) and loop conformations (residues 49 to 53) that may be exploited for selectivity.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Antagonistas del Ácido Fólico/química , Klebsiella pneumoniae/química , Tetrahidrofolato Deshidrogenasa/química , Trimetoprim/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Klebsiella pneumoniae/enzimología , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/genética , Resistencia al Trimetoprim/genéticaRESUMEN
Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.
Asunto(s)
Antagonistas del Ácido Fólico , Resistencia al Trimetoprim , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/análogos & derivados , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Plásmidos/genética , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Resistencia al Trimetoprim/genéticaRESUMEN
Resistance to therapeutics such as trimethoprim-sulfamethoxazole has become an increasing problem in strains of methicillin-resistant Staphylococcus aureus (MRSA). Clinically isolated trimethoprim-resistant strains reveal a double mutation, H30N/F98Y, in dihydrofolate reductase (DHFR). In order to develop novel and effective therapeutics against these resistant strains, we evaluated a series of propargyl-linked antifolate lead compounds for inhibition of the mutant enzyme. For the propargyl-linked antifolates, the F98Y mutation generates minimal (between 1.2- and 6-fold) losses of affinity and the H30N mutation generates greater losses (between 2.4- and 48-fold). Conversely, trimethoprim affinity is largely diminished by the F98Y mutation (36-fold) and is not affected by the H30N mutation. In order to elucidate a mechanism of resistance, we determined a crystal structure of a complex of this double mutant with a lead propargyl-linked antifolate. This structure suggests a resistance mechanism consistent both for the propargyl-linked class of antifolates and for trimethoprim that is based on the loss of a conserved water-mediated hydrogen bond.
Asunto(s)
Resistencia a la Meticilina/genética , Modelos Moleculares , Staphylococcus aureus/enzimología , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Resistencia al Trimetoprim/genética , Clonación Molecular , Cristalización , Antagonistas del Ácido Fólico/farmacología , Resistencia a la Meticilina/fisiología , Estructura Molecular , Mutación Missense/genética , Staphylococcus aureus/fisiología , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/farmacología , Resistencia al Trimetoprim/fisiologíaRESUMEN
Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Organofosfonatos/farmacología , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Fosfopiruvato Hidratasa/metabolismo , Pirrolidinonas/farmacología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fosfopiruvato Hidratasa/química , Conformación Proteica , Homología de Secuencia , Relación Estructura-ActividadRESUMEN
Raman difference spectroscopy is shown to provide a wealth of molecular detail on changes within bacterial cells caused by infusion of antibiotics or hydrogen peroxide. Escherichia coli strains paired with chloramphenicol, dihydrofolate reductase propargyl-based inhibitors, meropenem, or hydrogen peroxide provide details of the depletion of protein and nucleic acid populations in real time. Additionally, other reproducible Raman features appear and are attributed to changes in cell metabolite populations. An initial candidate for one of the metabolites involves population increases of citrate, an intermediate within the tricarboxyclic acid cycle. This is supported by the observation that a strain of E. coli without the ability to synthesize citrate, gltA, lacks an intense feature in the Raman difference spectrum that has been ascribed to citrate. The methodology for obtaining the Raman data involves infusing the drug into live cells, then washing, freezing, and finally lyophilizing the cells. The freeze-dried cells are then examined under a Raman microscope. The difference spectra [cells treated with drug] - [cells without treatment] are time-dependent and can yield population kinetics for intracellular species in vivo. There is a strong resemblance between the Raman difference spectra of E. coli cells treated with meropenem and those treated with hydrogen peroxide.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Espectrometría Raman , Cloranfenicol/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Peróxido de Hidrógeno/farmacología , Cinética , Meropenem/farmacología , MicroscopíaRESUMEN
Multidrug-resistant Enterobacteriaceae, notably Escherichia coli and Klebsiella pneumoniae, have become major health concerns worldwide. Resistance to effective therapeutics is often carried by class I and II integrons that can confer insensitivity to carbapenems, extended spectrum ß-lactamases, the antifolate trimethoprim, fluoroquinolones, and aminoglycosides. Specifically of interest to the study here, a prevalent gene (dfrA1) coding for an insensitive dihydrofolate reductase (DHFR) confers 190- or 1000-fold resistance to trimethoprim for K. pneumoniae and E. coli, respectively. Attaining inhibition of both the wild-type and resistant forms of the enzyme is critical for new antifolates. For several years, we have been developing the propargyl-linked antifolates (PLAs) as effective inhibitors against trimethoprim-resistant DHFR enzymes. Here, we show that the PLAs are active against both the wild-type and DfrA1 DHFR proteins. We report two high-resolution crystal structures of DfrA1 bound to potent PLAs. The structure-activity relationships and crystal structures will be critical in driving the design of broadly active inhibitors against wild-type and resistant DHFR.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Tetrahidrofolato Deshidrogenasa/química , Resistencia al Trimetoprim/efectos de los fármacos , Trimetoprim/farmacología , Proteínas Bacterianas/química , Cristalografía por Rayos X , Escherichia coli/enzimología , Humanos , Integrones , Klebsiella pneumoniae/enzimología , Relación Estructura-Actividad , beta-Lactamasas/metabolismoRESUMEN
Although classical, negatively charged antifolates such as methotrexate possess high affinity for the dihydrofolate reductase (DHFR) enzyme, they are unable to penetrate the bacterial cell wall, rendering them poor antibacterial agents. Herein, we report a new class of charged propargyl-linked antifolates that capture some of the key contacts common to the classical antifolates while maintaining the ability to passively diffuse across the bacterial cell wall. Eight synthesized compounds exhibit extraordinary potency against Gram-positive S. aureus with limited toxicity against mammalian cells and good metabolic profile. High resolution crystal structures of two of the compounds reveal extensive interactions between the carboxylate and active site residues through a highly organized water network.
RESUMEN
Understanding the structural basis of antibacterial resistance may enable rational design principles that avoid and subvert that resistance, thus leading to the discovery of more effective antibiotics. In this review, we explore the use of crystal structures to guide new discovery of antibiotics that are effective against resistant organisms. Structures of efflux pumps bound to substrates and inhibitors have aided the design of compounds with lower affinity for the pump or inhibitors that more effectively block the pump. Structures of ß-lactamase enzymes have revealed the mechanisms of action toward key carbapenems and structures of gyrase have aided the design of compounds that are less susceptible to point mutations.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Bacterianas/química , Descubrimiento de Drogas/métodos , Farmacorresistencia Bacteriana , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 µg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.
Asunto(s)
Antifúngicos , Candida albicans/efectos de los fármacos , Candida glabrata/efectos de los fármacos , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/farmacología , Morfinanos/síntesis química , Morfinanos/farmacología , Cromatografía Líquida de Alta Presión , Cristalización , Cristalografía por Rayos X , Antagonistas del Ácido Fólico/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Morfinanos/química , NADP/química , Solubilidad , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/efectos de los fármacos , Difracción de Rayos XRESUMEN
Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.