RESUMO
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.
Assuntos
Acinetobacter baumannii/enzimologia , Alquil e Aril Transferases/química , Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Descoberta de Drogas , Alquil e Aril Transferases/isolamento & purificação , Antibacterianos/química , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Cristalização , Escherichia coli , Expressão Gênica/genética , Modelos Moleculares , Conformação Proteica , Difração de Raios XRESUMO
A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , DNA Girase/metabolismo , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Knockout , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.
Assuntos
Citoplasma/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Trissacarídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular , Farmacorresistência Bacteriana Múltipla , Técnicas de Inativação de Genes , Maltose/genética , Maltose/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Óperon/genética , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Perileno/química , Polissacarídeos/metabolismo , Porinas/genética , Porinas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Regulon/genética , Trissacarídeos/químicaRESUMO
Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.
Assuntos
Antibacterianos/metabolismo , Enterobacteriaceae/metabolismo , Porinas/química , Porinas/metabolismo , beta-Lactamas/metabolismo , Antibacterianos/química , Cátions/metabolismo , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Difusão Facilitada , Glicina/metabolismo , Bibliotecas Digitais , Lipossomos/metabolismo , Concentração Osmolar , Multimerização Proteica , Eletricidade Estática , beta-Lactamas/químicaRESUMO
Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA ('poisoning') in an analogous fashion to fluoroquinolones, but without the requirement for the water-metal-ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase-DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.
Assuntos
Antibacterianos/química , DNA Girase/química , Pirazinas/química , Inibidores da Topoisomerase II/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/classificação , Ciprofloxacina/química , DNA/química , Clivagem do DNA , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Fluoroquinolonas/química , Magnésio/química , Modelos Moleculares , MutaçãoRESUMO
In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Imidazóis/farmacologia , Pirazinas/farmacologia , Quinazolinonas/farmacologia , Inibidores da Topoisomerase/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Antibacterianos/toxicidade , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Células Hep G2 , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Imidazóis/toxicidade , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Pirazinas/síntese química , Pirazinas/farmacocinética , Pirazinas/toxicidade , Quinazolinonas/síntese química , Quinazolinonas/farmacocinética , Quinazolinonas/toxicidade , Inibidores da Topoisomerase/síntese química , Inibidores da Topoisomerase/farmacocinética , Inibidores da Topoisomerase/toxicidadeRESUMO
A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.
Assuntos
Antibacterianos , Clivagem do DNA , DNA Girase , Tiofenos , Antibacterianos/química , Antibacterianos/metabolismo , Cristalografia por Raios X , DNA Girase/química , DNA Girase/metabolismo , Descoberta de Drogas , Modelos Moleculares , Tiofenos/química , Tiofenos/metabolismoRESUMO
Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Staphylococcus aureus/enzimologia , Relação Estrutura-AtividadeRESUMO
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Assuntos
Antibacterianos/química , Antineoplásicos/química , DNA Girase/química , Etoposídeo/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana , Etoposídeo/farmacologia , Fluoroquinolonas/farmacologia , Modelos Moleculares , Estrutura Molecular , Moxifloxacina , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologiaRESUMO
Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Microespectrofotometria , Análise de Célula Única/métodos , Antibacterianos/química , Bactérias/genética , Farmacorresistência Bacteriana , Estrutura Molecular , Oxirredução , Espécies Reativas de OxigênioRESUMO
The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.
Assuntos
Anti-Hipertensivos/síntese química , Benzimidazóis/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxidiazóis/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oxidiazóis/farmacocinética , Oxidiazóis/farmacologia , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.
Assuntos
Amidas/síntese química , Anti-Hipertensivos/síntese química , Indazóis/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridonas/síntese química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Indazóis/farmacocinética , Indazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Proteínas Serina-Treonina Quinases/química , Piridonas/farmacocinética , Piridonas/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
The mechanism of the catalytic, enantioselective addition of trichlorosilyl enolates to aldehydes has been investigated. Kinetic studies using ReactIR and rapid injection NMR (RINMR) spectroscopy have confirmed the simultaneous operation of dual mechanistic pathways involving either one or two phosphoramides bound to a siliconium ion organizational center. This mechanistic dichotomy was initially postulated on the basis of catalyst loading studies and nonlinear effects studies. This duality explains the difference in reactivity and stereoselectivity of various classes of phosphoramides. Determination of Arrhenius activation parameters revealed that aldol addition occurs through the reversible albeit unfavorable formation of an activated complex, and natural-abundance 13C NMR kinetic isotope effect (KIE) studies have determined that the turnover limiting step is the aldol addition. A thorough examination of a range of phosphoramides has established empirical structure-activity selectivity relationships. In addition, the effects of catalyst loading, rate of addition, solvents, and additives have been studied and together allow the formulation of a unified mechanistic picture for the aldol addition.
RESUMO
Studies toward the development of an enantioselective diazomethane-based cyclopropanation reagent derived from bis(oxazoline)palladium(II) complexes are reported. Several simple palladium chelates, 2 and 7, in addition to the novel carbon-bound complexes 15 were synthesized and evaluated in the cyclopropanation of various electron-deficient olefins. The X-ray crystal structure of aryl-bis(oxazoline)palladium complex 15c is described. Although all catalysts efficiently affected cyclopropanation, all products were racemic. An intriguing relationship between substitution on the oxazoline ring, particularly the commonly-derivatized 4-position, and catalyst efficiency was discovered. The results are rationalized by either partial or complete bis(oxazoline) decomplexation during the course of the reaction.