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1.
Proc Natl Acad Sci U S A ; 121(2): e2314101120, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38165935

RESUMO

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.


Assuntos
Anti-Infecciosos , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Animais , Camundongos , Espectinomicina/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Antibacterianos/farmacologia , Micobactérias não Tuberculosas , Anti-Infecciosos/farmacologia , Etilenos/farmacologia , Testes de Sensibilidade Microbiana
2.
PLoS Comput Biol ; 18(2): e1009855, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35143481

RESUMO

Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs' inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs' inhibition, while other PLAs remain unaffected by this resistance mechanism.


Assuntos
Antagonistas do Ácido Fólico , Infecções Estafilocócicas , Farmacorresistência Bacteriana/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , NADP/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/química , Trimetoprima/metabolismo , Trimetoprima/farmacologia
3.
Molecules ; 27(5)2022 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-35268619

RESUMO

Infections due to Gram-negative bacteria are increasingly dangerous due to the spread of multi-drug resistant strains, emphasizing the urgent need for new antibiotics with alternative modes of action. We have previously identified a novel class of antibacterial agents, thioacetamide-triazoles, using an antifolate targeted screen and determined their mode of action which is dependent on activation by cysteine synthase A. Herein, we report a detailed examination of the anti-E. coli structure-activity relationship of the thioacetamide-triazoles. Analogs of the initial hit compounds were synthesized to study the contribution of the aryl, thioacetamide, and triazole sections. A clear structure-activity relationship was observed generating compounds with excellent inhibition values. Substitutions to the aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems. Substitutions to the central thioacetamide linker section were more nuanced; the introduction of a methyl branch to the thioacetamide linker substantially decreased antibacterial activity, but the isomeric propionamide and N-benzamide systems retained activity. Changes to the triazole portion of the molecule dramatically decreased the antibacterial activity, further indicating that 1,2,3-triazole is critical for potency. From these studies, we have identified new lead compounds with desirable in-vitro ADME properties and in-vivo pharmacokinetic properties.


Assuntos
Escherichia coli , Triazóis , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tioacetamida , Triazóis/farmacologia
4.
Proc Natl Acad Sci U S A ; 112(3): 749-54, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25552560

RESUMO

Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structure-based protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.


Assuntos
Algoritmos , Antagonistas do Ácido Fólico/farmacologia , Proteínas/química , Resistência a Medicamentos/genética , Polimorfismo de Nucleotídeo Único , Staphylococcus aureus/enzimologia , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos
5.
J Am Chem Soc ; 137(28): 8983-90, 2015 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-26098608

RESUMO

While antifolates such as Bactrim (trimethoprim-sulfamethoxazole; TMP-SMX) continue to play an important role in treating community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), resistance-conferring mutations, specifically F98Y of dihydrofolate reductase (DHFR), have arisen and compromise continued use. In an attempt to extend the lifetime of this important class, we have developed a class of propargyl-linked antifolates (PLAs) that exhibit potent inhibition of the enzyme and bacterial strains. Probing the role of the configuration at the single propargylic stereocenter in these inhibitors required us to develop a new approach to nonracemic 3-aryl-1-butyne building blocks by the pairwise use of asymmetric conjugate addition and aldehyde dehydration protocols. Using this new route, a series of nonracemic PLA inhibitors was prepared and shown to possess potent enzyme inhibition (IC50 values <50 nM), antibacterial effects (several with MIC values <1 µg/mL) and to form stable ternary complexes with both wild-type and resistant mutants. Unexpectedly, crystal structures of a pair of individual enantiomers in the wild-type DHFR revealed that the single change in configuration of the stereocenter drove the selection of an alternative NADPH cofactor, with the minor α-anomer appearing with R-27. Remarkably, this cofactor switching becomes much more prevalent when the F98Y mutation is present. The observation of cofactor site plasticity leads to a postulate for the structural basis of TMP resistance in DHFR and also suggests design strategies that can be used to target these resistant enzymes.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Modelos Moleculares , Mutação Puntual , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Estereoisomerismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética
6.
Nat Commun ; 13(1): 3165, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672367

RESUMO

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.


Assuntos
Antibacterianos , Streptococcus pneumoniae , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Tolerância a Medicamentos , Humanos , Testes de Sensibilidade Microbiana
7.
J Antibiot (Tokyo) ; 74(6): 381-396, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33504919

RESUMO

Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs) through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the 2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+) and Gram (-) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the spectinamide and amSPCs. This study builds on the knowledge of the structure-activity relationships of spectinomycin analogs and is being used to aid the design of next-generation spectinomycins.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Ribossomos/efeitos dos fármacos , Espectinomicina/química , Antibacterianos/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Espectinomicina/análogos & derivados , Relação Estrutura-Atividade
8.
ACS Infect Dis ; 7(11): 3009-3024, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34699190

RESUMO

We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane's integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.


Assuntos
Acinetobacter baumannii , Antibacterianos , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana
9.
Clin Pharmacol Ther ; 109(4): 1000-1020, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33576025

RESUMO

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.


Assuntos
Técnicas Bacteriológicas/métodos , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana Múltipla/fisiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/fisiologia , Animais , Membrana Celular/fisiologia , Modelos Animais de Doenças , Humanos , Modelos Teóricos , Proteínas de Ligação às Penicilinas/fisiologia , beta-Lactamas/farmacologia
10.
ACS Infect Dis ; 6(3): 467-478, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31887254

RESUMO

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N-(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.


Assuntos
Cisteína Sintase/antagonistas & inibidores , Cisteína/biossíntese , Escherichia coli/efeitos dos fármacos , Tioacetamida/farmacologia , Triazóis/farmacologia , Antibacterianos/farmacologia , Antimetabólitos/farmacologia , Descoberta de Drogas , Escherichia coli/enzimologia , Ensaios de Triagem em Larga Escala , Redes e Vias Metabólicas/efeitos dos fármacos , Tioacetamida/química , Triazóis/química
11.
mBio ; 11(1)2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047131

RESUMO

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While ß-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a blaKPC-2 and several other ß-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular ß-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our ß-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of ß-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While ß-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of ß-lactams leads to high drug concentrations at their periplasmic target sites, allowing ß-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of ß-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five ß-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem ß-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of ß-lactams and combat carbapenem-resistant isolates.


Assuntos
Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacter cloacae/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamas/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Enterobacter cloacae/genética , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana/métodos
12.
ACS Infect Dis ; 5(11): 1896-1906, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31565920

RESUMO

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Antagonistas do Ácido Fólico/química , Staphylococcus aureus Resistente à Meticilina/enzimologia , Infecções Estafilocócicas/microbiologia , Tetra-Hidrofolato Desidrogenase/química , Trimetoprima/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Antagonistas do Ácido Fólico/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
13.
Methods Mol Biol ; 1529: 291-306, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27914058

RESUMO

Drug resistance in protein targets is an increasingly common phenomenon that reduces the efficacy of both existing and new antibiotics. However, knowledge of future resistance mutations during pre-clinical phases of drug development would enable the design of novel antibiotics that are robust against not only known resistant mutants, but also against those that have not yet been clinically observed. Computational structure-based protein design (CSPD) is a transformative field that enables the prediction of protein sequences with desired biochemical properties such as binding affinity and specificity to a target. The use of CSPD to predict previously unseen resistance mutations represents one of the frontiers of computational protein design. In a recent study (Reeve et al. Proc Natl Acad Sci U S A 112(3):749-754, 2015), we used our OSPREY (Open Source Protein REdesign for You) suite of CSPD algorithms to prospectively predict resistance mutations that arise in the active site of the dihydrofolate reductase enzyme from methicillin-resistant Staphylococcus aureus (SaDHFR) in response to selective pressure from an experimental competitive inhibitor. We demonstrated that our top predicted candidates are indeed viable resistant mutants. Since that study, we have significantly enhanced the capabilities of OSPREY with not only improved modeling of backbone flexibility, but also efficient multi-state design, fast sparse approximations, partitioned continuous rotamers for more accurate energy bounds, and a computationally efficient representation of molecular-mechanics and quantum-mechanical energy functions. Here, using SaDHFR as an example, we present a protocol for resistance prediction using the latest version of OSPREY. Specifically, we show how to use a combination of positive and negative design to predict active site escape mutations that maintain the enzyme's catalytic function but selectively ablate binding of an inhibitor.


Assuntos
Biologia Computacional/métodos , Resistência a Medicamentos/genética , Mutação , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Software , Algoritmos , Sequência de Aminoácidos , Bases de Dados Genéticas , Modelos Moleculares , Farmacogenética/métodos , Conformação Proteica , Navegador
14.
Cell Chem Biol ; 23(12): 1458-1467, 2016 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-27939900

RESUMO

Antibiotic resistance is a rapidly evolving health concern that requires a sustained effort to understand mechanisms of resistance and to develop new agents that overcome those mechanisms. The dihydrofolate reductase (DHFR) inhibitor, trimethoprim (TMP), remains one of the most important orally administered antibiotics. However, resistance through chromosomal mutations and mobile, plasmid-encoded insensitive DHFRs threatens the continued use of this agent. We are pursuing the development of new propargyl-linked antifolate (PLA) DHFR inhibitors designed to evade these mechanisms. While analyzing contemporary TMP-resistant clinical isolates of methicillin-resistant and sensitive Staphylococcus aureus, we discovered two mobile resistance elements, dfrG and dfrK. This is the first identification of these resistance mechanisms in the United States. These resistant organisms were isolated from a variety of infection sites, show clonal diversity, and each contain distinct resistance genotypes for common antibiotics. Several PLAs showed significant activity against these resistant strains by direct inhibition of the TMP resistance elements.

15.
J Med Chem ; 59(13): 6493-500, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27308944

RESUMO

Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme.


Assuntos
Antibacterianos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Trimetoprima/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
16.
ACS Med Chem Lett ; 7(7): 692-6, 2016 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-27437079

RESUMO

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.

17.
Future Microbiol ; 10(11): 1727-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26516790

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/química , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Cristalografia por Raios X , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica
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