RESUMO
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Assuntos
Antibacterianos/química , Antineoplásicos/química , Infecções por Escherichia coli/tratamento farmacológico , Neoplasias/tratamento farmacológico , Nucleotídeos/metabolismo , Ribonucleotídeo Redutases/química , Antibacterianos/uso terapêutico , Antineoplásicos/uso terapêutico , Biocatálise , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Humanos , Simulação de Acoplamento Molecular , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Nucleotídeos/química , Oxirredução , Estrutura Secundária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub-halicin-that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Aprendizado de Máquina , Tiadiazóis/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Animais , Antibacterianos/química , Quimioinformática/métodos , Clostridioides difficile/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiadiazóis/químicaRESUMO
Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.
Assuntos
Antibacterianos/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sítios de Ligação , Desenho de Fármacos , Resistência Microbiana a Medicamentos , Humanos , Modelos Biológicos , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/química , Ribossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened â¼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Animais , Antibacterianos/química , Escherichia coli , CamundongosRESUMO
After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.
Assuntos
Antibacterianos/metabolismo , Antineoplásicos/metabolismo , Bioquímica/história , Produtos Biológicos/metabolismo , Pesquisa Biomédica/história , Indústria Farmacêutica/história , Antibacterianos/química , Antineoplásicos/química , Bioquímica/tendências , Produtos Biológicos/química , Pesquisa Biomédica/tendências , Indústria Farmacêutica/tendências , Regulação da Expressão Gênica , História do Século XX , História do Século XXI , Humanos , Ligases/genética , Ligases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Resistência a Vancomicina/genética , Recursos HumanosRESUMO
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Streptomyces/química , Animais , Antibacterianos/química , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/química , Farmacorresistência Bacteriana , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos ICR , Microbiologia do Solo , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus pyogenes/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Avycaz combines an older cephalosporin antibiotic, ceftazidime, and the ß-lactamase inhibitor avibactam. Ceftazidime targets penicillin-binding proteins (PBPs) in the bacterial periplasm that are required for cell wall synthesis. Avibactam blocks ß-lactamases (ß-L) in the periplasm, which would otherwise inactivate the antibiotics resulting in drug resistance.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Bactérias/efeitos dos fármacos , Ceftazidima , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Inibidores de beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Ceftazidima/química , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Parede Celular/efeitos dos fármacos , Combinação de Medicamentos , Farmacorresistência Bacteriana , Humanos , Infecções Urinárias/tratamento farmacológico , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/uso terapêuticoRESUMO
The discovery of novel structural classes of antibiotics is urgently needed to address the ongoing antibiotic resistance crisis1-9. Deep learning approaches have aided in exploring chemical spaces1,10-15; these typically use black box models and do not provide chemical insights. Here we reasoned that the chemical substructures associated with antibiotic activity learned by neural network models can be identified and used to predict structural classes of antibiotics. We tested this hypothesis by developing an explainable, substructure-based approach for the efficient, deep learning-guided exploration of chemical spaces. We determined the antibiotic activities and human cell cytotoxicity profiles of 39,312 compounds and applied ensembles of graph neural networks to predict antibiotic activity and cytotoxicity for 12,076,365 compounds. Using explainable graph algorithms, we identified substructure-based rationales for compounds with high predicted antibiotic activity and low predicted cytotoxicity. We empirically tested 283 compounds and found that compounds exhibiting antibiotic activity against Staphylococcus aureus were enriched in putative structural classes arising from rationales. Of these structural classes of compounds, one is selective against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci, evades substantial resistance, and reduces bacterial titres in mouse models of MRSA skin and systemic thigh infection. Our approach enables the deep learning-guided discovery of structural classes of antibiotics and demonstrates that machine learning models in drug discovery can be explainable, providing insights into the chemical substructures that underlie selective antibiotic activity.
Assuntos
Antibacterianos , Aprendizado Profundo , Descoberta de Drogas , Animais , Humanos , Camundongos , Antibacterianos/química , Antibacterianos/classificação , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Redes Neurais de Computação , Algoritmos , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Modelos Animais de Doenças , Pele/efeitos dos fármacos , Pele/microbiologia , Descoberta de Drogas/métodos , Descoberta de Drogas/tendênciasRESUMO
Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.
Assuntos
Antibiose , Proteínas de Bactérias , Toxinas Bacterianas , Streptomyces , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibiose/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/ultraestrutura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Microscopia Crioeletrônica , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Lectinas/ultraestrutura , Testes de Sensibilidade Microbiana , Modelos Moleculares , Streptomyces/química , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces griseus/efeitos dos fármacos , Streptomyces griseus/genética , Streptomyces griseus/crescimento & desenvolvimento , Streptomyces griseus/metabolismoRESUMO
In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.
Assuntos
Antibacterianos , Bactérias , Produtos Biológicos , Animais , Humanos , Aminoglicosídeos/farmacologia , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/metabolismo , Antibióticos beta Lactam/química , Antibióticos beta Lactam/farmacologia , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Peptidil Transferases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Sideróforos/metabolismo , Sideróforos/química , Sideróforos/farmacologiaRESUMO
Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.
Assuntos
Antibacterianos , Staphylococcus aureus , Resistência beta-Lactâmica , beta-Lactamas , Humanos , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Resistência beta-Lactâmica/efeitos dos fármacos , beta-Lactamas/química , beta-Lactamas/farmacologia , Microscopia Crioeletrônica , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismoRESUMO
Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals.
Assuntos
Antibacterianos , Desenho de Fármacos , Porinas , Pseudomonas aeruginosa , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Estudos Retrospectivos , Eletricidade Estática , Ligação de Hidrogênio , Ácido Fusídico/metabolismo , Desenho de Fármacos/métodosRESUMO
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
Assuntos
Antibacterianos , Sítios de Ligação , RNA Polimerases Dirigidas por DNA , Escherichia coli , Mutação , Rifampina , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Quebras de DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Nucleotídeos/deficiência , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Rifampina/química , Rifampina/metabolismo , Rifampina/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.
Assuntos
Antibacterianos , Bactérias , Membrana Celular , Depsipeptídeos , Viabilidade Microbiana , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/citologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Difosfatos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Lipídeos/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Pirrolidinas/química , Açúcares/químicaRESUMO
The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Antibacterianos/química , Compostos Aza/química , Compostos Aza/farmacologia , Ciclo-Octanos/química , Ciclo-Octanos/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , beta-LactamasesRESUMO
Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenho de Fármacos , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Simulação de Dinâmica Molecular , Estrutura Secundária de ProteínaRESUMO
The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/classificação , Clindamicina/síntese química , Clindamicina/farmacologia , Descoberta de Drogas , Lincomicina/síntese química , Lincomicina/farmacologia , Metiltransferases/genética , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxepinas , Piranos , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Ribossomos/enzimologia , Inibidores da Topoisomerase II/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriocinas/química , Bacteriocinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Difração de Raios XRESUMO
Alternative to the conventional search for single-target, single-compound treatments, combination therapies can open entirely new opportunities to fight antibiotic resistance. However, combinatorial complexity prohibits experimental testing of drug combinations on a large scale, and methods to rationally design combination therapies are lagging behind. Here, we developed a combined experimental-computational approach to predict drug-drug interactions using high-throughput metabolomics. The approach was tested on 1,279 pharmacologically diverse drugs applied to the gram-negative bacterium Escherichia coli. Combining our metabolic profiling of drug response with previously generated metabolic and chemogenomic profiles of 3,807 single-gene deletion strains revealed an unexpectedly large space of inhibited gene functions and enabled rational design of drug combinations. This approach is applicable to other therapeutic areas and can unveil unprecedented insights into drug tolerance, side effects, and repurposing. The compendium of drug-associated metabolome profiles is available at https://zampierigroup.shinyapps.io/EcoPrestMet, providing a valuable resource for the microbiological and pharmacological communities.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Redes e Vias Metabólicas/efeitos dos fármacos , Medicamentos sob Prescrição/farmacologia , Antibacterianos/química , Quimioinformática/métodos , Combinação de Medicamentos , Interações Medicamentosas , Reposicionamento de Medicamentos/métodos , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Deleção de Genes , Internet , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Medicamentos sob Prescrição/químicaRESUMO
Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.