RESUMO
Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon.
Assuntos
Proteínas de Saccharomyces cerevisiae , DNA , Dano ao DNA , DNA Polimerase II/genética , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Ribonucleotídeos/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Despite the potency of most first-line anti-cancer drugs, nonadherence to these drug regimens remains high and is attributable to the prevalence of "off-target" drug effects that result in serious adverse events (SAEs) like hair loss, nausea, vomiting, and diarrhea. Some anti-cancer drugs are converted by liver uridine 5'-diphospho-glucuronosyltransferases through homeostatic host metabolism to form drug-glucuronide conjugates. These sugar-conjugated metabolites are generally inactive and can be safely excreted via the biliary system into the gastrointestinal tract. However, ß-glucuronidase (ßGUS) enzymes expressed by commensal gut bacteria can remove the glucuronic acid moiety, producing the reactivated drug and triggering dose-limiting side effects. Small-molecule ßGUS inhibitors may reduce this drug-induced gut toxicity, allowing patients to complete their full course of treatment. Herein, we report the discovery of novel chemical series of ßGUS inhibitors by structure-based virtual high-throughput screening (vHTS). We developed homology models for ßGUS and applied them to large-scale vHTS against nearly 400,000 compounds within the chemical libraries of the National Center for Advancing Translational Sciences at the National Institutes of Health. From the vHTS results, we cherry-picked 291 compounds via a multifactor prioritization procedure, providing 69 diverse compounds that exhibited positive inhibitory activity in a follow-up ßGUS biochemical assay in vitro. Our findings correspond to a hit rate of 24% and could inform the successful downstream development of a therapeutic adjunct that targets the human microbiome to prevent SAEs associated with first-line, standard-of-care anti-cancer drugs.
Assuntos
Antineoplásicos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Microbiota , Neoplasias , Antineoplásicos/efeitos adversos , Detecção Precoce de Câncer , Inibidores Enzimáticos/farmacologia , Glicoproteínas , HumanosRESUMO
The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Genes BRCA1/fisiologia , Humanos , Enzimas Multifuncionais/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismoRESUMO
The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.
Assuntos
Mimetismo Molecular , Receptor de Pregnano X/química , Animais , Células Cultivadas , Citocinas , Humanos , Inflamação , Intestinos , Ligantes , Camundongos , OrganoidesRESUMO
Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.
Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Glucuronidase/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/farmacologia , Bactérias/efeitos dos fármacos , Modelos Animais de Doenças , Disbiose/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Feminino , Glucuronidase/metabolismo , Humanos , Irinotecano/farmacologia , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológicoRESUMO
The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.
Assuntos
Dano ao DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Estresse Oxidativo/genética , Animais , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Topoisomerases Tipo I/metabolismo , Endonucleases/metabolismo , Domínios Proteicos/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Cells use the post-translational modification ADP-ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon-encoded mono-ADP-ribosylation cycle mediates response to host-induced oxidative stress. In this system, reversible mono ADP-ribosylation of a lipoylated target protein represses oxidative stress response. An NAD(+) -dependent sirtuin catalyzes the single ADP-ribose (ADPr) addition, while a linked macrodomain-containing protein removes the ADPr. Here we report the crystal structure of the sitruin-linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75-Å resolution. The monomeric SauMacro bears a previously unidentified Zn(2+) -binding site that putatively aids in substrate recognition and catalysis. An amino-terminal three-helix bundle motif unique to this class of macrodomain proteins provides a structural scaffold for the Zn(2+) site. Structural features of the enzyme further indicate a cleft proximal to the Zn(2+) binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target.
Assuntos
Proteínas de Bactérias/química , Sirtuínas/química , Staphylococcus aureus/química , Zinco/química , Cristalografia por Raios X , Domínios ProteicosRESUMO
The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.
Assuntos
Antineoplásicos/toxicidade , Inibidores Enzimáticos/toxicidade , Glucuronidase/antagonistas & inibidores , Glucuronidase/química , Microbiota/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/enzimologia , Camptotecina/análogos & derivados , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/toxicidade , Clostridium perfringens/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Escherichia coli/enzimologia , Glucuronidase/metabolismo , Irinotecano , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Streptococcus agalactiae/enzimologiaRESUMO
Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase ß (POLß). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA/química , DNA/genética , Animais , Progressão da Doença , Instabilidade Genômica , Humanos , Conformação de Ácido NucleicoRESUMO
Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.
Assuntos
Dano ao DNA , DNA/genética , RNA/genética , Ribonucleotídeos/genética , Animais , DNA/química , DNA/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismoRESUMO
It was appreciated early in drug discovery that the microbiota play an important role in the efficacy of therapeutic compounds. Indeed, the first antibiotic sulfa drugs were shown in the 1940s to be transformed by the bacteria that encode what we now call the intestinal microbiome. Here we briefly review the roles symbiotic bacteria play in the chemistry of human health, and we focus on the emerging appreciation that specific enzyme targets expressed by microbial symbiotes can be selectively disrupted to achieve clinical outcomes. We conclude that components of the microbiome should be considered 'druggable targets,' and we suggest that our rapidly evolving understanding of the chemical biology of mammalian-microbial symbiosis will translate into improved human health.
Assuntos
Microbiota/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Animais , Descoberta de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/microbiologia , Saúde , Humanos , Preparações Farmacêuticas/metabolismoRESUMO
Bacterial ß-glucuronidases expressed by the symbiotic intestinal microbiota appear to play important roles in drug-induced epithelial cell toxicity in the gastrointestinal (GI) tract. For the anticancer drug CPT-11 (irinotecan) and the nonsteroidal anti-inflammatory drug diclofenac, it has been shown that removal of the glucuronide moieties from drug metabolites by bacterial ß-glucuronidases in the GI lumen can significantly damage the intestinal epithelium. Furthermore, selective disruption of bacterial ß-glucuronidases by small molecule inhibitors alleviates these side effects, which, for CPT-11 {7-ethyl-10-[4-(1-piperidino)-1-piperidino]}, can be dose limiting. Here we characterize novel microbial ß-glucuronidase inhibitors that inhibit Escherichia coli ß-glucuronidase in vitro with Ki values between 180 nM and 2 µM, and disrupt the enzyme in E. coli cells, with EC50 values as low as 300 nM. All compounds are selective for E. coli ß-glucuronidase without inhibiting purified mammalian ß-glucuronidase, and they do not impact the survival of either bacterial or mammalian cells. The 2.8 Å resolution crystal structure of one inhibitor bound to E. coli ß-glucuronidase demonstrates that it contacts and orders only a portion of the "bacterial loop" present in microbial, but not mammalian, ß-glucuronidases. The most potent compound examined in this group was found to protect mice against CPT-11-induced diarrhea. Taken together, these data advance our understanding of the chemical and structural basis of selective microbial ß-glucuronidase inhibition, which may improve human drug efficacy and toxicity.
Assuntos
Camptotecina/análogos & derivados , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Glicoproteínas/farmacologia , Animais , Camptotecina/toxicidade , Bovinos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Feminino , Irinotecano , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The human nuclear xenobiotic receptor PXR recognizes a range of potentially harmful drugs and endobiotic chemicals but must complex with the nuclear receptor RXRα to control the expression of numerous drug metabolism genes. To date, the structural basis and functional consequences of this interaction have remained unclear. Here we present 2.8-Å-resolution crystal structures of the heterodimeric complex formed between the ligand-binding domains of human PXR and RXRα. These structures establish that PXR and RXRα form a heterotetramer unprecedented in the nuclear receptor family of ligand-regulated transcription factors. We further show that both PXR and RXRα bind to the transcriptional coregulator SRC-1 with higher affinity when they are part of the PXR/RXRα heterotetramer complex than they do when each ligand-binding domain is examined alone. Furthermore, we purify the full-length forms of each receptor from recombinant bacterial expression systems and characterize their interactions with a range of direct and everted repeat DNA elements. Taken together, these data advance our understanding of PXR, the master regulator of drug metabolism gene expression in humans, in its functional partnership with RXRα.
Assuntos
Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Receptor de Pregnano X , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
Xenobiotic compounds undergo a critical range of biotransformations performed by the phase I, II, and III drug-metabolizing enzymes. The oxidation, conjugation, and transportation of potentially harmful xenobiotic and endobiotic compounds achieved by these catalytic systems are significantly regulated, at the gene expression level, by members of the nuclear receptor (NR) family of ligand-modulated transcription factors. Activation of NRs by a variety of endo- and exogenous chemicals are elemental to induction and repression of drug-metabolism pathways. The master xenobiotic sensing NRs, the promiscuous pregnane X receptor and less-promiscuous constitutive androstane receptor are crucial to initial ligand recognition, jump-starting the metabolic process. Other receptors, including farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4 alpha, peroxisome proliferator activated receptor, glucocorticoid receptor, liver X receptor, and RAR-related orphan receptor, are not directly linked to promiscuous xenobiotic binding, but clearly play important roles in the modulation of metabolic gene expression. Crystallographic studies of the ligand-binding domains of nine NRs involved in drug metabolism provide key insights into ligand-based and constitutive activity, coregulator recruitment, and gene regulation. Structures of other, noncanonical transcription factors also shed light on secondary, but important, pathways of control. Pharmacological targeting of some of these nuclear and atypical receptors has been instituted as a means to treat metabolic and developmental disorders and provides a future avenue to be explored for other members of the xenobiotic-sensing NRs.
Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Xenobióticos/metabolismo , Xenobióticos/farmacologia , Animais , Expressão Gênica , Humanos , Inativação Metabólica , Ligantes , Modelos Moleculares , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Relação Estrutura-Atividade , Xenobióticos/farmacocinéticaRESUMO
Disruption of the unusual thiol-based redox homeostasis mechanisms in Staphylococcus aureus represents a unique opportunity to identify new metabolic processes and new targets for intervention. Targeting uncommon aspects of CoASH biosynthetic and redox functions in S. aureus, the antibiotic CJ-15,801 has recently been demonstrated to be an antimetabolite of the CoASH biosynthetic pathway in this organism; CoAS-mimetics containing α,ß-unsaturated sulfone and carboxyl moieties have also been exploited as irreversible inhibitors of S. aureus coenzyme A-disulfide reductase (SaCoADR). In this work we have determined the crystal structures of three of these covalent SaCoADR-inhibitor complexes, prepared by inactivation of wild-type enzyme during turnover. The structures reveal the covalent linkage between the active-site Cys43-S(γ) and C(ß) of the vinyl sulfone or carboxyl moiety. The full occupancy of two inhibitor molecules per enzyme dimer, together with kinetic analyses of the wild-type/C43S heterodimer, indicates that half-sites-reactivity is not a factor during normal catalytic turnover. Further, we provide the structures of SaCoADR active-site mutants; in particular, Tyr419'-OH plays dramatic roles in directing intramolecular reduction of the Cys43-SSCoA redox center, in the redox asymmetry observed for the two FAD per dimer in NADPH titrations, and in catalysis. The two conformations observed for the Ser43 side chain in the C43S mutant structure lend support to a conformational switch for Cys43-S(γ) during its catalytic Cys43-SSCoA/Cys43-SH redox cycle. Finally, the structures of the three inhibitor complexes provide a framework for design of more effective inhibitors with therapeutic potential against several major bacterial pathogens.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Coenzima A/química , Coenzima A/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Mutação , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Multimerização Proteica , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial ß-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli ß-D-glucuronidase. Using a previously characterized novel bacteria-specific ß-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC50 â¼164 nM). We next hypothesized that pharmacologic inhibition of bacterial ß-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 µg per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the C(max), half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial ß-D-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Glucuronidase/antagonistas & inibidores , Enteropatias/induzido quimicamente , Enteropatias/enzimologia , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Diclofenaco/farmacocinética , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , Enterócitos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Glucuronidase/metabolismo , Glicoproteínas/farmacologia , Enteropatias/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Úlcera/induzido quimicamente , Úlcera/enzimologia , Úlcera/metabolismoRESUMO
The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial ß-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial ß-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial ß-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Camptotecina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Glucuronidase/antagonistas & inibidores , Glucuronidase/farmacologia , Animais , Antineoplásicos Fitogênicos/metabolismo , Bactérias Anaeróbias/efeitos dos fármacos , Camptotecina/metabolismo , Camptotecina/toxicidade , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Cristalografia por Raios X , Diarreia/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Feminino , Glucuronidase/química , Glucuronidase/isolamento & purificação , Glucuronidase/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Irinotecano , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Conformação ProteicaRESUMO
Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (âGlcNAc-malate) and the BaBshB deacetylase (âGlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.
Assuntos
Bacillus anthracis/enzimologia , Cisteína/biossíntese , Cisteína/metabolismo , Bacillus anthracis/metabolismo , Sítios de Ligação , Boroidretos , Cisteína/análogos & derivados , Cisteína/química , Glucosamina/análogos & derivados , Glucosamina/biossíntese , Glucosamina/metabolismo , Glicopeptídeos , Glicosiltransferases/biossíntese , Glicosiltransferases/metabolismo , Inositol , Liases Intramoleculares , Peso Molecular , Oxirredução , Compostos de Sulfidrila/metabolismo , Difosfato de Uridina/biossíntese , Difosfato de Uridina/metabolismoRESUMO
4',6-diamidino-2-phenylindole (DAPI), netropsin, and pentamidine are minor groove binders that have terminal -C(NH2)2+ groups. The hydration changes that accompany their binding to the minor groove of the (AATT)2 sequence have been studied using the osmotic stress technique with fluorescence spectroscopy. The affinity of DAPI for the binding site decreases with the increasing osmolality of the solution, resulting in acquisition of 35+/-1 waters upon binding. A competition fluorescence assay was utilized to measure the binding constants and hydration changes of the other two ligands, using the DNA-DAPI complex as the fluorescence reporter. Upon their association to the (AATT)2 binding site, netropsin and pentamidine acquire 26+/-3 and 34+/-2 additional waters of hydration, respectively. The hydration changes are discussed in the context of the terminal functional groups of the ligands and conformational changes in the DNA.