RESUMO
The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.
Assuntos
Citocromo P-450 CYP11B2/antagonistas & inibidores , Imidazóis/farmacologia , Piridinas/farmacologia , Animais , Cricetulus , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacocinética , Macaca mulatta , Masculino , Microssomos Hepáticos/metabolismo , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Ratos Wistar , Esteroide 11-beta-Hidroxilase/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.
Assuntos
Anti-Hipertensivos/farmacologia , Citocromo P-450 CYP11B2/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Indazóis/farmacologia , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Inibidores da Aromatase/síntese química , Inibidores da Aromatase/farmacocinética , Inibidores da Aromatase/farmacologia , Linhagem Celular , Cricetulus , Inibidores do Citocromo P-450 CYP2D6/síntese química , Inibidores do Citocromo P-450 CYP2D6/farmacocinética , Inibidores do Citocromo P-450 CYP2D6/farmacologia , Humanos , Indazóis/síntese química , Indazóis/farmacocinética , Macaca mulatta , Masculino , Ratos Sprague-Dawley , Estereoisomerismo , Esteroide 11-beta-Hidroxilase/antagonistas & inibidoresRESUMO
Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.
Assuntos
Apolipoproteínas/sangue , Lipoproteínas HDL/sangue , Insuficiência Renal Crônica/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Estudos de Coortes , Estudos Transversais , Feminino , Variação Genética , Genótipo , Humanos , Lipoproteínas HDL/genética , Masculino , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/genética , Fatores de RiscoRESUMO
RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.
Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/genética , Cromatografia Líquida de Alta Pressão/métodos , Variação Genética , Nefropatias/sangue , Lipoproteínas HDL/sangue , Lipoproteínas HDL/genética , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Apolipoproteína L1 , Genótipo , Humanos , Nefropatias/diagnóstico , Nefropatias/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Ácidos Carboxílicos/química , Inibidores Enzimáticos/química , Pirazóis/química , Tiofenos/química , Oxirredutases do Álcool/metabolismo , Animais , Sítios de Ligação , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacocinética , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Rim/enzimologia , Rim/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Estrutura Terciária de Proteína , Pirazóis/síntese química , Pirazóis/uso terapêutico , Ratos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/uso terapêuticoRESUMO
Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Acetamidas/farmacologia , Acetamidas/toxicidade , Adamantano , Aminobenzoatos , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Aminoglicosídeos/toxicidade , Anilidas , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Linezolida , Lipídeos/biossíntese , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Oxazolidinonas/farmacologia , Oxazolidinonas/toxicidade , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 µM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Administração Oral , Animais , Concentração Inibidora 50 , Masculino , Estrutura Molecular , Ratos , Ratos Wistar , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive "blood pressure high" (BPH), normotensive "blood pressure normal" (BPN), and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. To distinguish in the kidney signature genes whose differential expression pattern may cause changes in blood pressure from those genes whose differential expression pattern results from changes in blood pressure, we integrated phenotype-associated genes into Genetic Bayesian networks. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension.
Assuntos
Perfilação da Expressão Gênica , Hipertensão/genética , Miocárdio/metabolismo , Animais , Aorta/metabolismo , Pressão Sanguínea/genética , Modelos Animais de Doenças , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Biológicos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.
RESUMO
BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Doadores de Óxido Nítrico/farmacologia , Triazenos/farmacologia , Adolescente , Adulto , Idoso , Animais , GMP Cíclico/metabolismo , Cães , Humanos , Técnicas In Vitro , Túbulos Renais Proximais/citologia , Masculino , Pessoa de Meia-Idade , Doadores de Óxido Nítrico/uso terapêutico , Triazenos/uso terapêutico , Adulto JovemRESUMO
Invertebrate glutamate-gated chloride channels (GluCls) are important targets for anthelmintics and insecticides such as ivermectin. To facilitate screening for novel GluCl modulators, the Caenorhabditis elegans GluCl alpha2beta channel was chosen as a surrogate for parasite channels not yet cloned, and an inducible stable human embryonic kidney cell line was generated. Functional expression of the alpha2 and beta subunits was confirmed by whole-cell voltage clamp assays. Using this cell line, a high-throughput assay was developed that detects membrane potential changes associated with the activation of GluCls. In this assay, membrane depolarization was quantified via changes in fluorescence resonance energy transfer between two membrane-associated dyes. Robust and reproducible signals were detected in response to addition of glutamate or ivermectin. This assay was used for the screening of over 180,000 samples from natural and synthetic sources.
Assuntos
Bioensaio/métodos , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , Ácido Glutâmico/farmacologia , Ativação do Canal Iônico/fisiologia , Rim/fisiologia , Técnicas de Patch-Clamp/métodos , Espectrometria de Fluorescência/métodos , Animais , Caenorhabditis elegans , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação do Canal Iônico/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Robótica/métodosRESUMO
We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.
RESUMO
Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.
Assuntos
Angiotensinogênio/metabolismo , Pressão Sanguínea/genética , Hipertensão/metabolismo , Fígado/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/genética , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hipertensão/genética , Hipertensão/fisiopatologia , Fígado/fisiopatologia , Masculino , Nanopartículas , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-DawleyRESUMO
l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.
RESUMO
To identify the genes and pathways that underlie cardiovascular and metabolic phenotypes we performed an integrated analysis of a mouse C57BL/6JxA/J F2 (B6AF2) cross by relating genome-wide gene expression data from adipose, kidney, and liver tissues to physiological endpoints measured in the population. We have identified a large number of trait QTLs including loci driving variation in cardiac function on chromosomes 2 and 6 and a hotspot for adiposity, energy metabolism, and glucose traits on chromosome 8. Integration of adipose gene expression data identified a core set of genes that drive the chromosome 8 adiposity QTL. This chromosome 8 trans eQTL signature contains genes associated with mitochondrial function and oxidative phosphorylation and maps to a subnetwork with conserved function in humans that was previously implicated in human obesity. In addition, human eSNPs corresponding to orthologous genes from the signature show enrichment for association to type II diabetes in the DIAGRAM cohort, supporting the idea that the chromosome 8 locus perturbs a molecular network that in humans senses variations in DNA and in turn affects metabolic disease risk. We functionally validate predictions from this approach by demonstrating metabolic phenotypes in knockout mice for three genes from the trans eQTL signature, Akr1b8, Emr1, and Rgs2. In addition we show that the transcriptional signatures for knockout of two of these genes, Akr1b8 and Rgs2, map to the F2 network modules associated with the chromosome 8 trans eQTL signature and that these modules are in turn very significantly correlated with adiposity in the F2 population. Overall this study demonstrates how integrating gene expression data with QTL analysis in a network-based framework can aid in the elucidation of the molecular drivers of disease that can be translated from mice to humans.
Assuntos
Doenças Cardiovasculares/genética , Sistema Cardiovascular , Cruzamentos Genéticos , Locos de Características Quantitativas , Animais , Pressão Sanguínea , Composição Corporal , Colesterol/metabolismo , Estudos de Coortes , Eletrocardiografia/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , FenótipoRESUMO
Emergence of bacterial resistance is a major issue for all classes of antibiotics; therefore, the identification of new classes is critically needed. Recently we reported the discovery of platensimycin by screening natural product extracts using a target-based whole-cell strategy with antisense silencing technology in concert with cell free biochemical validations. Continued screening efforts led to the discovery of platencin, a novel natural product that is chemically and biologically related but different from platensimycin. Platencin exhibits a broad-spectrum Gram-positive antibacterial activity through inhibition of fatty acid biosynthesis. It does not exhibit cross-resistance to key antibiotic resistant strains tested, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, and vancomycin-resistant Enterococci. Platencin shows potent in vivo efficacy without any observed toxicity. It targets two essential proteins, beta-ketoacyl-[acyl carrier protein (ACP)] synthase II (FabF) and III (FabH) with IC50 values of 1.95 and 3.91 microg/ml, respectively, whereas platensimycin targets only FabF (IC50 = 0.13 microg/ml) in S. aureus, emphasizing the fact that more antibiotics with novel structures and new modes of action can be discovered by using this antisense differential sensitivity whole-cell screening paradigm.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Aminofenóis/farmacologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Policíclicos/farmacologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Aminofenóis/química , Antibacterianos/química , Inibidores Enzimáticos/química , Viabilidade Microbiana/efeitos dos fármacos , Estrutura Molecular , Compostos Policíclicos/químicaRESUMO
Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5' mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Antibacterianos/farmacologia , Bactérias/enzimologia , Produtos Biológicos/química , Inibidores Enzimáticos/farmacologia , Antibacterianos/isolamento & purificação , Desenho de Fármacos , Ácidos Graxos/biossíntese , Testes de Sensibilidade Microbiana , RNA Antissenso/farmacologia , RNA Mensageiro/química , Relação Estrutura-AtividadeRESUMO
Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Ácidos Graxos/biossíntese , Bactérias/enzimologia , Cátions Bivalentes/farmacologia , Ditiotreitol/farmacologia , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Concentração Inibidora 50 , Cinética , Mercaptoetanol/farmacologia , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
The emergence of bacterial resistance to antibiotics is a major health problem and, therefore, it is critical to develop new antibiotics with novel modes of action. FtsZ, a tubulin-like GTPase, plays an essential role in bacterial cell division, and its homologs are present in almost all eubacteria and archaea. During cell division, FtsZ forms polymers in the presence of GTP that recruit other division proteins to make the cell division apparatus. Therefore, inhibition of FtsZ polymerization will prevent cells from dividing, leading to cell death. Using a fluorescent FtsZ polymerization assay, the screening of >100,000 extracts of microbial fermentation broths and plants followed by fractionation led to the identification of viriditoxin, which blocked FtsZ polymerization with an IC50 of 8.2 microg/ml and concomitant GTPase inhibition with an IC50 of 7.0 microg/ml. That the mode of antibacterial action of viriditoxin is via inhibition of FtsZ was confirmed by the observation of its effects on cell morphology, macromolecular synthesis, DNA-damage response, and increased minimum inhibitory concentration as a result of an increase in the expression of the FtsZ protein. Viriditoxin exhibited broad-spectrum antibacterial activity against clinically relevant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, without affecting the viability of eukaryotic cells.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Naftóis/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Resistência Microbiana a Medicamentos , Enterococcus faecium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fluoresceína , Corantes Fluorescentes , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Naftóis/química , Naftóis/isolamento & purificação , Polímeros/química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Histamine has been shown to play a role in arthropod vision; it is the major neurotransmitter of arthropod photoreceptors. Histamine-gated chloride channels have been identified in insect optic lobes. We report the first isolation of cDNA clones encoding histamine-gated chloride channel subunits from the fruit fly Drosophila melanogaster. The encoded proteins, HisCl1 and HisCl2, share 60% amino acid identity with each other. The closest structural homologue is the human glycine alpha3 receptor, which shares 45 and 43% amino acid identity respectively. Northern hybridization analysis suggested that hisCl1 and hisCl2 mRNAs are predominantly expressed in the insect eye. Oocytes injected with in vitro transcribed RNA, encoding either HisCl1 or HisCl2, produced substantial chloride currents in response to histamine but not in response to GABA, glycine, and glutamate. The histamine sensitivity was similar to that observed in insect laminar neurons. Histamine-activated currents were not blocked by picrotoxinin, fipronil, strychnine, or the H2 antagonist cimetidine. Co-injection of both hisCl1 and hisCl2 RNAs resulted in expression of a histamine-gated chloride channel with increased sensitivity to histamine, demonstrating coassembly of the subunits. The insecticide ivermectin reversibly activated homomeric HisCl1 channels and, more potently, HisCl1 and HisCl2 heteromeric channels.