RESUMO
In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Mycobacterium tuberculosis/metabolismo , Ágar/química , Antibacterianos/química , Domínio Catalítico , Química Farmacêutica/métodos , Técnicas de Química Combinatória , Dissulfetos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Transporte de Elétrons , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/metabolismo , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Pseudomonas aeruginosa/metabolismoRESUMO
New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new spiropyrimidinetriones (SPTs), DNA gyrase inhibitors having activity against drug-resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB. While the clinical candidate zoliflodacin has progressed to phase 3 trials for the treatment of gonorrhea, compounds herein demonstrated higher inhibitory potency against Mtb DNA gyrase (e.g., compound 42 with IC50 = 2.0) and lower Mtb minimum inhibitor concentrations (0.49 µM for 42). Notably, 42 and analogues showed selective Mtb activity relative to representative Gram-positive and Gram-negative bacteria. DNA gyrase inhibition was shown to involve stabilization of double-cleaved DNA, while on-target activity was supported by hypersensitivity against a gyrA hypomorph. Finally, a docking model for SPTs with Mtb DNA gyrase was developed, and a structural hypothesis was built for structure-activity relationship expansion.
Assuntos
Mycobacterium tuberculosis , Inibidores da Topoisomerase II , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antituberculosos/química , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , DNA Girase/genética , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/uso terapêuticoRESUMO
Phenotypic whole cell high-throughput screening of a â¼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.
Assuntos
Antituberculosos/farmacologia , Parede Celular/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Sulfonamidas/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Descoberta de Drogas , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
Assuntos
Antituberculosos/química , Pirimidinonas/química , Animais , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meia-Vida , Humanos , Ferro/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microssomos/metabolismo , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Pirazóis/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.
Assuntos
Antituberculosos , Mycobacterium tuberculosis , Animais , Antituberculosos/farmacologia , Desenho de Fármacos , Camundongos , Relação Estrutura-AtividadeRESUMO
In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic-stage Plasmodium chabaudi. Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 h postinfection, followed by strong increases in natural killer (NK) cell-associated and major histocompatibility complex class I-related transcripts by 32 h postinfection. We showed by flow cytometry that the observed elevation in NK cell-associated transcripts was the result of a dramatic increase in the proportion of NK cells in the blood during infection. Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection. Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice. These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells.
Assuntos
Expressão Gênica/imunologia , Células Matadoras Naturais/imunologia , Malária/imunologia , Transferência Adotiva , Animais , Proliferação de Células , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Interferons/biossíntese , Interferons/imunologia , Malária/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Plasmodium chabaudi/imunologiaRESUMO
The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.
Assuntos
Variação Genética/genética , Genoma de Protozoário , Repetições de Microssatélites/genética , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Animais , DNA de Protozoário/análise , Genes de Protozoários , Proteínas de Protozoários/genéticaRESUMO
Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum. More specifically, we applied bioinformatic tools to a previously characterized micro-array data set from drug-treated asexual stage cultures (Gunasekera et al., submitted). Cluster analysis of 600 drug responsive genes identified only a single 5' motif, GAGAGAA. Two additional 5' motifs, ACTATAAAGA and TGCAC, were also shared among loci displaying patterns of coordinate expression across varying asexual growth stages. Secondly and most importantly, the functional relevance of each motif was tested in two independent assays-transient transfection and gel-retardation experiments. The GAGAGAA and TGCAC motifs were both active in the former. The GAGAGAA and ACTATAAAGA elements formed specific RNA-protein, but not DNA-protein complexes in gel shift assays, suggesting a key level of control at the RNA level. This is the first report of functionally characterized motifs in P. falciparum that were uncovered following clustering analysis of its asexual stage transcriptome. Together, both the bioinformatic and functional data reported here imply that multiple forms of gene regulation, including post-transcriptional control, may be important in the malarial system.
Assuntos
Plasmodium falciparum/genética , Animais , Antimaláricos/farmacologia , Sequência de Bases , Cloroquina/farmacologia , DNA de Protozoário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes de Protozoários , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Plasmodium falciparum/efeitos dos fármacos , Subunidades Proteicas , TransfecçãoRESUMO
Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas.
Assuntos
Malária Falciparum/classificação , Repetições de Microssatélites , Plasmodium falciparum/genética , Animais , Genótipo , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/classificação , Proteínas de Protozoários/genéticaRESUMO
We followed parasite genotypes of 75 patients for 42 days after treatment of uncomplicated malaria with chloroquine + sulfadoxine-pyrimethamine in Kampala, Uganda. Infections were complex (mean, 2.88 strains) and followed three patterns: 27% of patients eliminated all strains and remained parasite-free, 48% had a long aparasitemic interval followed by reappearance of original strains after 3-33 days (mean, 9.2 days), and 25% failed to clear original strains and required therapy after 3-35 days (mean, 17 days). These results highlight the complexity of malaria in Africa and have implications for efficacy trials, because missing late reappearances of strains could lead to misclassification of outcomes.
Assuntos
Antimaláricos/administração & dosagem , Cloroquina/administração & dosagem , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Pirimetamina/administração & dosagem , Sulfadoxina/administração & dosagem , Adolescente , Adulto , Animais , Criança , Pré-Escolar , DNA de Protozoário/análise , Intervalo Livre de Doença , Combinação de Medicamentos , Resistência a Medicamentos/genética , Quimioterapia Combinada , Feminino , Genótipo , Humanos , Lactente , Malária Falciparum/etiologia , Malária Falciparum/mortalidade , Malária Falciparum/parasitologia , Masculino , Área Carente de Assistência Médica , Reação em Cadeia da Polimerase , Uganda/epidemiologiaRESUMO
Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Genes MDR/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Antimaláricos/uso terapêutico , Primers do DNA , DNA de Protozoário/análise , Variação Genética , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Senegal/epidemiologiaRESUMO
MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Mapeamento de Interação de Proteínas , Multimerização Proteica , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/química , Espectroscopia de Ressonância de Spin Eletrônica , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P. falciparum. Instead, cell cycle changes appear to have a more pronounced effect on gene expression; only a fraction of the drug responsive loci (approximately 5%) were shared between two separate starting cultures that varied in staging profile in the current study, as well as a previous published analysis using SAGE technology [Gunasekera, A.M., Patankar, S., Schug, J., Eisen, G.,Wirth, D.F., 2003. Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Molecular Microbiology 50, 1229-1239]. These findings are important to report, given the striking contrast to similar studies in other model eukaryotic organisms.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Genoma de Protozoário/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , RNA de Protozoário/efeitos dos fármacos , Animais , Northern Blotting , Regulação da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA de Protozoário/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacosRESUMO
To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus. The pfcrt T76 allele was present not only in all in vivo resistant isolates, 89% of in vitro resistant isolates, but also in 35% of in vitro sensitive isolates. The pfmdr1 N86Y polymorphism did not correlate with in vitro or in vivo CQ resistance. Our data suggest that the pfcrt T76 allele alone is required but not a sufficient predictor for in vivo CQ resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antiparasitários/farmacologia , Cloroquina/farmacologia , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana/genética , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adolescente , Adulto , Animais , DNA de Protozoário/análise , Resistência Microbiana a Medicamentos/genética , Feminino , Marcadores Genéticos , Humanos , Masculino , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Parasitária , Plasmodium falciparum/genética , Polimorfismo de Fragmento de Restrição , Senegal , Análise de Sequência de DNARESUMO
The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasite's response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug-resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed. The aim of this study was to map the 5' UTR of pfmdr1, and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs. RT-PCR and 5'-RACE mapping showed that the 5' UTR has a length of 1.94 kb. A putative promoter has been identified via transient transfection. Northern analysis revealed a 2.1- to 2.7-fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression. 3D7 parasites were subsequently treated with experimentally derived IC50 concentrations of mefloquine, quinine and pyrimethamine. pfmdr1 transcript levels specifically increased 2.5-fold at 6 h in mefloquine-treated parasites and threefold in parasites treated with quinine for 30 min. There was no evidence of transcript induction in pyrimethamine-treated parasites. This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.