RESUMO
One potentially rich source of possible targets for antifungal therapy are those Candida albicans genes deemed essential for growth under the standard culture (i.e., in vitro) conditions; however, these genes are largely unexplored as drug targets because essential genes are not experimentally amenable to conventional gene deletion and virulence studies. Using tetracycline-regulatable promoter-based conditional mutants, we investigated a murine model of candidiasis in which repressing essential genes in the host was achieved. By adding doxycycline to the drinking water starting 3 days prior to (dox - 3D) or 2 days post (dox + 2D) infection, the phenotypic consequences of temporal gene inactivation were assessed by monitoring animal survival and fungal burden in prophylaxis and acute infection settings. Of 177 selected conditional shut-off strains tested, the virulence of 102 was blocked under both repressing conditions, suggesting that the corresponding genes are essential for growth and survival in a murine host across early and established infection periods. Among these genes were those previously identified as antifungal drug targets (i.e., FKS1, ERG1, and ERG11), verifying that this methodology can be used to validate potential new targets. We also identify genes either conditionally essential or dispensable for in vitro growth but required for survival and virulence, including those in late stage ergosterol synthesis, or early steps in fatty acid or riboflavin biosynthesis. This study evaluates the role of essential genes with respect to pathogen virulence in a large-scale, systems biology context, and provides a general method for gene target validation and for uncovering unexpected antimicrobial targets.
Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/metabolismo , Proteínas Fúngicas/biossíntese , Viabilidade Microbiana , Fatores de Virulência/biossíntese , Animais , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Candida albicans/genética , Candidíase/tratamento farmacológico , Candidíase/genética , Modelos Animais de Doenças , Doxiciclina/farmacologia , Desenho de Fármacos , Ergosterol/biossíntese , Ergosterol/genética , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/fisiologia , Camundongos , Riboflavina/biossíntese , Riboflavina/genética , Fatores de Virulência/genéticaRESUMO
The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.
Assuntos
Carbolinas/química , Carbolinas/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/biossíntese , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/biossíntese , Células Th2/metabolismo , Animais , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Macaca fascicularis , Camundongos , Inibidores da Agregação Plaquetária/farmacologia , Ligação Proteica/imunologia , Ratos , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/fisiologia , Ovinos , Especificidade da Espécie , Células Th2/efeitos dos fármacosRESUMO
A new series of indole amide acting as hCRTH2 receptor ligands had been explored and are described herein. Several amide derivatives displaying low nanomolar activity in hCRTH2 binding and whole blood assays were identified. They were found to behave as a full antagonists, exhibiting good selectivity over related prostaglandin receptors. Also, prototypical compounds in this novel series which displayed acceptable CYP profiles and were orally bioavailable in rats were identified.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Indóis/síntese química , Indóis/farmacologia , Receptores de Dopamina D2/agonistas , Amidas/química , Animais , Indóis/química , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
A new class of 7-azaindole analogs of MK-7246 as potent and selective CRTH2 antagonists is reported. The SAR leading to the identification of the optimal azaindole regioisomer as well as the pharmacokinetics and off-target activities of the most potent antagonists are disclosed.
Assuntos
Indóis/química , Indóis/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Animais , Carbolinas/química , Humanos , Indóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.
Assuntos
Carbolinas/química , Pneumopatias/tratamento farmacológico , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Carbolinas/farmacocinética , Carbolinas/uso terapêutico , Humanos , Macaca mulatta , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Relação Estrutura-AtividadeRESUMO
The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.
Assuntos
Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/genética , Genes Essenciais/genética , Staphylococcus aureus/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , RNA Antissenso/genética , Staphylococcus aureus/efeitos dos fármacosRESUMO
Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors.
Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica , Genes Essenciais , Genes Fúngicos , Regiões Promotoras Genéticas , Animais , Antifúngicos/uso terapêutico , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Nitrato Redutases/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , RNA Mensageiro/análise , Recombinação Genética , Esterol 14-Desmetilase , Transcrição Gênica , Virulência/genética , Virulência/fisiologiaRESUMO
Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Desenho de Fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico , Candida albicans/genética , Candida albicans/metabolismo , DNA Fúngico/análise , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismoRESUMO
Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Alelos , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Candida albicans/metabolismo , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Misturas Complexas/farmacologia , Desoxiadenosinas/metabolismo , Desoxiadenosinas/farmacologia , Farmacorresistência Fúngica , Fermentação , Heterozigoto , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Poliadenilação/efeitos dos fármacos , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Resultado do TratamentoRESUMO
Mechanism-of-action (MOA) studies of bioactive compounds are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compound's MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compounds affecting purine metabolism. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metabolism. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochemical and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Aspergillus fumigatus/enzimologia , Candida albicans/enzimologia , Diazo-Oxo-Norleucina/farmacologia , Farmacorresistência Fúngica , Eletroforese em Gel de Poliacrilamida , IMP Desidrogenase/antagonistas & inibidores , Isoxazóis/farmacologia , Testes de Sensibilidade Microbiana , Ácido Micofenólico/farmacologia , Purinas/metabolismo , Ribonucleosídeos/farmacologiaRESUMO
Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to ß-lactam antibiotics paired with ß-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as ß-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with ß-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 Å crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the ß-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to ß-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , beta-Lactamas/farmacologia , Animais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Cristalografia por Raios X , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Redes Reguladoras de Genes/genética , Guanosina Difosfato , Imipenem/farmacologia , Staphylococcus aureus Resistente à Meticilina/citologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Mutação/genética , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Tiazóis/química , Tiazóis/farmacologia , Virulência/efeitos dos fármacos , beta-Lactamas/uso terapêuticoRESUMO
Antibiotic drug resistance among hospital and community acquired methicillin resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current therapeutics. We describe a chemical genetic strategy using antisense interference to broadly identify new drug targets that potentiate the effects of existing antibiotics against both etiological classes of MRSA infection. Further, we describe the resulting chemical genetic interaction networks and highlight the prominent and overlapping target sets that restore MRSA susceptibility to penicillin, cephalosporins, and carbapenems. Pharmacological validation of this approach is the potent synergy between a known inhibitor to a member of this genetic potentiation network (GlmS) and a broad set of ß-lactam antibiotics against methicillin resistant Staphylococci. Developing drug-like leads to these targets may serve as rational and effective combination agents when paired with existing ß-lactam antibiotics to restore their efficacy against MRSA.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , beta-Lactamas/farmacologia , Antibacterianos/química , DNA Antissenso/química , DNA Antissenso/metabolismo , Farmacorresistência Bacteriana , Peptidoglicano/biossíntese , beta-Lactamas/químicaRESUMO
The balance between saturated and unsaturated fatty acids plays a crucial role in determining the membrane fluidity. In the diploid fungal pathogen Candida albicans, the gene for fatty acid Delta9 desaturase, OLE1, is essential for viability. Using a reverse genetic approach, termed the fitness test, we identified a group of structurally related synthetic compounds that induce specific hypersensitivity of the OLE1(+/-) strain. Genetic repression of OLE1 and chemical inhibition by two selected compounds, ECC145 and ECC188, resulted in a marked decrease in the total unsaturated fatty acids and impaired hyphal development. The resulting auxotroph of both was suppressed by the exogenous monounsaturated fatty acids (16:1Delta9 and 18:1Delta9). These correlations suggest that both compounds affect the level of unsaturated fatty acids, likely by impairing Ole1p directly or indirectly. However, the residual levels of monounsaturated fatty acids (MUFAs) resulted from chemical inhibition were significantly higher than OLE1 repression, indicating even partial inhibition of MUFAs is sufficient to stop cellular proliferation. Although the essentiality of OLE1 was suppressed by MUFAs in vitro, we demonstrated that it was required for virulence in a murine model of systemic candidiasis even when the animals were supplemented with a high fat diet. Thus, the fungal fatty acid desaturase is an attractive antifungal drug target. Taking advantage of the inhibitors and the relevant conditional shut-off strains, we validated several chemical genetic interactions observed in the fitness test profiles that reveal novel genetic interactions between OLE1/unsaturated fatty acids and other cellular processes.
Assuntos
Antifúngicos/farmacologia , Candida albicans/genética , Ácidos Graxos Insaturados/biossíntese , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Animais , Antifúngicos/química , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/microbiologia , Candidíase/mortalidade , Cerulenina/farmacologia , Análise por Conglomerados , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/efeitos dos fármacos , Hifas/genética , Hifas/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estrutura Molecular , Mutação , Estearoil-CoA Dessaturase , Taxa de Sobrevida , Tiazóis/química , Tiazóis/farmacologia , Fatores de Tempo , Triazóis/química , Triazóis/farmacologia , Virulência/genéticaRESUMO
We constructed three sets of plasmids for use in Aspergillus niger. These plasmids were assembled using various combinations of a series of modular DNA cassettes that included a selectable marker, pyrG, derived from Aspergillus nidulans; two promoter regions for directing protein expression; a cassette derived from the AMA1 replicator sequence to support autonomous replication; and a reporter gene based on the A. niger lacA gene. One set included integrating and autonomously replicating plasmids for the expression of homologous and heterologous proteins. The second was a set of autonomously replicating plasmids, with a secreted beta-galactosidase encoding reporter gene, for studying gene regulation events. The third set included pyrG-derived gene-blaster cassettes suitable for genome manipulation by targeted gene replacement.
Assuntos
Aspergillus niger/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Vetores Genéticos , Plasmídeos/genética , Proteínas/metabolismo , Aspergillus nidulans/genética , Replicação do DNA , DNA Fúngico , Genes Reporter , Regiões Promotoras Genéticas , beta-Galactosidase/metabolismoRESUMO
Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.