RESUMO
Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.
Assuntos
Antifúngicos/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/microbiologia , Citocromos b/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antimicina A/metabolismo , Doença de Chagas/genética , Citocromos b/genética , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/imunologia , Genômica , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Consumo de Oxigênio/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/metabolismoRESUMO
Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Colorimetria/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Doenças Negligenciadas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas , Tripanossomicidas/química , Xantina/química , Xantina/farmacologiaRESUMO
Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.
Assuntos
Carboxiliases/metabolismo , Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Ativação Enzimática , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity.
Assuntos
Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Administração Oral , Animais , Descoberta de Drogas , Ensaio de Imunoadsorção Enzimática , Humanos , Quinolinas/administração & dosagem , Quinolinas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Continuing studies on tetrahydroquinoline glucocorticoid receptor anti-inflammatory agents lead to the identification of several tetrahydroquinolin-3-yl carbamates that exhibited steroid-like activity in in vitro transrepression assays with reduced transactivation of phosphoenol pyruvate carboxykinase (PEPCK), a key enzyme in the gluconeogenesis pathway.
Assuntos
Carboxiliases/metabolismo , Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Ativação EnzimáticaRESUMO
We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure-activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists.
Assuntos
Quinolinas/química , Receptores de Glucocorticoides/agonistas , Avaliação Pré-Clínica de Medicamentos , Ligação Proteica , Quinolinas/síntese química , Quinolinas/farmacologia , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure-activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity (K(i)=1.5nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Indóis/farmacologia , Quinolinas/farmacologia , Receptores de Glucocorticoides/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Indóis/síntese química , Indóis/química , Ligantes , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Steroidal glucocorticoids are widely prescribed for the treatment of a variety of inflammatory and autoimmune diseases. Although they are effective, the side-effects associated with chronic glucocorticoid treatment, such as osteoporosis and hyperglycemia, can severely limit their long-term use. Hence, there is a need to develop new effective anti-inflammatory agents for systemic use which are dissociated from their unwanted side effects.
Assuntos
Anti-Inflamatórios/química , Receptores de Glucocorticoides/efeitos dos fármacos , Desenho de Fármacos , Glucocorticoides/química , Humanos , Ligantes , Relação Estrutura-AtividadeRESUMO
Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.
Assuntos
Antineoplásicos/síntese química , Benzopiranos/síntese química , Mieloma Múltiplo/tratamento farmacológico , Quinolinas/síntese química , Receptores de Glucocorticoides/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Ligação Competitiva , Dexametasona/farmacologia , Humanos , Camundongos , Antagonistas de Receptores de Mineralocorticoides , Modelos Moleculares , Mieloma Múltiplo/patologia , Quinolinas/química , Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Mineralocorticoides/agonistas , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure-activity relationships for this series of compounds are presented.
Assuntos
Crisenos/farmacologia , Receptores de Glucocorticoides/efeitos dos fármacos , Crisenos/síntese química , Crisenos/química , Relação Estrutura-AtividadeRESUMO
Enamide 4 was studied for its effectiveness as a polyene precursor in biomimetic cyclizations. While most conventional Lewis acids were poor cyclization promoters, FeCl(3).6H(2)O initiated the conversion of 4 into tricycles 6 and 7 in excellent yield. The two isomeric products result from the cyclization of intermediate aldehyde 5 by either a chair or boat B-ring transition state. These results suggest that enamides may be incorporated into polyene precursors for the construction of larger azapolycycles such as azasteroids.