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1.
ACS Infect Dis ; 3(7): 512-526, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28350440

RESUMO

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (Tb). Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.


Assuntos
Adenosilmetionina Descarboxilase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Adenosilmetionina Descarboxilase/genética , Adenosilmetionina Descarboxilase/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Cães , Inibidores Enzimáticos/química , Expressão Gênica , Humanos , Cinética , Células Madin Darby de Rim Canino , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Modelos Biológicos , Testes de Sensibilidade Parasitária , Permeabilidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tripanossomicidas/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
2.
J Med Chem ; 58(15): 5930-41, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26226049

RESUMO

Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Humanos , Estereoisomerismo
3.
J Med Chem ; 56(4): 1739-47, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23363003

RESUMO

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity relationship study of small molecules designed to inhibit HIF-2α-ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor-protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α-ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Mutação , Oxidiazóis/síntese química , Oxidiazóis/química , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
4.
Nat Chem Biol ; 9(4): 271-6, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23434853

RESUMO

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors, such as renal cell carcinomas.


Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Antineoplásicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química
5.
Cell ; 149(4): 753-67, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22579281

RESUMO

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Proteínas de Ligação a RNA/análise , RNA/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Sistema Livre de Células , Grânulos Citoplasmáticos/química , Células-Tronco Embrionárias/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células NIH 3T3 , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Testículo/citologia , Testículo/metabolismo , Difração de Raios X
6.
J Am Chem Soc ; 133(5): 1428-37, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21210688

RESUMO

Degeneration of the hippocampus is associated with Alzheimer's disease and occurs very early in the progression of the disease. Current options for treating the cognitive symptoms associated with Alzheimer's are inadequate, giving urgency to the search for novel therapeutic strategies. Pharmacologic agents that safely enhance hippocampal neurogenesis may provide new therapeutic approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apoptotic cell death, and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile, and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or photo-cross-linking.


Assuntos
Carbazóis/química , Carbazóis/farmacologia , Descoberta de Drogas/métodos , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Animais , Carbazóis/uso terapêutico , Carbazóis/toxicidade , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Células HeLa , Humanos , Masculino , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/toxicidade , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 19(14): 3825-7, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19410457

RESUMO

Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, inhibitors of Wnt response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure-activity relationship studies of these compounds.


Assuntos
Aminoquinolinas/química , Imidas/química , Proteínas Wnt/antagonistas & inibidores , Animais , Proteína Axina , Proteínas Repressoras/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Cauda , Proteínas Wnt/metabolismo , Peixe-Zebra/metabolismo
8.
J Biol Chem ; 284(15): 9899-907, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19196710

RESUMO

Polyamines are small organic cations found in all cells, and the biosynthetic pathway is well described in eukaryotes and Escherichia coli. The characterized pathway uses decarboxylated S-adenosylmethionine as the aminopropyl group donor to form spermidine from putrescine by the key enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We report here the in vivo characterization of an alternative polyamine biosynthetic pathway from Vibrio cholerae, the causative agent of human cholera. The pathway uses aspartate beta-semialdehyde as the aminopropyl group donor and consists of a fused protein containing l-2,4-diaminobutyrate aminotransferase and l-2,4-diaminobutyrate decarboxylase, a carboxynorspermidine dehydrogenase (CANSDH), and a carboxynorspermidine decarboxylase (CANSDC). We show that in V. cholerae, this pathway is required for synthesis of both sym-norspermidine and spermidine. Heterologous expression of the V. cholerae pathway in E. coli results in accumulation of the nonnative polyamines diaminopropane and sym-norspermidine. Genetic deletion of the V. cholerae CANSDC led to accumulation of carboxynorspermidine, whereas deletion of either CANSDC or the putative CANSDH led to loss of sym-norspermidine and spermidine. These results allowed unambiguous identification of the gene encoding CANSDH. Furthermore, deletion of either CANSDH or CANSDC led to a 50-60% reduction in growth rate of planktonic cells and severely reduced biofilm formation, which could be rescued by exogenously supplied sym-norspermidine but not spermidine. The pathway was not required for infectivity in a mouse model of V. cholerae infection. Notably, the alternative polyamine biosynthetic pathway is widespread in bacteria and is likely to play a previously unrecognized role in the biology of these organisms.


Assuntos
Biofilmes , Poliaminas/metabolismo , Vibrio cholerae/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Animais , Carboxiliases/metabolismo , Cromatografia Líquida de Alta Pressão , Escherichia coli/metabolismo , Camundongos , Modelos Biológicos , Modelos Químicos , Modelos Genéticos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo
9.
Proc Natl Acad Sci U S A ; 105(16): 6063-8, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18420817

RESUMO

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Coração/efeitos dos fármacos , Hidrazonas/farmacologia , Miocárdio/citologia , Regeneração/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , Cromossomos Artificiais Bacterianos/genética , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Embrionárias/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Coração/fisiologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Hidrazonas/química , Hidrazonas/isolamento & purificação , Luciferases de Vaga-Lume/genética , Camundongos , Miocárdio/metabolismo , Proteínas Nucleares/genética , Ratos , Transativadores/genética , Fatores de Transcrição/genética , Tropomiosina/genética
10.
Assay Drug Dev Technol ; 5(6): 769-83, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18078379

RESUMO

Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Domínios PDZ/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Transferência Ressonante de Energia de Fluorescência , Indicadores e Reagentes , Luz , Luminescência , Oxigênio/química , Oxigênio/efeitos da radiação , Ratos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/efeitos da radiação , Proteínas Recombinantes/química , Estreptavidina/química , Especificidade por Substrato
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