RESUMO
To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.
Assuntos
Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (Ki â¼ 25 µM) and antimycobacterial activity (MIC80) â¼ 40-80 µM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.
Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Sequência de Aminoácidos , Antituberculosos/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/efeitos dos fármacos , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Two novel series of oxazepine and diazepine based HSP90 inhibitors are reported. This effort relied on structure based design and isothermal calorimetry to identify small drug like macrocycles. Computational modelling was used to build into a solvent exposed pocket near the opening of the ATP binding site, which led to potent inhibitors of HSP90 (25-30).
Assuntos
Amidas/química , Benzodiazepinas/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Indóis/química , Oxazepinas/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Amidas/metabolismo , Amidas/farmacologia , Animais , Azepinas/química , Benzamidas/química , Benzamidas/metabolismo , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacologia , Sítios de Ligação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Proteínas de Choque Térmico HSP90/metabolismo , Indóis/metabolismo , Indóis/farmacologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Oxazepinas/metabolismo , Oxazepinas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismoRESUMO
HSP90 continues to be a target of interest for neurodegeneration indications. Selective knockdown of the HSP90 cytosolic isoforms α and ß is sufficient to reduce mutant huntingtin protein levels in vitro. Chemotype-dependent binding conformations of HSP90α/ß appear to strongly influence isoform selectivity. The rational design of HSP90α/ß inhibitors selective versus the mitochondrial (TRAP1) and endoplasmic reticulum (GRP94) isoforms offers a potential mitigating strategy for mechanism-based toxicities. Better tolerated HSP90 inhibitors would be attractive for targeting chronic neurodegenerative diseases such as Huntington's disease.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Doenças Neurodegenerativas/tratamento farmacológico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Isoformas de Proteínas/antagonistas & inibidoresRESUMO
Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27âa highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Elongases de Ácidos Graxos/administração & dosagem , Pirazóis/farmacologia , Adrenoleucodistrofia/tratamento farmacológico , Adrenoleucodistrofia/patologia , Amidas/química , Animais , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Pirazóis/química , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Pictet-Spengler condensation of aldehydes or alpha-keto-esters with 4-(2-anilinophenyl)-7-azaindole (11) or deazapurine (12) gave high yields of the 3,4-fused cyclic compounds. SAR studies, by varying the substituted benzaldehyde components, lead to the discovery of a series of potent JAK2 kinase inhibitors.
Assuntos
Indóis/química , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Purinas/química , Benzaldeídos/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Janus Quinase 2/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
Constitutive activation of the EPO/JAK2 signaling cascade has recently been implicated in a variety of myeloproliferative disorders including polycythemia vera, essential thrombocythemia and myelofibrosis. In an effort to uncover therapeutic potential of blocking the EPO/JAK2 signaling cascade, we sought to discover selective inhibitors that block the kinase activity of JAK2. Herein, we describe the discovery and structure based optimization of a novel series of 2-amino-pyrazolo[1,5-a]pyrimidines that exhibit potent inhibition of JAK2.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.
Assuntos
Homeostase , Mucosa Intestinal/metabolismo , Espaço Intracelular/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Actomiosina/metabolismo , Animais , Células CACO-2 , Doença Crônica , Homeostase/efeitos dos fármacos , Humanos , Inflamação/patologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/patologia , Camundongos , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Bibliotecas de Moléculas Pequenas/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Drug resistant tuberculosis (TB) infections are on the rise and antibiotics that inhibit Mycobacterium tuberculosis through a novel mechanism could be an important component of evolving TB therapy. Protein kinase A (PknA) and protein kinase B (PknB) are both essential serine-threonine kinases in M. tuberculosis. Given the extensive knowledge base in kinase inhibition, these enzymes present an interesting opportunity for antimycobacterial drug discovery. This study focused on targeting both PknA and PknB while improving the selectivity window over related mammalian kinases. Compounds achieved potent inhibition (Ki ≈ 5 nM) of both PknA and PknB. A binding pocket unique to mycobacterial kinases was identified. Substitutions that filled this pocket resulted in a 100-fold differential against a broad selection of mammalian kinases. Reducing lipophilicity improved antimycobacterial activity with the most potent compounds achieving minimum inhibitory concentrations ranging from 3 to 5 µM (1-2 µg/mL) against the H37Ra isolate of M. tuberculosis.
RESUMO
While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Compostos Heterocíclicos com 2 Anéis/química , Janus Quinase 3/antagonistas & inibidores , Valina/análogos & derivados , Animais , Linhagem Celular , Bases de Dados de Compostos Químicos , Cães , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/imunologia , Haplorrinos , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Janus Quinase 2/química , Janus Quinase 3/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-Atividade , Valina/química , Valina/farmacocinética , Valina/farmacologiaRESUMO
A structure-based drug design strategy was used to optimize a novel benzolactam series of HSP90α/ß inhibitors to achieve >1000-fold selectivity versus the HSP90 endoplasmic reticulum and mitochondrial isoforms (GRP94 and TRAP1, respectively). Selective HSP90α/ß inhibitors were found to be equipotent to pan-HSP90 inhibitors in promoting the clearance of mutant huntingtin protein (mHtt) in vitro, however with less cellular toxicity. Improved tolerability profiles may enable the use of HSP90α/ß selective inhibitors in treating chronic neurodegenerative indications such as Huntington's disease (HD). A potent, selective, orally available HSP90α/ß inhibitor was identified (compound 31) that crosses the blood-brain barrier. Compound 31 demonstrated proof of concept by successfully reducing brain Htt levels following oral dosing in rats.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Doença de Huntington/tratamento farmacológico , Animais , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/química , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.
Assuntos
Compostos Aza/química , Compostos Aza/farmacologia , Indóis/química , Indóis/farmacologia , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Compostos Aza/síntese química , Compostos Aza/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Eritropoetina/metabolismo , Indóis/síntese química , Indóis/farmacocinética , Janus Quinase 2/metabolismo , Lactamas Macrocíclicas/síntese química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacocinética , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.