RESUMO
Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Motivos de Aminoácidos , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.
Assuntos
Benzamidas/farmacologia , Receptores Opioides kappa/antagonistas & inibidores , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Animais , Arrestinas/metabolismo , Benzamidas/química , Células CHO , Técnicas de Química Sintética , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Naltrexona/análogos & derivados , Naltrexona/química , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides kappa/genética , Sulfonamidas/química , Tetra-Hidroisoquinolinas/química , beta-ArrestinasRESUMO
The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).
Assuntos
Aminopeptidases/antagonistas & inibidores , Antimaláricos/química , Glutamil Aminopeptidase/antagonistas & inibidores , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Catepsina L/química , Análise por Conglomerados , Desenho de Fármacos , Eritrócitos/parasitologia , Fasciola hepatica/enzimologia , Glutamil Aminopeptidase/química , Humanos , Concentração Inibidora 50 , Cinética , Peptídeos/metabolismo , Plasmodium falciparum/enzimologia , Proteínas Recombinantes/química , Bibliotecas de Moléculas Pequenas/química , Software , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.
RESUMO
There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Rejeição de Enxerto/tratamento farmacológico , Janus Quinases/antagonistas & inibidores , Pirimidinas/síntese química , Pirróis/síntese química , Animais , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Monoterpenos Cicloexânicos , Cães , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ativação Linfocitária/efeitos dos fármacos , Macaca fascicularis , Masculino , Modelos Moleculares , Monoterpenos/síntese química , Monoterpenos/farmacocinética , Monoterpenos/farmacologia , Piperidinas/síntese química , Piperidinas/farmacocinética , Piperidinas/farmacologia , Ligação Proteica , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Distribuição TecidualRESUMO
Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.
Assuntos
Azetidinas/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Cetolídeos/química , Cetolídeos/farmacologia , Infecções Respiratórias/tratamento farmacológico , Animais , Bactérias/efeitos dos fármacos , Infecções Comunitárias Adquiridas/tratamento farmacológico , Suscetibilidade a Doenças , Descoberta de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Cetolídeos/efeitos adversos , Cetolídeos/síntese química , Cetolídeos/uso terapêutico , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
PF-956980 is a selective inhibitor of JAK3, related in structure to CP-690550, a compound being evaluated in clinical trials for rheumatoid arthritis and prevention of allograft rejection. PF-956980 has been evaluated against a panel of 30 kinases, and found to have nanomolar potency against only JAK3. Cellular and whole blood activity of this compound parallels its potency and selectivity in enzyme assays. It was effective in vivo at inhibiting the delayed type hypersensivity reaction in mice. We compared 2 commercially available JAK3 inhibitors (WHI-P131 and WHI-P154) in the same panel of biochemical and cellular assays and found them to be neither potent nor selective for JAK3. Both were found to be nanomolar inhibitors of the EGF receptor family of kinases. As these compounds have been used in numerous publications in the transplant and autoimmune disease literature, their specificity should be considered when interpreting these results.
Assuntos
Inibidores Enzimáticos/farmacologia , Janus Quinase 3/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Artrite Reumatoide/tratamento farmacológico , Ensaios Clínicos como Assunto , Inibidores Enzimáticos/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Humanos , Cinética , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêuticoRESUMO
Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.