RESUMO
Interference with DNA damage checkpoints has been demonstrated preclinically to be a highly effective means of increasing the cytotoxicity of a number of DNA-damaging cancer therapies. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA may proceed into premature mitosis followed by cell death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 functionality leads to abrogation of DNA damage checkpoints and has been shown preclinically to enhance the activity of many standard of care chemotherapeutic agents. LY2603618 is a potent and selective small molecule inhibitor of Chk1 protein kinase activity in vitro (IC(50) = 7 nM) and the first selective Chk1 inhibitor to enter clinical cancer trials. Treatment of cells with LY2603618 produced a cellular phenotype similar to that reported for depletion of Chk1 by RNAi. Inhibition of intracellular Chk1 by LY2603618 results in impaired DNA synthesis, elevated H2A.X phosphorylation indicative of DNA damage and premature entry into mitosis. When HeLa cells were exposed to doxorubicin to induce a G2/M checkpoint arrest, subsequent treatment with LY2603618 released the checkpoint, resulting in cells entering into metaphase with poorly condensed chromosomes. Consistent with abrogation of the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 colon cancer cells were more sensitive to gemcitabine when also treated with LY2603618, while wild-type TP53 HCT116 cells were not sensitized by LY2603618 to gemcitabine. Treatment of Calu-6 human mutant TP53 lung cancer cell xenografts with gemcitabine resulted in a stimulation of Chk1 kinase activity that was inhibited by co-administration of LY2603618. By all criteria, LY2603618 is a highly effective inhibitor of multiple aspects of Chk1 biology.
Assuntos
Antineoplásicos/farmacologia , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Doxorrubicina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/uso terapêutico , Proteína Supressora de Tumor p53/genética , GencitabinaRESUMO
We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.
Assuntos
Isoquinolinas/farmacologia , Dor/tratamento farmacológico , Pró-Fármacos/farmacologia , Receptores de Ácido Caínico/antagonistas & inibidores , Tetrazóis/farmacologia , Administração Oral , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Isoquinolinas/química , Masculino , Dados de Sequência Molecular , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Receptores de Ácido Caínico/química , Relação Estrutura-Atividade , Tetrazóis/químicaRESUMO
Adenoviruses can cause infectious diarrheal disease or respiratory infections in humans; 2 recent reports have indicated probable human infection with simian adenoviruses (SAdVs). To assess the possibility of animal-to-human transmission of SAdVs, we tested fecal samples from asymptomatic rhesus macaques housed in 5 primate facilities in the United States and cultured 23 SAdV isolates. Of these, 9 were purified and completely sequenced; 3 SAdV samples from the American Type Culture Collection (SAdV-6, SAdV-18, and SAdV-20) were also completely sequenced. The sequence of SAdV-18 was closely related to that of human adenovirus F across the whole genome, and the new isolates were found to harbor 2 fiber genes similar to those of human adenovirus (HAdV) strains HAdV-40 and HAdV-41, which can cause infectious diarrhea. The high prevalence of adenoviruses in fecal samples from asymptomatic rhesus macaques and the similarity of the isolates to human strains indicates the possibility of animal-to-human transmission of SAdVs.
Assuntos
Infecções por Adenoviridae/veterinária , Adenovirus dos Símios/isolamento & purificação , Fezes/virologia , Macaca mulatta/virologia , Doenças dos Macacos/transmissão , Zoonoses/transmissão , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/transmissão , Infecções por Adenoviridae/virologia , Adenovirus dos Símios/classificação , Adenovirus dos Símios/genética , Sequência de Aminoácidos , Animais , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Dados de Sequência Molecular , Doenças dos Macacos/epidemiologia , Doenças dos Macacos/virologia , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Estados Unidos/epidemiologia , Zoonoses/epidemiologia , Zoonoses/virologiaRESUMO
LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRß, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.
Assuntos
Acetanilidas/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Acetanilidas/síntese química , Acetanilidas/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cães , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.
Assuntos
Adenovirus dos Símios/genética , Técnicas de Transferência de Genes/instrumentação , Vetores Genéticos/genética , Adenovirus dos Símios/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Vetores Genéticos/fisiologia , Humanos , Dados de Sequência Molecular , Replicação ViralRESUMO
The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.
Assuntos
Ligantes , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tiofenos/química , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/patologia , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ligação Proteica , Relação Estrutura-Atividade , Tiofenos/metabolismo , Tiofenos/farmacologia , Tiofenos/uso terapêuticoRESUMO
We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.
Assuntos
Anticorpos Biespecíficos , Doenças Autoimunes/tratamento farmacológico , Fator Ativador de Células B/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fator Ativador de Células B/imunologia , Feminino , Células HEK293 , Células HT29 , Humanos , Interleucina-17/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
In our continuing effort to expand the SAR of the quinoline domain of dihydropyrrolopyrazole series, we have discovered compound 15d, which demonstrated the antitumor efficacy with oral bioavailability. This effort also demonstrated that the PK/PD in vivo target inhibition paradigm is an effective approach to assess potential for antitumor efficacy. The dihydropyrrolopyrazole inhibitor 15d (LY2109761) is representative of a novel series of antitumor agents.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Pirróis/síntese química , Pirróis/química , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo I , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-abl/química , Trifosfato de Adenosina/química , Sítio Alostérico , Cinética , Cadeias de Markov , Ácido Mirístico/química , Conformação Proteica , TermodinâmicaRESUMO
Transforming growth factor-ß (TGFß) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFß receptor I (TGFßRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFßRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFß-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFß-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects. Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFß-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFß pathway inhibitors.
RESUMO
Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/síntese química , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Animais , Compostos Bicíclicos com Pontes/farmacocinética , Cristalografia por Raios X , AMP Cíclico/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacocinética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenciclidina/antagonistas & inibidores , Fenciclidina/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
The successful use of any adenoviral vectors is predicated upon the use of a serotype that is not neutralized by circulating antibodies. However, efforts to develop a diverse repertoire of serologically distinct adenovirus vectors may be hindered by the necessity to generate cell lines to allow for the successful propagation of vectors deleted of essential genes. A strategy to construct chimeric adenoviruses whereby the rescue and propagation of an E1-deleted HAdV-B-derived adenoviral vector can be achieved using existing cell lines such as HEK 293 is reported. It is further shown that this strategy may be more widely applicable.
Assuntos
Adenovirus dos Símios/genética , DNA Viral/genética , Vetores Genéticos , Genoma Viral , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/imunologia , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/imunologia , Adenovirus dos Símios/classificação , Adenovirus dos Símios/imunologia , Animais , Linhagem Celular , Estudos de Viabilidade , Humanos , Modelos Genéticos , Testes de Neutralização , Pan troglodytes/virologia , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Sorotipagem , TransfecçãoRESUMO
Novel dihydropyrrolopyrazole-substituted benzimidazoles were synthesized and evaluated in vitro as inhibitors of transforming growth factor-beta type I receptor (TGF-beta RI), TGF-beta RII, and mixed lineage kinase-7 (MLK-7). These compounds were found to be potent TGF-beta RI inhibitors and selective versus TGF-beta RII and MLK-7 kinases. Benzimidazole derivative 8b was active in an in vivo target (TGF-beta RI) inhibition assay.
Assuntos
Benzimidazóis/síntese química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Pirróis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Vison , Estrutura Terciária de Proteína , Pirazóis/química , Pirazóis/farmacologia , Pirróis/química , Pirróis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts.
Assuntos
Dislipidemias/tratamento farmacológico , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Sítios de Ligação , Cricetinae , Cristalografia por Raios X , Dieta Hiperlipídica , Feminino , Ligantes , Lipídeos/sangue , Mesocricetus , Modelos Moleculares , Conformação Proteica , Ratos , Ratos Wistar , Receptores de Glucocorticoides/genética , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacologiaRESUMO
The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas ras/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Cães , Feminino , Meia-Vida , Humanos , Masculino , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacocinética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genéticaRESUMO
As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.
Assuntos
Compostos Bicíclicos com Pontes/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Compostos de Espiro/farmacologia , Animais , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/metabolismo , Cristalografia por Raios X , Humanos , Masculino , Modelos Moleculares , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Compostos de Espiro/química , Compostos de Espiro/metabolismoRESUMO
Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/química , Receptores de Glutamato Metabotrópico/agonistas , Triazóis/química , Regulação Alostérica , Animais , Ligação Competitiva , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Cães , Agonismo Parcial de Drogas , Humanos , Masculino , Camundongos , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Estereoisomerismo , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path for further insights into structure-based ligand design.
Assuntos
Anti-Inflamatórios não Esteroides/química , Benzoxepinas/química , Receptores de Glucocorticoides/química , Sulfonamidas/química , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Benzoxepinas/farmacocinética , Benzoxepinas/farmacologia , Sítios de Ligação , Carragenina , Linhagem Celular , Doença Crônica , Colágeno , Cristalografia por Raios X , Desenho de Fármacos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Ligantes , Masculino , Modelos Moleculares , Conformação Molecular , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologiaRESUMO
In order to elucidate the relative importance of neutralizing determinants on each of the three major adenoviral capsid components, we have generated chimeric vectors where the hexon protein, or the fiber protein, or both hexon and fiber proteins of one serotype (Simian Adenovirus 24/Pan 7) have been replaced by those of another (Simian Adenovirus 23/Pan 6). The effect of each replacement was evaluated by neutralization assays and by attempted vector re-administration into mice. Both hexon and fiber were found to harbor neutralization epitopes although in vivo transduction was more severely affected by anti-hexon antibodies.
Assuntos
Adenovirus dos Símios/genética , Adenovirus dos Símios/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Vetores Genéticos , Animais , Anticorpos Antivirais , Antígenos Virais/genética , Quimera/genética , Quimera/imunologia , Epitopos/genética , Imunização , Técnicas In Vitro , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Testes de NeutralizaçãoRESUMO
The nuclear receptor (NR) superfamily comprises approximately 50 members that are responsible for regulating a number of physiologic processes in humans, including metabolism, homeostasis, and reproduction. Included in the superfamily are the receptors for steroids, lipophilic vitamins, bile acids, retinoids, and various fatty acids. NRs exert their action as transcription factors that directly bind to the promoters of target genes and regulate their rate of transcription. To modulate transcription, however, NRs must recruit a number of accessory coregulators known as corepressors and coactivators. These coregulators harbor a variety of activities, such as the ability to modify chromatin structure, interact with basal transcriptional machinery, and modify RNA splicing. Recent studies have revealed that the pharmacological characteristics of various NR ligands are regulated by their ability to modulate the coregulator interaction profile of an NR.