RESUMEN
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.
Asunto(s)
Ligandos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Tiofenos/química , Animales , Artritis/inducido químicamente , Artritis/tratamiento farmacológico , Artritis/patología , Sitios de Unión , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Femenino , Semivida , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Simulación de Dinámica Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Unión Proteica , Relación Estructura-Actividad , Tiofenos/metabolismo , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas c-abl/química , Adenosina Trifosfato/química , Sitio Alostérico , Cinética , Cadenas de Markov , Ácido Mirístico/química , Conformación Proteica , TermodinámicaRESUMEN
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.
Asunto(s)
Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/farmacología , Agonistas de Aminoácidos Excitadores/síntesis química , Agonistas de Aminoácidos Excitadores/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Animales , Compuestos Bicíclicos con Puentes/farmacocinética , Cristalografía por Rayos X , AMP Cíclico/farmacología , Agonistas de Aminoácidos Excitadores/farmacocinética , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Masculino , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenciclidina/antagonistas & inhibidores , Fenciclidina/farmacología , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Receptores de Glutamato Metabotrópico/agonistas , Triazoles/química , Regulación Alostérica , Animales , Unión Competitiva , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Calcio/metabolismo , AMP Cíclico/metabolismo , Perros , Agonismo Parcial de Drogas , Humanos , Masculino , Ratones , Modelos Moleculares , Actividad Motora/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Estereoisomerismo , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/química , Pirimidinas/farmacología , Proteínas ras/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral/efectos de los fármacos , Técnicas de Química Sintética , Perros , Femenino , Semivida , Humanos , Masculino , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacocinética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genéticaRESUMEN
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.
Asunto(s)
Compuestos Bicíclicos con Puentes/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Compuestos de Espiro/farmacología , Animales , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismo , Cristalografía por Rayos X , Humanos , Masculino , Modelos Moleculares , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Compuestos de Espiro/química , Compuestos de Espiro/metabolismoRESUMEN
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.
Asunto(s)
Isoquinolinas/farmacología , Dolor/tratamiento farmacológico , Profármacos/farmacología , Receptores de Ácido Kaínico/antagonistas & inhibidores , Tetrazoles/farmacología , Administración Oral , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Isoquinolinas/química , Masculino , Datos de Secuencia Molecular , Profármacos/química , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Kaínico/química , Relación Estructura-Actividad , Tetrazoles/químicaRESUMEN
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.
Asunto(s)
Acetanilidas/uso terapéutico , Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Acetanilidas/síntesis química , Acetanilidas/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Perros , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirroles/farmacología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/química , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteínas Quinasas/química , Pirazoles/síntesis química , Pirazoles/química , Pirroles/síntesis química , Pirroles/química , Ratas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Estereoisomerismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.