RESUMO
Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.
Assuntos
Descoberta de Drogas , Ligantes , Estudos Prospectivos , Estudos Retrospectivos , TermodinâmicaRESUMO
Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.
Assuntos
Ciclofilinas/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Lactamas/farmacologia , Cristalografia por Raios X , Ciclofilinas/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lactamas/síntese química , Lactamas/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
New spiroindolinone antagonists of CRTH2 are described. Following identification of insufficient stability in human plasma as an important liability of the lead compounds, replacement of the spirosuccinimide core with a spirohydantoin or spiropyrrolidinone structure has yielded a compound that is fully stable in human plasma and with good potency in a human whole blood assay (IC50 = 69 nM) but shows a much lower oral bioavailability (6-9% in rodents) than the earlier compounds. Successive optimization aimed at restoring an acceptable oral bioavailability has yielded compound (S)-17a, which exhibits both stability in human plasma and a good oral bioavailability in rat (37%) and mouse (39%). This compound is also active in a mouse model of ovalbumin-induced lung inflammation following oral dosing at 30 mg/kg.
RESUMO
Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (K i < 10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C19 > 1 µM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development.
RESUMO
New phenoxyacetic acid antagonists of CRTH2 are described. Following the discovery of a hit compound by a focused screening, high protein binding was identified as its main weakness. Optimization aimed at reducing serum protein binding led to the identification of several compounds that showed not only excellent affinities for the receptor (41 compounds with K(i) < 10 nM) but also excellent potencies in a human whole blood assay (IC(50) < 100 nM; PGD2-induced eosinophil shape change). Additional optimization of the PK characteristics led to the identification of several compounds suitable for in vivo testing. Of these, 19k and 19s were tested in two different pharmacological models (acute FITC-mediated contact hypersensitivity and ovalbumin-induced eosinophilia models) and found to be active after oral dosing (10 and 30 mg/kg).
Assuntos
Acetatos/síntese química , Alcinos/síntese química , Antialérgicos/síntese química , Anti-Inflamatórios/síntese química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Sulfonas/síntese química , Acetatos/farmacocinética , Acetatos/farmacologia , Administração Oral , Alcinos/farmacocinética , Alcinos/farmacologia , Animais , Antialérgicos/farmacocinética , Antialérgicos/farmacologia , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Ligação Competitiva , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Forma Celular , Quimiotaxia de Leucócito , Dermatite de Contato/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Eosinófilos/fisiologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Ovalbumina/imunologia , Fenoxiacetatos , Ligação Proteica , Eosinofilia Pulmonar/tratamento farmacológico , Eosinofilia Pulmonar/imunologia , Ensaio Radioligante , Ratos , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Sulfonas/farmacologiaRESUMO
Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll-like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1-p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE-induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR-ASK1-p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.
Assuntos
Doenças Desmielinizantes/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Encefalopatias/tratamento farmacológico , Encefalopatias/genética , Encefalopatias/imunologia , Encefalopatias/metabolismo , Quimiocinas/imunologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/imunologia , Modelos Animais de Doenças , Encefalite , Inibidores Enzimáticos/administração & dosagem , Feminino , Regulação da Expressão Gênica , Doença de Hashimoto/tratamento farmacológico , Doença de Hashimoto/genética , Doença de Hashimoto/imunologia , Doença de Hashimoto/metabolismo , Humanos , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.
Assuntos
Diferenciação Celular/fisiologia , Redes Reguladoras de Genes , Oligodendroglia/fisiologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Dexametasona/farmacologia , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Fosfatases de Especificidade Dupla/fisiologia , Inativação Gênica , Estudo de Associação Genômica Ampla , Camundongos , Proteína Básica da Mielina/biossíntese , Neurogênese/fisiologia , Oligodendroglia/citologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase . Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.
Assuntos
Quimiotaxia/efeitos dos fármacos , Colite Ulcerativa/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Animais , Colite Ulcerativa/tratamento farmacológico , Citoesqueleto/metabolismo , Feminino , Técnicas In Vitro , Leucócitos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Monócitos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/efeitos adversos , Transdução de Sinais , Tioglicolatos/farmacologiaRESUMO
In the course of a high throughput screening, a series of pyrazole compounds were identified with luteinizing hormone receptor (LH-R) agonist activity. A focused pyrazole library was produced by solid-phase synthesis and key pyrazole regioisomers were obtained selectively in solution. Evaluation of those compounds in a cAMP assay in CHO cells transfected with h-LH receptor allowed us to propose a structure-activity relationship model for this series and led to the identification of the first low molecular weight molecule with in vitro activity in a Leydig cells assay (ED(50)=1.31 microM) and in vivo in a model of testosterone induction in rats (significant effect at 32 mpk ip).