RESUMO
Compound 1 ((4-amino-3,5-dichlorophenyl)-1-(4-methylpiperidin-1-yl)-4-(2-nitroimidazol-1-yl)-1-oxobutane-2-sulfonamido) was discovered to be a 690nM antagonist of human CCR10 Ca2+ flux. Optimization delivered (2R)-4-(2-cyanopyrrol-1-yl)-S-(1H-indol-4-yl)-1-(4-methylpiperidin-1-yl)-1-oxobutane-2-sulfonamido (eut-22) that is 300 fold more potent a CCR10 antagonist than 1 and eliminates potential toxicity, mutagenicity, and drug-drug-interaction liabilities often associated with nitroaryls and anilines. eut-22 is highly selective over other GPCR's, including a number of other chemokine receptors. Finally, eut-22 is efficacious in the murine DNFB model of contact hypersensitivity. The efficacy of this compound provides further evidence for the role of CCR10 in dermatological inflammatory conditions.
Assuntos
Amidas/farmacologia , Dermatite de Contato/tratamento farmacológico , Dinitrofluorbenzeno/toxicidade , Modelos Animais de Doenças , Receptores CCR10/antagonistas & inibidores , Amidas/química , Amidas/uso terapêutico , Animais , Ácidos Carboxílicos/química , Linhagem Celular , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC(50) = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (-93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to -39 ± 4 mm Hg on day 4 at T(max)); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic α(1A) and α(2A) was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0-92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC(50) <0.14 nM) without relevant α(1A) and α(2A) inhibition and no adverse cardiovascular effects in vivo.
Assuntos
Azepinas/farmacologia , Benzimidazóis/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Descoberta de Drogas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were optimized for cellular potency and kinase selectivity. This led to the identification of compound 24, BIX 02565, an attractive candidate for use in vitro and in vivo to explore the role of RSK as a target for the treatment heart failure.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/química , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Amidas/química , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , Nitrogênio/química , Relação Estrutura-AtividadeRESUMO
A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subsequent kinase selectivity optimization.
Assuntos
Azepinas/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/química , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Amidas/química , Azepinas/farmacologia , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Indóis/síntese química , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , Nitrogênio/química , Relação Estrutura-AtividadeRESUMO
We have identified two novel MEK5 inhibitors, BIX02188 and BIX02189, which inhibited catalytic function of purified, MEK5 enzyme. The MEK5 inhibitors blocked phosphorylation of ERK5, without affecting phosphorylation of ERK1/2 in sorbitol-stimulated HeLa cells. The compounds also inhibited transcriptional activation of MEF2C, a downstream substrate of the MEK5/ERK5 signaling cascade, in a cellular trans-reporter assay system. These inhibitors offer novel pharmacological tools to better characterize the role of the MEK5/ERK5 pathway in various biological systems.
Assuntos
Compostos de Anilina/farmacologia , Indóis/farmacologia , MAP Quinase Quinase 5/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Compostos de Anilina/isolamento & purificação , Células HeLa , Humanos , Indóis/isolamento & purificação , Proteínas de Domínio MADS/antagonistas & inibidores , Proteínas de Domínio MADS/genética , MAP Quinase Quinase 5/metabolismo , Fatores de Transcrição MEF2 , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fatores de Regulação Miogênica/antagonistas & inibidores , Fatores de Regulação Miogênica/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/isolamento & purificação , Sorbitol/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.
Assuntos
Benzimidazóis/administração & dosagem , Benzimidazóis/química , Benzimidazóis/síntese química , Química Farmacêutica/métodos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Administração Oral , Animais , Benzimidazóis/farmacologia , Complexo CD3/biossíntese , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Relação Estrutura-Atividade , Linfócitos T/citologiaRESUMO
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.
Assuntos
Adenosina Trifosfatases/metabolismo , Bioensaio/métodos , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Corantes Fluorescentes/química , Humanos , CinéticaRESUMO
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.
Assuntos
Benzimidazóis/síntese química , Inibidores Enzimáticos/síntese química , Imunossupressores/síntese química , Isoquinolinas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Administração Oral , Animais , Anticorpos Monoclonais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Complexo CD3/imunologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Interleucina-2/sangue , Isoquinolinas/química , Isoquinolinas/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.
Assuntos
Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Isoquinolinas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Sítios de Ligação , Complexo CD3/imunologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacologia , Interleucina-2/biossíntese , Isoquinolinas/química , Isoquinolinas/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Benzimidazole 1 was identified as a selective inhibitor of ITK by high throughput screening. Hit-to-lead studies defined the SAR at all three substituents. Reversing the amide linkage at C6 led to 16, with a fivefold improvement of potency. This enhancement is rationalized by the conformational preference of the substituent. A model for the binding of the benzimidazoles to the ATP-binding site of ITK is proposed.
Assuntos
Benzimidazóis/química , Química Farmacêutica/métodos , Inibidores de Proteínas Quinases/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Benzimidazóis/síntese química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Modelos Químicos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with IC50s as low as 10 nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the p-loop and the hinge region of MK2a.
Assuntos
Carbolinas/química , Carbolinas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.