RESUMEN
The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.
Asunto(s)
Antiinflamatorios/farmacología , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/farmacología , Complejos Multiproteicos/metabolismo , Péptidos/farmacología , Factor de Transcripción ReIA/metabolismo , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/química , Complejos Multiproteicos/antagonistas & inhibidores , Péptidos/química , Péptidos/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidoresRESUMEN
A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.
Asunto(s)
Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinonas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Animales , Artritis/tratamiento farmacológico , Artritis Experimental , Células CACO-2 , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Masculino , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Pirimidinonas/química , Ratas , Ratas Endogámicas Lew , Relación Estructura-ActividadRESUMEN
The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.
Asunto(s)
Benzamidas/síntesis química , Benzamidas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Pironas/síntesis química , Pironas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Benzamidas/química , Modelos Animales de Enfermedad , Perros , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Estructura Molecular , Piridonas , Pironas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Piridonas/síntesis química , Piridonas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Masculino , Microsomas Hepáticos/enzimología , Estructura Molecular , Piridazinas/química , Piridazinas/farmacología , Piridonas/química , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).
Asunto(s)
Aminopiridinas/síntesis química , Asma/tratamiento farmacológico , Quinasa I-kappa B/antagonistas & inhibidores , Niacinamida/análogos & derivados , Inhibidores de Proteínas Quinasas/síntesis química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Pirazoles/síntesis química , Administración por Inhalación , Aminopiridinas/química , Aminopiridinas/farmacología , Unión Competitiva , Diseño de Fármacos , Células HEK293 , Humanos , Indazoles/química , Indazoles/metabolismo , Indazoles/farmacología , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/metabolismo , Ácidos Isonicotínicos/farmacología , Microsomas Hepáticos/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Terapia Molecular Dirigida , Niacinamida/síntesis química , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacología , Fenetilaminas/metabolismo , Bloqueadores de los Canales de Potasio/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Relación Estructura-Actividad , Sulfonamidas/metabolismoRESUMEN
NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.
Asunto(s)
Quinasa I-kappa B/metabolismo , Animales , Células Cultivadas , Simulación por Computador , Descubrimiento de Drogas/métodos , Humanos , Quinasa I-kappa B/biosíntesis , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Indazoles/química , Indazoles/metabolismo , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Homología Estructural de ProteínaRESUMEN
The structure based drug design, synthesis and structure-activity relationship of a series of C6 sulfur linked triazolopyridine based p38 inhibitors are described. The metabolic deficiencies of this series were overcome through changes in the C6 linker from sulfur to methylene, which was predicted by molecular modeling to be bioisosteric. X-ray of the ethylene linked compound 61 confirmed the predicted binding orientation of the scaffold in the p38 enzyme.
Asunto(s)
Benzamidas/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Triazoles/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Benzamidas/síntesis química , Benzamidas/farmacología , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Microsomas Hepáticos/metabolismo , Modelos Químicos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Ratas , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The synthesis, structure-activity relationship and modeling of a series of 5-substituted-N-aryl pyridazinone based p38alpha inhibitors are described. In comparing the series to the similar N-aryl pyridinone series, it was found that the pyridazinones maintained a weaker interaction to the p38 enzyme, and therefore showed generally weaker binding than the pyridinones.
Asunto(s)
Antiinfecciosos/química , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Sitios de Unión , Simulación por Computador , Descubrimiento de Drogas , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/síntesis química , Piridazinas/farmacología , Relación Estructura-ActividadRESUMEN
Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38alpha kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Dominio Catalítico , Humanos , Modelos Moleculares , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.
Asunto(s)
Enfermedades Gastrointestinales/inducido químicamente , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Enfermedades Linfáticas/inducido químicamente , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Linfocitos B/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colon/efectos de los fármacos , Colon/patología , Perros , Femenino , Enfermedades Gastrointestinales/patología , Hemorragia Gastrointestinal/inducido químicamente , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Lineales , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Enfermedades Linfáticas/patología , Macaca fascicularis , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismo , Pruebas de Toxicidad Aguda , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.
Asunto(s)
Benzamidas/farmacología , Biología Computacional , Inhibidores de Proteínas Quinasas/farmacología , Pironas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Benzamidas/química , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Fosforilación , Inhibidores de Proteínas Quinasas/química , Piridonas , Pironas/química , Especificidad por Sustrato , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Benzamidas/uso terapéutico , Pironas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adolescente , Adulto , Animales , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/enzimología , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Benzamidas/sangre , Benzamidas/química , Benzamidas/farmacología , Densidad Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Línea Celular , Citocinas/biosíntesis , Citocinas/sangre , Dinoprostona/biosíntesis , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Lipopolisacáridos/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/enzimología , Monocitos/inmunología , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteoclastos/inmunología , Piridonas , Pironas/sangre , Pironas/química , Pironas/farmacología , Ratas , Ratas Endogámicas Lew , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Síndrome de Respuesta Inflamatoria Sistémica/enzimología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Adulto JovenRESUMEN
The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38alpha. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.
Asunto(s)
Antiinflamatorios/química , Inhibidores de Proteínas Quinasas/química , Piridonas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Piridonas/síntesis química , Piridonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.
RESUMEN
p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α-MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1ß expression by promoting IL-1ß mRNA degradation. Thus, IL-1ß is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α-MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.
Asunto(s)
Inflamasomas/metabolismo , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal , Animales , Artritis/patología , Huesos/patología , Citocinas/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Articulaciones/patología , Masculino , Ratones , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad del ARN , Ratas Endogámicas LewRESUMEN
PH-797804 ((aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamde) is a diarylpyridinone inhibitor of p38 mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. Due to steric constraints imposed by the pyridinone carbonyl group and the 6- and 6'-methyl substituents of PH-797804, rotation around the connecting bond of the pyridinone and the N-phenyl ring is restricted. Density functional theory predicts a remarkably high rotational energy barrier of >30â kcal mol(-1), corresponding to a half-life of more than one hundred years at room temperature. This gives rise to discrete conformational spaces for the N-phenylpyridinone group, and as a result, two atropic isomers that do not interconvert under ambient conditions. Molecular modeling studies predict that the two isomers should differ in their binding affinity for p38α kinase; whereas the atropic S (aS) isomer binds favorably, the opposite aR isomer incurs significant steric interference with p38α kinase. The two isomers were subsequently identified and separated by chiral chromatography. IC50 values from p38α kinase assays confirm that one atropisomer is >100-fold more potent than the other. It was ultimately confirmed by small-molecule X-ray diffraction that the more potent atropisomer, PH-797804, is the aS isomer of the racemic pair. Extensive pharmacological characterization supports that PH-797804 carries most activity both inâ vitro and inâ vivo, and it has a stability profile compatible with oral formulation and delivery options.