Shaun R. Selness

Anti-Inflammatory Properties of a Novel N-Phenyl Pyridinone Inhibitor of p38 Mitogen-Activated Protein Kinase: Preclinical-to-Clinical Translation

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 α isoform of human p38 MAP kinase, exhibiting a Ki = 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 E2, 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-α 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.

Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

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.

SD0006: A Potent, Selective and Orally Available Inhibitor of p38 Kinase

SD0006 is a diarylpyrazole that was prepared as an inhibitor of p38 kinase-α (p38α). In vitro, SD0006 was selective for p38α kinase over 50 other kinases screened (including p38γ and p38δ with modest selectivity over p38β). Crystal structures with p38α show binding at the ATP site with additional residue interactions outside the ATP pocket unique to p38α that can confer advantages over other ATP competitive inhibitors. Direct correlation between inhibition of p38α activity and that of lipopolysaccharide-stimulated TNFα release was established in cellular models and in vivo, including a phase 1 clinical trial. Potency (IC50) for inhibiting tumor necrosis factor-α (TNFα) release, in vitro and in vivo, was <200 nmol/l. In vivo, SD0006 was effective in the rat streptococcal-cell-wall-induced arthritis model, with dramatic protective effects on paw joint integrity and bone density as shown by radiographic analysis. In the murine collagen-induced arthritis model, equivalence was demonstrated to anti-TNFα treatment. SD0006 also demonstrated good oral anti-inflammatory efficacy with excellent cross-species correlation between the rat, cynomolgus monkey, and human. SD0006 suppressed expression of multiple proinflammatory proteins at both the transcriptional and translational levels. These properties suggest SD0006 could provide broader therapeutic efficacy than cytokine-targeted monotherapeutics.

Novel Insights into the Cellular Mechanisms of the Anti-inflammatory Effects of NF-κB Essential Modulator Binding Domain Peptides

The classical nuclear factor κB (NF-κB) signaling pathway is under the control of the IκB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-κB 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-κB 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 κBα (IκBα). 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.

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase.

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.

Discovery and characterization of atropisomer PH-797804, a p38 MAP kinase inhibitor, as a clinical drug candidate.

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 >30 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 in vitro and in vivo, and it has a stability profile compatible with oral formulation and delivery options.

Discovery of N-substituted pyridinones as potent and selective inhibitors of p38 kinase.

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.

Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

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.

Acute Lymphoid and Gastrointestinal Toxicity Induced by Selective p38α Map Kinase and Map Kinase–Activated Protein Kinase-2 (MK2) Inhibitors in the Dog

Exposure to moderately selective p38α 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 p38α 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 p38α MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38α MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Continued exploration of the triazolopyridine scaffold as a platform for p38 MAP kinase inhibition

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.