A unique p38α MAPK–MK2 pathway inhibitor, CDD-450, is used to uncover the function of this protein complex in inflammasome priming signals. Importantly, CDD-450 is as efficacious as global p38α MAPK inhibitors in decreasing inflammation in disease models.
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.
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 Nphenyl pyridinone p38 MAP kinase inhibitor benzamide [3-[3-bromo-4-[(2,4-PH-797804 is an ATP-competitive, readily reversible inhibitor of the ␣ isoform of human p38 MAP kinase, exhibiting a K i ϭ 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-␣ 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 antiinflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.Rheumatoid arthritis (RA) is an aggressive autoimmune disease involving complex interactions among T cells, macrophages, synoviocytes, and other immune cells (Firestein, 2003). Analysis of synovial fluid and tissue from RA patients implicated key cytokines, including TNF-␣, IL-1, and IL-6 in the pathogenesis of the disease (Firestein et al., 1990). Further studies shed light on the complex networks within which these cytokines function and the delicate balance between a pro-or an anti-inflammatory outcome (McInnes and ABBREVIATIONS: RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL, interleukin; COX, cyclooxygenase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH 2 -terminal kinase; benzamide,methoxy]-6-methyl-2-oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (Ϫ)-(9CI); SCW, streptococcal cell wall; LPS, lipopolysaccharide; MKK, mitogen-activated protein kinase kinase; EGFRP, epidermal growth factor receptor peptide; GST, glutathione transferase; RASF, rheumatoid arthritis synovial fibroblast(s); MK-2, mitogen-activated protein kinase-act...
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.
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