P38α Mitogen-activated Protein Kinase Inhibitors Research Articles

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 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.

Fluorescence polarization binding assay to develop inhibitors of inactive p38α mitogen-activated protein kinase

Development of inhibitors that target inactive kinase conformations is becoming a more attractive approach to kinase inhibitor research. The major advantage of this methodology is that targeting the inactive conformation reduces competition with high intracellular adenosine triphosphate (ATP) concentrations. p38α Mitogen-activated protein kinase (MAPK) signaling has been identified as the principal mediator of inflammation associated with a spectrum of disorders (e.g., arthritis, Alzheimer’s disease, various malignancies). To allow identification and development of p38α MAPK inhibitors that preferentially bind to the inactive conformation, a novel fluorescence polarization-based binding assay is presented. The assay is homogeneous, requires low amounts of the kinase and fluoroprobe, and does not rely on radioactivity. It may, therefore, offer an inexpensive alternative to current p38α MAPK inhibitor screening methods. The validation of the system with known p38α MAPK inhibitors confirmed that the binding assay, rather than the conventional enzyme activity assay, correlates with cellular efficacy. Finally, we show that pyridinyl imidazoles that potently bind to the inactive p38α MAPK prevent activation of p38 MAPK in living cells, suggesting that pyridinyl imidazoles other than SB203580 are able to induce the DFG-out conformation that is incompatible with activation (where DFG is a single-letter amino acid code for the aspartate–phenylalanine–glycine sequence at the start of the activation loop).

Part 2: Structure–activity relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38α mitogen-activated protein kinase

A novel class of pyrazolopyridazine p38α mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC 50 values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38α inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38α/β over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED 50 of ca. 0.08 mg/kg.

Pyridinylquinoxalines and Pyridinylpyridopyrazines as Lead Compounds for Novel p38α Mitogen-Activated Protein Kinase Inhibitors

Various substituted 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)quinoxalines and 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)pyridopyrazines were synthesized as novel p38 alpha MAP kinase inhibitors via different short synthetic strategies with high variation possibilities. The formation of the quinoxaline/pyridopyrazine core was achieved from alpha-diketones and o-phenylenediamines/alpha-diaminopyridines under microwave irradiation. Introduction of an amino moiety at the pyridine C2 position of the 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)quinoxalines led to compounds showing potent enzyme inhibition down to the double-digit nanomolar range (6f; IC(50) = 81 nM). Replacement of the quinoxaline core with pyrido[2,3-b]pyrazine gave compound 9e with superior p38 alpha MAP kinase inhibition (IC(50) = 38 nM).

P38α Mitogen-Activated Protein Kinase Inhibitors: Optimization of a Series of Biphenylamides to Give a Molecule Suitable for Clinical Progression

p38alpha MAP kinase is a key anti-inflammatory target for rheumatoid arthritis, influencing biosynthesis of pro-inflammatory cytokines TNFalpha and IL-1beta at a translational and transcriptional level. In this paper, we describe how we have optimized a series of novel p38alpha/beta inhibitors using crystal structures of our inhibitors bound to p38alpha, classical medicinal chemistry, and modeling of virtual libraries to derive a molecule suitable for progression into clinical development.

Role of the Hydrogen Bonding Heteroatom−Lys53 Interaction between the p38α Mitogen-Activated Protein (MAP) Kinase and Pyridinyl-Substituted 5-Membered Heterocyclic Ring Inhibitors

In the framework of investigating the role of heteroatoms in pyridinyl-substituted 5-membered (hetero)cycles as potential p38alpha MAP kinase inhibitor scaffolds, cyclopentene, pyrrole, furan, and imidazole analogues were synthesized and tested with respect to their ability to inhibit p38alpha MAP kinase. The vicinal pyridine/4-fluorophenyl pharmacophore was conserved, such as in the prototypical imidazole inhibitor SB203580. The strength of the HB interaction was calculated and compared to the biological data.

The p38α mitogen-activated protein kinase as a central nervous system drug discovery target

Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38α mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38α MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38α MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38α MAPK in neurodegenerative disorders.

Inhibition of p38α Mitogen-Activated Protein Kinase Prevents the Development of Osteolytic Bone Disease, Reduces Tumor Burden, and Increases Survival in Murine Models of Multiple Myeloma

The bone microenvironment plays a critical role in supporting the growth and survival of multiple myeloma as well as in the development of osteolytic bone disease. Signaling through p38alpha mitogen-activated protein kinase (MAPK) mediates synthesis of multiple myeloma cell growth factors, and its inhibition reduces proliferation in vitro. However, it is unclear whether targeting p38alpha MAPK prevents multiple myeloma growth and the development of bone disease in vivo. In this study, we determined whether SCIO-469, a selective p38alpha MAPK inhibitor, inhibits multiple myeloma growth and prevents bone disease in the 5T2MM and 5T33MM models. SCIO-469 decreased constitutive p38alpha MAPK phosphorylation of both 5T2MM and 5T33MM cells in vitro. This was associated with decreased DNA synthesis and an induction of apoptosis when the cells were cultured with bone marrow stromal cells. Treatment of C57Bl/KaLwRij mice bearing 5T33MM cells with SCIO-469 inhibited p38alpha MAPK phosphorylation and was associated with a significant decrease in serum paraprotein, an almost complete reduction in tumor cells in the bone marrow, a decrease in angiogenesis, and a significant increase in disease-free survival. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in myeloma bone disease characterized by increased osteoclast occupation of the bone surface, reduced cancellous bone, and the development of osteolytic bone lesions. Treatment of 5T2MM-injected mice with SCIO-469 reduced this development of bone disease. Together, these data show that targeting p38alpha MAPK with SCIO-469 decreases myeloma burden in vivo, in addition to preventing the development of myeloma bone disease.

A Selective p38α Mitogen-Activated Protein Kinase Inhibitor Reverses Cartilage and Bone Destruction in Mice with Collagen-Induced Arthritis

Destruction of cartilage and bone is a poorly managed hallmark of human rheumatoid arthritis (RA). p38 Mitogen-activated protein kinase (MAPK) has been shown to regulate key proinflammatory pathways in RA, including tumor necrosis factor alpha, interleukin (IL)-1beta, and cyclooxygenase-2, as well as the process of osteoclast differentiation. Therefore, we evaluated whether a p38alpha MAPK inhibitor, indole-5-carboxamide (SD-282), could modulate cartilage and bone destruction in a mouse model of RA induced with bovine type II collagen [collagen-induced arthritis (CIA)]. In mice with early disease, SD-282 treatment significantly improved clinical severity scores, reduced bone and cartilage loss, and reduced mRNA levels of proinflammatory genes in paw tissue, including IL-1beta, IL-6, and cyclooxygenase-2. Notably, SD-282 treatment of mice with advanced disease resulted in significant improvement in clinical severity scoring and paw swelling, a reversal in bone and cartilage destruction as assessed by histology, bone volume fraction and thickness, and three-dimensional image analysis. These changes were accompanied by reduced osteoclast number and lowered levels of serum cartilage oligomeric matrix protein, a marker of cartilage breakdown. Thus, in a model of experimental arthritis associated with significant osteolysis, p38alpha MAPK inhibition not only attenuates disease progression but also reverses cartilage and bone destruction in mice with advanced CIA disease.

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38α MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFα-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFα-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFα-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1α as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Selective inhibition of p38α mitogen-activated protein kinase improves cardiac function and reduces myocardial apoptosis in rat model of acute myocardial injury induced by L-NAME/angiotensin II

p38 mitogen-activated protein kinase (MAPK) is activated during heart disease. However, whether p38 MAPK inhibition is cardioprotective still remain unclear. In a model of acute myocardial injury induced by Nω-nitro-L-arginine methyl ester (L-NAME)/angiotensin II (Ang II), we investigated the cardioprotective effects of selective p38α MAPK inhibition using a synthesized inhibitor, SD-282. Rats were treated with L-NAME (40 mg/kg/day) in drinking water plus 1% salt for 14 days along with Ang II (0.5 mg/kg/day) for 3 days. SD-282 (60 mg/kg) was administrated orally, twice a day for 4 days, starting a day prior to Ang II administration. The cardioprotective effects of p38α MAPK inhibition were evaluated by improvement of cardiac function, reduction of inflammatory cell infiltration and cardiomyocytes apoptosis. It demonstrated that the p38α MAPK inhibitor, SD-282 improved cardiac function indicated by increase in stroke volume (SV), cardiac output (CO), ejection fraction (EF), stroke work (SW) and decrease in arterial elastance (Ea). (Table) Tabled 1 Cardiac Function of Rats Treated With or Without SD-282 Group HR (bpm) SV (ul) CO (ul/min) EF (%) SW (mmHg∗ul) Ea (mmHg/ul) n Control 340±27 8.5±0.8 2902±314 30.5±1.5 677.7±47.5 17.1±1.8 6 Vehicle 269±30 3.1±1.1 766±265 14.4±3.5 276.4±181 55.4±16.4 9 SD-282 284±14 6.5±2.6 1831±723 21.6±6.8 549.8±274 29.5±19.6 9 p value ns <0.01 <0.001 <0.01 <0.001 <0.001 Open table in a new tab

Perioperative pain: other: SCIO-469, a novel p38a MAPK inhibitor, shows analgesic efficacy in acute post-surgical dental pain

SCIO-469 is a selective p38α mitogen activated protein kinase (MAPK) inhibitor. This double-blind, randomized study compared the analgesic efficacy, safety and tolerability of pre- and post-operatively administered oral SCIO-469 vs. placebo in post-surgical dental pain; in addition, as a positive control, ibuprofen 400 mg was compared to placebo. Subjects (n=263) undergoing extraction of at least one impacted third molar were enrolled to receive pre-operative treatment with 150 mg, 300 mg, or 210 mg SCIO-469, ibuprofen, or placebo 60 min before surgery. One hour after first incision, subjects received either 90 mg SCIO-469 (210 mg group only) or placebo. The primary endpoint was median time to first rescue medication for each group using the Kaplan-Meier product limit estimator. All SCIO-469 groups had a significantly longer time to rescue medication compared to placebo; pre-and post-operative treatment of 210+90 mg SCIO 469 had the greatest difference: 8.1 vs 4.1 hours for placebo (p=0.003). The positive standard, ibuprofen 400 mg, showed significant increase in time to rescue medication vs placebo (p=0.04). For the 150 mg and 210+90 mg groups, pain intensity at hours 2 to 24 was significantly reduced (p<0.05) compared to placebo. A significantly greater percentage of these subjects rated SCIO-469 treatment as “good” to “excellent” vs placebo (p<0.04). Overall there were no safety concerns identified and treatment at all SCIO-469 doses was well tolerated with the exception of a higher frequency of dizziness compared to placebo or ibuprofen; these events were mild or moderate in intensity. These results are the first clinical demonstration of significant anti-nociceptive effects in acute pain for a p38α MAPK inhibitor.

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.