P38α Mitogen-activated Protein Kinase Inhibitors Research Articles

Design, Synthesis, and Molecular Docking of Novel Miscellaneous Chalcones as p38α Mitogen-Activated Protein Kinase Inhibitors.

New chalcones were synthesized and evaluated to serve as p38-α type of mitogen-activated protein kinase (MAPK) inhibitors. According to the National Cancer Institute, the findings indicated that at a 10 μM dosage, compounds 3a and 6 were the most active among all the compounds examined, with mean growth inhibition% of 94.83 and 58.49, respectively. In 5-dose testing, they showed anticancer activity in the micro-molar range with GI50 in the range of 1.41-46.1 and 2.07-31.3 μM, respectively. Besides, powerful activity, especially against the leukaemia cell lines and good selectivity to cancer cells compared to normal PCS-800-017 with a selectivity index=12.41 and 23.77, respectively. Compounds 3a and 6 inhibited p38α MAPK with IC50 values of 0.1462±0.0063 and 0.4356±0.0189 μM, correspondingly. 3a showed good inhibition for HL-60(TB) cells and induced cell cycle arrest in HL-60(TB) cells at the G2/M phase. Besides, it elevated the total apoptosis by 14.68-fold and increased the caspase-3 level by 3.52-fold compared with doxorubicin, which raised it by 4.30-fold, inducing apoptosis by acting as caspase-dependent inducers. These results suggest that 3a is a promising antiproliferative and p38α MAPK inhibitor, confirmed by molecular docking with high compatibility 3a with the p38α MAPK binding site.

CNS-Active p38α MAPK Inhibitors for the Management of Neuroinflammatory Diseases: Medicinal Chemical Properties and Therapeutic Capabilities.

During the last two decades, many p38α mitogen-activated protein kinase (p38α MAPK) inhibitors have been developed and tested in preclinical/clinical studies for the treatment of various disorders, especially problems with the origin of inflammation. Previous studies strongly suggest the involvement of the p38α MAPK pathway in the pathogenesis of neurodegenerative disorders. Despite the significant progress made in this field, so far no studies have focused on p38α MAPK inhibitors that have the capability to be used for the treatment of neurodegenerative disorders. In the present review, we evaluated a wide range of well-known p38α MAPK inhibitors (more than 140 small molecules) by measuring key physicochemical parameters to identify those capable of successfully crossing the blood-brain barrier (BBB). As a result, we identify about 50 naturally occurring and synthetic p38α MAPK inhibitors with high potential to cross the BBB, which can be further explored in the future for the treatment of neurodegenerative disorders. In addition, a detailed analysis of the previously released X-ray crystal structure of the inhibitors in the active site of the p38α MAPK enzyme revealed that some residues such as Met109 play a critical role in the occurrence of effective interactions by constructing strong H-bonds. This study can encourage scientists to focus more on the design, production, and biological evaluation of new central nervous system (CNS)-active p38α MAPK inhibitors in the future.

Multi-stage virtual screening of natural products against p38α mitogen-activated protein kinase: predictive modeling by machine learning, docking study and molecular dynamics simulation

p38α is a mitogen-activated protein kinase (MAPK), and the signaling pathways involved are closely related to the inflammation, apoptosis and differentiation of cells, which also makes it an attractive target for drug discovery. With the high efficiency and low cost, virtual screening technology is becoming an indispensable part of drug development. In this study, a novel multi-stage virtual screening method based on machine learning, molecular docking and molecular dynamics simulation was developed to identify p38α MAPK inhibitors from natural products in ZINC database, which improves the prediction accuracy by considering and utilizing both ligand and receptor information compared to any individual approach. Ultimately, we screened out two candidate inhibitors with acceptable ADMET properties (ZINC4260400 and ZINC8300300). Among the generated machine learning models, Random Forest (RF) and Support Vector Machine (SVM) performed better, with the area under the receiver operating characteristic curve (AUC) values of 0.932 and 0.931 on the test set, as well as 0.834 and 0.850 on the external validation set. In addition, the results of molecular docking and ADMET prediction showed that two compounds with appropriate pharmacokinetic properties had binding free energies less than −8.0 kcal/mol for the target protein, and the results of molecular dynamics simulations further confirmed that they were stable during the process of inhibition.

Integration of In Silico Strategies for Drug Repositioning towards P38α Mitogen-Activated Protein Kinase (MAPK) at the Allosteric Site.

P38α mitogen-activated protein kinase (p38α MAPK), one of the p38 MAPK isoforms participating in a signaling cascade, has been identified for its pivotal role in the regulation of physiological processes such as cell proliferation, differentiation, survival, and death. Herein, by shedding light on docking- and 100-ns dynamic-based screening from 3210 FDA-approved drugs, we found that lomitapide (a lipid-lowering agent) and nilotinib (a Bcr-Abl fusion protein inhibitor) could alternatively inhibit phosphorylation of p38α MAPK at the allosteric site. All-atom molecular dynamics simulations and free energy calculations including end-point and QM-based ONIOM methods revealed that the binding affinity of the two screened drugs exhibited a comparable level as the known p38α MAPK inhibitor (BIRB796), suggesting the high potential of being a novel p38α MAPK inhibitor. In addition, noncovalent contacts and the number of hydrogen bonds were found to be corresponding with the great binding recognition. Key influential amino acids were mostly hydrophobic residues, while the two charged residues including E71 and D168 were considered crucial ones due to their ability to form very strong H-bonds with the focused drugs. Altogether, our contributions obtained here could be theoretical guidance for further conducting experimental-based preclinical studies necessary for developing therapeutic agents targeting p38α MAPK.

Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors

Stress-induced p38α mitogen-activated protein (MAP) kinase activation modulates cytokine overproduction and is associated with neuroinflammation and neurodegeneration. As a potential therapeutic approach, novel Skepinone-based p38α MAP kinase inhibitors were optimized to cross the blood-brain barrier via either amino acid transporters or hydrophobic diffusion. To enhance absorption from the oral route, we used methyl ester prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (43; p38α, IC50 = 5.5 nM) and 4-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC50 = 12 nM) had brain-to-plasma ratios of 1.4 and 4.4, respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (p38α, IC50 = 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

Identification of an allosteric and Smad3-selective inhibitor of p38αMAPK using a substrate-based approach

p38α mitogen activated protein kinase (MAPK) plays important roles in multiple cellular functions by phosphorylating a wide variety of substrates, and therefore, p38α MAPK has been considered as an important drug target. In this study, we designed peptide-based inhibitors for p38α MAPK, which can only inhibit the Smad3 phosphorylation specifically, by targeting the KIM binding site of p38α MAPK. Peptide 6 showed a significant inhibitory potency for the Smad3 phosphorylation by p38α MAPK. Peptide 6 showed no ATP dependency, and did not inhibit the phosphorylation of other substrates by p38α MAPK. The discovery of peptide 6 by targeting the KIM binding site likely provide an opportunity for the discovery of a novel class of allosteric and substrate-specific p38α MAPK inhibitors.

A selectivity study of polysubstituted pyridinylimidazoles as dual inhibitors of JNK3 and p38α MAPK based on 3D-QSAR, molecular docking, and molecular dynamics simulation

Within the mitogen-activated protein kinase (MAPK) 1 family, the two kinases of c-Jun N-terminal kinase 3 (JNK3) and p38α MAPK have emerged in the last decades as particularly attractive therapeutic targets due to their implication in several neurodegenerative pathologic conditions. In this study, the structure and activity relationship of 60 dual JNK3/p38α MAPK inhibitors was explored; three-dimensional quantitative structure-activity relationship (3D-QSAR), including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis fields (CoMSIA), was performed. From the data we got, the 3D-QSAR model (CoMFAJNK3 with q2 is 0.642, r2 is 0.958; CoMSIAJNK3 with q2 is 0.660, r2 is 0.963; CoMFAp38α with q2 is 0.605, r2 is 0.980; CoMSIAp38α with q2 is 0.608, r2 is 0.970) had good predictability. Molecular docking further revealed the binding mode of inhibitors to JNK3/p38α MAPK. The results of 3D-QSAR, molecular docking, and molecular dynamics simulation also provided guidance for the discovery of new dual inhibitors of JNK3 and p38α MAPK. Finally, 10 novel compounds with good potential activity and ADME/T profile were designed. Molecular dynamics simulation results validated that Met149/Lys 93/Gln 155 (JNK3) and Met109/Lys53 (p38α) located in the active site play a key role for novel dual inhibitors.

P38α MAP kinase inhibitors to overcome EGFR tertiary C797S point mutation associated with osimertinib in non-small cell lung cancer (NSCLC): emergence of fourth-generation EGFR inhibitor

The third-generation EGFR (epidermal growth factor receptor) inhibitors selectively and irreversibly target EGFR-T790M and other activating EGFR mutations. Osimertinib is the only FDA-approved third-generation inhibitor, which has a good potency against the EGFR-T790M mutant with minimal toxicities and excellent selectivity for wild-type EGFR. EGFR tertiary Cys797 to Ser797 (C797S) point mutation emanate rapidly after the treatment of osimertinib, which is an undruggable mutation to all three existing generation drugs. Recently, trisubstituted imidazoles were reported based on an off-target hit of a p38α MAPK (mitogen-activated protein kinase) inhibitor as the fourth-generation EGFR-TKIs to overcome the C797S resistance by inhibiting the clinically relevant triple mutant kinase L858R/T790M/C797 EGFR. Here, we are reporting the clinical trial p38α MAPK kinase inhibitors SD-06, Amgen 16, RWJ67657 and SCIO-323 as L858R/T790M/C797S EGFR TK inhibitors to overcome the problem of drug resistance in non-small cell lung cancer (NSCLC). Communicated by Ramaswamy H. Sarma

Co-crystal structure determination and cellular evaluation of 1,4-dihydropyrazolo[4,3-c] [1,2] benzothiazine 5,5-dioxide p38α MAPK inhibitors

p38α mitogen-activated protein kinase (MAPK) is an attracting pharmacological target in inflammatory diseases and cancer. Searching for new and more efficient p38-MAPK inhibitors, two recently developed pyrazolobenzothiazine-based (COXP4M12 and COXH11) compounds were investigated in this study using a cellular model of p38 activation. This consisted of HT29 human colorectal adenocarcinoma cells exposed to H2O2 or lipopolysaccharide (LPS).Immunoblot data confirmed the inhibitory effect of COXP4M12 and COXH11 on p38 substrate phosphorylation (MAPK-APK2 and ATF2 transcription factor). Compound cytotoxicity was very low and apparent efficacy of these inhibitors was comparable with that of SB203580, a commercially available type I inhibitor of p38. All these compounds also inhibit upstream kinases that promote p38-MAPK phosphorylation and co-activate the stress-activated protein kinase JNK, while ERK1/2 MAPK phosphorylation was unaffected.Compound-target kinase interaction was investigated by means of co-crystallization experiments that provided further structural and molecular insight on the inhibitory mechanism and optimization strategy of this new class of p38-MAPK inhibitors.

Human p38α mitogen-activated protein kinase in the Asp168-Phe169-Gly170-in (DFG-in) state can bind allosteric inhibitor Doramapimod

Doramapimod (BIRB-796) is widely recognized as one of the most potent and selective type II inhibitors of human p38α mitogen-activated protein kinase (MAPK); however, the understanding of its binding mechanism remains incomplete. Previous studies indicated high affinity of the ligand to a so-called allosteric pocket revealed only in the ‘out’ state of the DFG motif (i.e. Asp168-Phe169-Gly170) when Phe169 becomes fully exposed to the solvent. The possibility of alternative binding in the DFG-in state was hypothesized, but the molecular mechanism was not known. Methods of bioinformatics, docking and long-time scale classical and accelerated molecular dynamics have been applied to study the interaction of Doramapimod with the human p38α MAPK. It was shown that Doramapimod can bind to the protein even when the Phe169 is fully buried inside the allosteric pocket and the kinase activation loop is in the DFG-in state. Orientation of the inhibitor in such a complex is significantly different from that in the known crystallographic complex formed by the kinase in the DFG-out state; however, the Doramapimod’s binding is followed by the ligand-induced conformational changes, which finally improve accommodation of the inhibitor. Molecular modelling has confirmed that Doramapimod combines the features of type I and II inhibitors of p38α MAPK, i.e. can directly and indirectly compete with the ATP binding. It can be concluded that optimization of the initial binding in the DFG-in state and the final accommodation in the DFG-out state should be both considered at designing novel efficient type II inhibitors of MAPK and homologous proteins.Communicated by Ramaswamy H. Sarma

Structural Optimization of a Pyridinylimidazole Scaffold: Shifting the Selectivity from p38α Mitogen-Activated Protein Kinase to c-Jun N-Terminal Kinase 3.

Starting from known p38α mitogen-activated protein kinase (MAPK) inhibitors, a series of inhibitors of the c-Jun N-terminal kinase (JNK) 3 was obtained. Altering the substitution pattern of the pyridinylimidazole scaffold proved to be effective in shifting the inhibitory activity from the original target p38α MAPK to the closely related JNK3. In particular, a significant improvement for JNK3 selectivity could be achieved by addressing the hydrophobic region I with a small methyl group. Furthermore, additional structural modifications permitted to explore structure–activity relationships. The most potent inhibitor 4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)-N-(4-morpholinophenyl)pyridin-2-amine showed an IC50 value for the JNK3 in the low triple digit nanomolar range and its binding mode was confirmed by X-ray crystallography.

An isoform-selective p38α mitogen-activated protein kinase inhibitor rescues early entorhinal cortex dysfunctions in a mouse model of Alzheimer's disease.

Neuroinflammation is a fundamental mechanism in Alzheimer's disease (AD) progression. The stress-induced activation of the p38α mitogen-activated protein kinase (MAPK) leads to increased production of proinflammatory cytokines and neurodegeneration. We investigated the effects of an isoform selective p38α MAPK inhibitor, MW01-18-150SRM (MW150), administered at 2.5mg/kg/d (i.p.; 14days) on early entorhinal cortex (EC) alterations in an AD mouse model carrying human mutations of the amyloid precursor protein (mhAPP). We used electrophysiological analyses with long-term potentiation induction in EC-containing brain slices and EC-relevant associative memory tasks. We found that MW150 was capable of rescuing long-term potentiation in 2-month old mhAPP mice. Acute delivery of MW150 to brain slices was similarly effective in rescuing long-term potentiation, with a comparable efficacy to that of the widely used multikinase inhibitor SB203580. MW150-treated mhAPP mice demonstrated improved ability to discriminate novel associations between objects and their position/context. Our findings suggest that the selective inhibition of the stress-activated p38α MAPK with MW150 can attenuate the EC dysfunctions associated with neuroinflammation in an early stage of AD progression.

A Diverse and Versatile Regiospecific Synthesis of Tetrasubstituted Alkylsulfanylimidazoles as p38α Mitogen-Activated Protein Kinase Inhibitors.

An alternative strategy for the synthesis of 1-aryl- and 1-alkyl-2-methylsulfanyl-4-(4-fluorophenyl)-5-(pyridin-4-yl)imidazoles as potential p38α mitogen-activated protein kinase inhibitors is reported. The regioselective N-substitution of the imidazole ring was achieved by treatment of α-aminoketones with different aryl or alkyl isothiocyanates. In contrast to previously published synthesis routes starting from 2-amino-4-methylpyridine, the presented route is characterized by a higher flexibility and a lower number of steps. This strategy was also applied to access 1-alkyl-2-methylsulfanyl-5-(4-fluorophenyl)-4-(pyridin-4-yl)imidazoles in six steps starting from 2-chloro-4-methylpyridine.

A Comprehensive Structural Overview of p38α Mitogen-Activated Protein Kinase in Complex with ATP-Site and Non-ATP-Site Binders.

Herein we review all the currently available ATP-site and non-ATP-site ligands bound to p38α mitogen-activated protein kinase (MAPK) available in the RCSB Protein Data Bank (PDB). The co-crystallized inhibitors have been classified into different families according to their experimental binding mode and chemical structure, and the ligand-protein interactions are discussed using the most representative compounds. This systematic structural analysis could provide some take-home lessons for drug discovery programs aimed at the rational identification and optimization of new p38α MAPK inhibitors.

From 2-Alkylsulfanylimidazoles to 2-Alkylimidazoles: An Approach towards Metabolically More Stable p38α MAP Kinase Inhibitors.

In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine) undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38α mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group resulted in 2-alkylimidazole derivatives 1 and 2, respectively, having a remarkably improved metabolic stability. The 2-alkylimidazole analogs 1 and 2 showed 20% and 10% biotransformation after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold increased binding affinity towards the target kinase as well as similar in vitro potency and ex vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. For example, 2-alkylimidazole 2, the analog of LN950, inhibits both the p38α MAP kinase as well as the LPS-stimulated tumor necrosis factor-α release from human whole blood in the low double-digit nanomolar range.

Discovery of naphthyl-N-acylhydrazone p38α MAPK inhibitors with in vivo anti-inflammatory and anti-TNF-α activity.

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti-inflammatory profile of new naphthyl-N-acylhydrazone derivatives using animal models. Although all tested compounds (3a-d) have been characterized as p38α MAPK inhibitors and have showed in vivo anti-inflammatory action, LASSBio-1824 (3b) presented the best performance as p38α MAPK inhibitor, with IC50 =4.45μm, and also demonstrated to be the most promising anti-inflammatory prototype, with good in vivo anti-TNF-α profile after oral administration.

Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase

Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors.

Oxidants induce a corticosteroid-insensitive phosphorylation of histone 3 at serine 10 in monocytes.

Oxidative stress enhances inflammation and reduces the effectiveness of corticosteroids, but the inflammatory signalling pathways induced by oxidants remain ill-defined. Phosphorylation of histone 3 at serine 10 (H3-Pser10) marks out a subset of inflammatory genes for transcription, several of which are induced in oxidant-associated inflammation. However, the influence of oxidants or of corticosteroids on this modification remains unknown. We assessed the regulation of H3-Pser10 by oxidants and lipopolysaccharide (LPS) in human blood monocytes and lung macrophages and the effectiveness of its abolition in controlling inflammatory gene expression in cells from asthmatic subjects compared to corticosteroids alone. Both oxidants and LPS promoted the induction of H3-Pser10 which was unaffected by corticosteroids. The induction of H3-Pser10 was mediated through p38α mitogen-activated protein kinase (MAPK) and IκB kinase 2 (IKK-2) signalling. Consequently, inhibitors of p38α MAPK or IKK-2 used in combination with dexamethasone were more effective at controlling inflammatory gene expression from monocytes and lung macrophages from asthmatic patients than the corticosteroid alone. Therefore, reduction of H3-Pser10 by inhibition of p38α MAPK or of IKK-2 may provide greater anti-inflammatory control than corticosteroids alone in oxidant-associated inflammation such as severe asthma.

A Comprehensive Structural Overview of p38α MAPK in Complex with Type I Inhibitors.

p38α mitogen-activated protein kinase (MAPK) is a well-recognized therapeutic target for the treatment of autoimmune and inflammatory diseases. Over the past two decades, tremendous efforts have been focused on the discovery and development of small-molecule p38α MAPK inhibitors, although currently no drugs targeting this protein are clinically available. Therefore, the identification of novel chemotypes that are able to inhibit p38α MAPK function is still of great therapeutic significance. With the objective to support drug discovery programs aimed at identifying new immunomodulators acting on p38α MAPK, herein we present a complete overview of the available crystal structures of this protein in complex with ATP-site type I inhibitors. The 85 available complexes are classified by chemotype and experimental binding mode, and the ligand-protein interactions are discussed using the most representative inhibitors. The type and frequency of key inhibitor features are analyzed to give a final summary of the chemical requirements of promising p38α MAPK inhibitors. The proposed pharmacophore can be exploited to enhance the opportunities to identify novel type I inhibitors of p38α MAPK.

Design, Synthesis, and Biological Evaluation of Tetra-Substituted Thiophenes as Inhibitors of p38α MAPK.

p38α mitogen-activated protein kinase (MAPK) plays a role in several cellular processes and consequently has been a therapeutic target in inflammatory diseases, cancer, and cardiovascular disease. A number of known p38α MAPK inhibitors contain vicinal 4-fluorophenyl/4-pyridyl rings connected to either a 5- or 6-membered heterocycle. In this study, a small library of substituted thiophene-based compounds bearing the vicinal 4-fluorophenyl/4-pyridyl rings was designed using computational docking as a visualisation tool. Compounds were synthesised and evaluated in a fluorescence polarisation binding assay. The synthesised analogues had a higher binding affinity to the active phosphorylated form of p38α MAPK than the inactive nonphosphorylated form of the protein. 4-(2-(4-fluorophenyl)thiophen-3-yl)pyridine had a Ki value of 0.6 μm to active p38α MAPK highlighting that substitution of the core ring to a thiophene retains affinity to the enzyme and can be utilised in p38α MAPK inhibitors. This compound was further elaborated using a substituted phenyl ring in order to probe the second hydrophobic pocket. Many of these analogues exhibited low micromolar affinity to active p38α MAPK. The suppression of neonatal rat fibroblast collagen synthesis was also observed suggesting that further development of these compounds may lead to potential therapeutics having cardioprotective properties.