P38α MAP Kinase Research Articles

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Spectral (vibrational, fluorescence), electronic features and molecular docking studies of 3-(5‑chloro-benzofuran-3-ylmethyl)-5-(4‑methoxy-phenyl)-4H-[1,2,4] triazole using experimental and DFT methods

This study examine the experimental and computational properties of 3-(5‑chloro-benzofuran-3-ylmethyl)-5-(4‑methoxy-phenyl)-4H-[1,2,4] triazole (CBT) based on the density functional theory (DFT). All the computational calculations for the investigated structure were carried out at the theoretical level B3LYP/6–311++G (d, p). The experimental and computational infrared spectra of the molecule were investigated, and complete vibrational assignments were performed on the basis of the potential energy distribution (PED) of the vibrational modes, Additionally, these compounds' solvatochromic behavior has been inspected, and values of the excited-state dipole moment are higher than the ground state for CBT and it is more polar in the excited state than in the ground state. Along with these studies, we have also conducted electronic structure characteristics. The energy gap of CBT was computed from Frontier Molecular Orbital (FMO) and it was observed at 4.91 eV Molecular Electrostatic Potential (MEP) gives the information about the charge distribution on the molecule, and Fukui function analyses have supported to find out the electronic properties of the molecule. Drug likeness studies shows that, HBA and HBD results are 4 and 1, respectively and these were agreed with the standard values. Molecular docking analysis has been conducted against inflammation species such as COX-2, α-Glucosidase, p38α MAP kinase, and EGFR with CBT. These results show good inhibitory activity against COX-2 receptor.

TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells.

A conditional knock-in mouse was generated in which the TAK1 catalytic subunit was largely replaced by the kinase-inactive TAK1[D175A] mutant in immune cells. The activation of p38α MAP kinase, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the canonical IKK complex induced by stimulation with several TLR-activating ligands was reduced in bone marrow-derived macrophages (BMDM) from TAK1[D175A] mice. TLR signalling in TAK1[D175A] BMDM was catalysed by the residual wild-type TAK1 in these cells because it was abolished by either of two structurally unrelated TAK1 inhibitors (NG25 and 5Z-7-oxozeaenol) whose off-target effects do not overlap. The secretion of inflammatory mediators and production of the mRNAs encoding these cytokines induced by TLR ligation was greatly reduced in peritoneal neutrophils or BMDM from TAK1[D175A] mice. The Pam3CSK4- or LPS-stimulated activation of MAP kinases and the canonical IKK complex, as well as cytokine secretion, was also abolished in TAK1 knock-out human THP1 monocytes or macrophages. The results establish that TAK1 protein kinase activity is required for TLR-dependent signalling and cytokine secretion in myeloid cells from mice. We discuss possible reasons why other investigators, studying myeloid mice with a conditional knock-out of TAK1 or a different conditional kinase-inactive knock-in of TAK1, reported TAK1 to be a negative regulator of LPS-signalling and cytokine production in mouse macrophages and neutrophils.

Inhibitors and Activators of the p38 Mitogen-Activated MAP Kinase (MAPK) Family as Drugs to Treat Cancer and Inflammation.

The p38 MAP kinases are a sub-family of the broad group of mitogen-activated serinethreonine protein kinases. The best-characterised, most widely expressed, and most targeted by drugs is p38α MAP kinase. This review briefly summarises the place of p38α MAP kinase in cellular signalling and discusses the structures and activity profiles of representative examples of the major classes of inhibitors and activators (both synthetic compounds and natural products) of this enzyme. Primary screening was direct in vitro inhibition of isolated p38α enzyme.

Computer-aided drug design applied to a series of pyridinyl imidazole derivatives targeting p38α MAP kinase: 2D-QSAR, docking, MD simulation, and ADMET investigations

The p38 mitogen-activated protein kinase (MAPK) is a crucial target for chronic inflammation.

Structural Diversity and Role of Phytochemicals against P38-α Mitogen-activated Protein Kinase and Epidermal Growth Factor Receptor Kinase Domain: A Privileged Computational Approach

Computational databases and tools in recent times have been proved to provide an essential aid for anticancer studies in the field of oncology. Molecular docking studies facilitate the structural diversity of plant-derived phytomolecules having anticancer properties against receptor proteins involved in cancer signaling pathways. The current study involves the investigation of phytocompounds-agasthisflavone, anacardic acid, zoapatanolide A, a purified product of the plant extract Amarogopinois546 were subjected to docking studies on p38-α MAPK and EGFR Kinase domain. The effectiveness of this study was evaluated by comparing the docking interactions of a standard drug, doxorubicin against the receptor molecules. The docking study is analyzed by compound estimated with lowest binding energy is considered to have the highest affinity towards the active site of the receptor proteins. The isolated plant compound Amarogopinois546 displayed the least binding score with a large number of hydrogen bonds and hydrophobic interactions towards the P38α MAP kinase receptor in comparison with the EGFR kinase domain. This preliminary result can strongly be supported for carrying out experimental evaluation in near future.

P38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. In males, microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. In females, the effect of p38α deficiency was divergent, exhibiting a unique transcriptional profile that included an upregulation of tissue protective genes, and a small subset of inflammatory genes that were also upregulated in males. Taken together, these results reveal a p38α-dependent sex-specific molecular pathway in microglia that is protective in CNS autoimmunity in males, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus suggesting design of future sex-specific therapeutic approaches.

Phenolic Profiling and Therapeutic Potential of Certain Isolated Compounds from Parkia roxburghii against AChE Activity as well as GABAA α5, GSK-3β, and p38α MAP-Kinase Genes.

Parkia roxburghii belongs to the family Mimosaceae; it has been used since ancient times as a cure for different health complications; such as inflammatory and gynecological diseases and hemiplegia. In this investigation, a reversed-phase-high-performance liquid chromatography (RP-HPLC) profile was carried out for P. roxburghii; also, the isolated bioactive compounds including quercetin, catechin, and biochaninA were individually and/or in combination investigated for their inhibitory effects on scopolamine-induced memory impairments in mice, implying that they have the ability to reduce the neurodegenerative effects of scopolamine and thus could be employed as a more effective therapeutic agent in the treatment of Alzheimer’s disease (AD) in humans. The possible interactions of Parkia flavonoids with acetylcholinesterase (AChE), γ-aminobutyric acid A receptor, alpha5 (GABAA α5), glycogen synthase kinase-3 (GSK-3), p38 mitogen-activated protein kinase (p38MAP-kinase), signal-regulated kinase (ERK), and protein-serine/threonine kinase (Akt) were then determined using molecular docking.

Binding mechanism of inhibitors to p38α MAP kinase deciphered by using multiple replica Gaussian accelerated molecular dynamics and calculations of binding free energies

The p38α MAP Kinase has been an important target of drug design for treatment of inflammatory diseases and cancers. This work applies multiple replica Gaussian accelerated molecular dynamics (MR-GaMD) simulations and the molecular mechanics generalized Born surface area (MM-GBSA) method to probe the binding mechanism of inhibitors L51, R24 and 1AU to p38α. Dynamics analyses show that inhibitor bindings exert significant effect on conformational changes of the active helix α2 and the conserved DFG loop. The rank of binding free energies calculated with MM-GBSA not only agrees well with that determined by the experimental IC50 values but also suggests that mutual compensation between the enthalpy and entropy changes can improve binding of inhibitors to p38α. The analyses of free energy landscapes indicate that the L51, R24 and 1AU bound p38α display a DFG-out conformation. The residue-based free energy decomposition method is used to evaluate contributions of separate residues to the inhibitor-p38α binding and the results imply that residues V30, V38, L74, L75, I84, T106, H107, L108, M109, L167, F169 and D168 can be utilized as efficient targets of potent inhibitors toward p38α.

P38 MAP kinase signaling in microglia plays a sex-specific protective role in CNS autoimmunity and regulates microglial transcriptional states

Abstract Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. MS is three times more common in women, yet more severe in men. The mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T cells, but CNS-infiltrating myeloid cells are key effector cells driving pathology. The role of CNS-resident microglial cells is less clear. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. Here, we sought to define the mechanisms responsible, using genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Single-cell RNAseq revealed a diversity of microglial states, connected by distinct convergent transcriptional trajectories. Microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglia, with downregulation of regulatory genes such as Atf3, Rgs1, and Btg2, and upregulation of inflammatory genes such as Cd74, Trem2, and MHC class I and II. These results reveal a p38α-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, suggesting that autoimmunity in males and females may be driven by distinct cellular and molecular pathways.

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

The discovery and evaluation of 3-amino-2(1H)-pyrazinones as a novel series of selective p38α MAP kinase inhibitors

The discovery and optimisation of a novel series of potent and selective p38α inhibitors is described. Evaluating the structure-activity relationship of an aminoalkyl substituent at the 3 position of the 2(1H)-pyrazinone core, p38α potency was increased 20000-fold. The most advanced compound (25) demonstrated excellent in vivo properties suitable for an inhaled route of administration.

Bioisosteric Replacement of Arylamide-Linked Spine Residues with N-Acylhydrazones and Selenophenes as a Design Strategy to Novel Dibenzosuberone Derivatives as Type I 1/2 p38α MAP Kinase Inhibitors.

The recent disclosure of type I 1/2 inhibitors for p38α MAPK demonstrated how the stabilization of the R-spine can be used as a strategy to greatly increase the target residence time (TRT) of inhibitors. Herein, for the first time, we describe N-acylhydrazone and selenophene residues as spine motifs, yielding metabolically stable inhibitors with high potency on enzymatic, NanoBRET, and whole blood assays, improved metabolic stability, and prolonged TRT.

Design and Synthesis of Novel 1-hydroxy-2,4,5-triaryl Imidazole Derivatives as Anti-cytokine Agents.

Among recent advances in the identification of anti-inflammation agents, anti-cytokines (like Interleukin-1), related to p38 MAPK families play an important role; Here in we designed new effective and low toxic anti-cytokine agents based on 1-hydroxy-2,4,5-triaryl imidazole derivatives. The reaction of oximoinoketone intermediate with ten different aromatic aldehyde and ammonium acetate in refluxing acetic acid condition give imidazole derived product, the IL-1β inhibitory assay were performed on Human PBMCs (peripheral blood mononuclear cells) using an enzyme-linked immunosorbent assay (ELISA) kit and then in computational part the binding mode of the best compound was accomplished by docking in Crystal structure of p38 MAP kinase (PDB ID: 1A9U) compared with SB202190 as standard drug. All compounds were synthesized and evaluated in biological assay showing the inhibitory activity from 28% to 82% compared to SB202190 and binding mode analysis revealed that the hydrogen-bond interactions with residues (Met109, Val30) were key point in inhibitor binding. Compound 5g clearly proved the best inhibitory action and could be further utilized for designing newer anti-cytokine agents and p38α MAP kinase potentially inhibitory action.

Conformational selection vs. induced fit: insights into the binding mechanisms of p38α MAP Kinase inhibitors.

The conformational dynamics of a kinase's activation loop have been challenging to assess due to the activation loop's intrinsic flexibility. To directly probe the conformational equilibrium of the activation loop of mitogen-activated protein kinase p38α, we present an approach based on site-directed spin labeling, electron paramagnetic resonance (EPR) distance restraints, and multilateration. We demonstrate that the activation loop of apo p38α resides in a highly flexible equilibrium state and we reveal that binding of small molecules significantly alters this equilibrium and the populated sub-states.

Promiscuity analysis of a kinase panel screen with designated p38 alpha inhibitors

Protein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome.

Synthesis and biological evaluation of novel benzo[c][1,2,5]thiadiazol-5-yl and thieno[3,2-c]- pyridin-2-yl imidazole derivatives as ALK5 inhibitors.

Transforming growth factor (TGF-β), a key mediator of tumor growth and metastasis, has been recognized as an important cancer drug target. A series of benzo[c][1,2,5]thiadiazol-5-yl imidazoles (14a-g) and thieno[3,2-c]-pyridin-2-yl imidazoles (20a-g) were designed, synthesized, and evaluated for their activin receptor-like kinase 5 (ALK5) activities. Among these compounds, 14c showed the highest activity (IC50 = 0.008 μM) against ALK5 kinase, which was 16.1-fold and 1.8-fold higher than those of positive control compounds LY-2157299 (IC50 = 0.129 μM) and EW-7197 (IC50 = 0.014 μM), respectively. Compound 14g (350) showed the highest selectivity index of ALK5 against p38α MAP kinase, which was significantly higher than that of positive control compounds LY-2157299 (4) and EW-7197 (211). The inhibitory effects of compound 14c on TGF-β-induced Smad signaling and cell motility were studied in SPC-A1, HepG2 and HUVEC cells using western blot analysis and wound healing assay. ADMET prediction analysis showed that compounds 14c and 14g had good pharmacokinetics and drug-likeness behaviors.

Dibenzepinones, dibenzoxepines and benzosuberones based p38α MAP kinase inhibitors: Their pharmacophore modelling, 3D-QSAR and docking studies

In the present study, a series of dibenzepinones, dibenzoxepines, and benzosuberones targeting p38α MAP kinase were subjected to pharmacophore modelling, 3D-QSAR and molecular docking studies. The IC50 values for these 67 compounds ranged between 0.003 and 6.80 μM. A five-point model (DDHHR.8) was generated using these compounds. This model was found to be statistically significant and was found to have high correlation (R2 = 0.98), cross-validation coefficient (Q2 = 0.95) and F (330) values at six component PLS factor. Tests were performed to ascertain the efficacy of the generated model. These tests included external validation, Tropsha's test for predictive ability, Y-randomisation test and domain of applicability (APD). In order to check the restrictivity of the model, enrichment studies were performed with inactive compounds by using decoy set molecules. To evaluate the effectiveness of the docking protocol, the co-crystallised ligand was extracted from the ligand-binding domain of the protein and was re-docked into the same position. Both the conformers were then superimposed, suggesting satisfactory docking parameters with an RMSD value of less than 1.0 Å (0.853 Å). A 10 ns molecular dynamics simulation confirmed the docking results of the 3UVP-ligand complex and the presumed active conformation. The outcome of the present study provides insight into the molecular features that promote bioactivity and can be exploited for the prediction of novel potent p38α MAP kinase inhibitors before carrying out their synthesis and anticancer evaluation.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

P38 MAP kinase signaling in microglia plays a sex-specific protective role in CNS autoimmunity

Abstract Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is 3 times more common in women, but the underlying mechanisms remain unknown. MS is initiated by autoreactive T helper cells, but CNS-infiltrating inflammatory myeloid cells are the main effector cells driving disease pathology. The role of CNS-resident macrophage-like microglial cells is unclear. We have previously shown that genetic ablation of p38α MAP kinase (p38) in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE). Strikingly, protection was seen in female mice, and not males. Here, we sought to precisely define the mechanisms responsible. To this end, we used multiple genetic approaches and bone marrow chimeras to ablate p38 in microglial cells, peripheral myeloid cells, or both. Deletion of p38 in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38 in the periphery was protective in both sexes, while deletion of p38 in microglia exacerbated EAE only in males. Transcriptional profiling of microglia isolated from the CNS at peak of EAE revealed male-specific p38-dependent transcriptional modules with anti-inflammatory/tissue reparatory functionality. Taken together, these results reveal the presence of a p38-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, suggesting that autoimmunity in males and females may be driven by distinct cellular and molecular pathways, thus informing the design of novel future sex-specific therapeutic approaches.