Mitogen-activated Protein Kinase Inhibitor Research Articles

Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons

Deficits in axonal transport are one of the earliest pathological outcomes in several models of amyotrophic lateral sclerosis (ALS), including SOD1G93A mice. Evidence suggests that rescuing these deficits prevents disease progression, stops denervation, and extends survival. Kinase inhibitors have been previously identified as transport enhancers, and are being investigated as potential therapies for ALS. For example, inhibitors of p38 mitogen-activated protein kinase and insulin growth factor receptor 1 have been shown to rescue axonal transport deficits in vivo in symptomatic SOD1G93A mice. In this work, we investigated the impact of RET, the tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), as a modifier of axonal transport. We identified the fundamental interplay between RET signalling and axonal transport in both wild-type and SOD1G93A motor neurons in vitro. We demonstrated that blockade of RET signalling using pharmacological inhibitors and genetic knockdown enhances signalling endosome transport in wild-type motor neurons and uncovered a divergence in the response of primary motor neurons to GDNF compared with cell lines. Finally, we showed that inhibition of the GDNF-RET signalling axis rescues in vivo transport deficits in early symptomatic SOD1G93A mice, promoting RET as a potential therapeutic target in the treatment of ALS.

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC50 = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca2+]i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

New biomarker paves the way for a clinical trial for neurodegeneration in Langerhans cell histiocytosis.

Neurodegeneration in Langerhans cell histiocytosis (ND-LCH) is a major clinical issue requiring urgent resolution. Sveijer et al. showed that plasma neurofilament light protein is a promising biomarker for screening patients with ND-LCH and determining the therapeutic effect of a mitogen-activated protein kinase inhibitor. Therefore, this can be a powerful tool for conducting clinical trials for ND-LCH. Commentary on: Sveijer M, von Bahr Greenwood T, Jädersten M, Kvedaraite E, Zetterberg H, Blennow K, et al. Screening for neurodegeneration in Langerhans cell histiocytosis with neurofilament light in plasma. Br J Haematol. 2022;198:722-729.

Pharmacokinetics, Pharmacodynamics and Antiviral Efficacy of the MEK Inhibitor Zapnometinib in Animal Models and in Humans.

The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib (12.5 mg/kg p. o. twice daily, 8 h interval). Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (Cmax) was reached between 2–4 h after administration with an elimination half-life of 4–5 h in dogs, 8 h in mice or hamsters and 19 h in human subjects. Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 μg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%–80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 μg/ml zapnometinib in further clinical studies.

Abstract 5333: Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition

Abstract Background: Oncogenic KRAS is present in almost all cases of pancreatic ductal adenocarcinoma (PDAC). However, targeting KRAS or its canonical signaling cascades, especially the mitogen-activated protein kinase (MAPK) pathway, remains clinically unsuccessful. Targeting ERK kinases has recently emerged as a promising therapeutic strategy and combinatorial strategies should be developed. Aim: Identify adaptive mechanisms to ERK inhibition that can be co-targeted to achieve effective tumor inhibition in multiple patient-derived xenograft (PDX) models. Methods: Reverse-phase protein array (RPPA) was used in early-passage patient-derived cell lines (PDCLs) to identify potential resistance mechanisms. These were confirmed using RNA interference and overexpression in PDAC cell lines and PDCLs. The promising combinations were tested in 30 early-passaged PDAC PDXs. Results: RPPA showed dramatic downregulation of DUSP4 and DUSP6 phosphatases following MEK and ERK inhibition, which coincided with upregulation of phospho-HER2 and -HER3. Knockdown of DUSP6, but not DUSP4, was sufficient in phosphorylation of HER2. Conversely, overexpression of DUSP6 curbed HER2 and ERK activation. Downregulation of DUSP4 and DUSP6 induced by ulixertinib, an ERK inhibitor now in clinical development, was reversed by bortezomib, suggesting DUSP4 and DUSP6 are proteosomally degraded. Combined ulixertinib plus PI3K inhibitor copanlisib, or pan-HER inhibitor afatinib slowed but did not arrest PDX tumor growth in vivo, and addition of gemcitabine was required to achieve tumor regression or durable growth arrest. Alternatively, ulixertinib or MEK inhibitor (trametinib) in combination with trastuzumab deruxtecan (DS-8201a), an anti-HER2 conjugated topoisomerase I inhibitor, were extremely effective, leading to complete and durable tumor regression for all tested PDX models. We showed that upregulation of HER2 expression following MEK or ERK inhibitor treatment provides a conduit for enhanced internalization of DS-8201a. Conclusions: Our study provided novel mechanistic insight on how PDAC cells evade MAPK inhibition via enhancing HER2 signaling. We demonstrated that the combination of MEK or ERK inhibitor plus DS-8201a is extremely effective, leading to complete tumor regression in multiple PDAC PDX models. This combination should be advanced as a clinical trial for PDAC patients. Keys: DS-8201a, DUSP6, HER2, KRAS, ulixertinib, pancreatic ductal adenocarcinoma Citation Format: Ashenafi Shiferaw Bulle, Kuljeet Seehar, Sapana Bansod, Yali Chen, Chen Hung-Po, Paarth B. Dodhiawala, Lin Li, Vikas Somani, Jacqueline Mudd, Ryan C. Fields, Deborah Knoerzer, Andrea Wang-Gillam, Lim Kian-Huat. Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5333.

Abstract 6366: A novel small molecule inhibitor of MAPK and PI3K ameliorates disease phenotypes in myelofibrosis

Abstract Myelofibrosis is a myeloproliferative neoplasm (MPN) caused primarily by mutations in hematopoietic stem and progenitor cells (HSPCs) that activate Janus kinase 2 (JAK2). Central roles for JAK2 signaling in myelofibrosis (MF) have been established, leading to marked extramedullary hematopoiesis, splenomegaly, and bone marrow fibrosis, but the use of JAK inhibitors has shown limited ability to produce durable remissions in most patient populations. Bypass or resistance mechanisms to JAK inhibition have been linked to concomitant activation of compensatory phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways, and while genetic and functional studies have not fully defined the mechanisms for transformation and maintenance of the proliferative state in MF, targeting multiple signaling pathways will be required to alter the disease course and improve therapeutic outcomes. Treatments using kinase inhibitor combinations remain a challenge clinically, and trials have struggled to create positive balance between gains in survival, therapeutic efficacy, and dose-limiting toxicity. A multi-functional kinase inhibitor was developed to deliver consistent and synergistic dosing ratios against multiple targets simultaneously, thereby blocking compensatory oncogenic signaling pathways while minimizing the prospect for adverse effects. Using synthetic medicinal chemistry, we present a potent and selective, orally bioavailable, single-molecule multi-targeted kinase inhibitor (LP-182) against MAPK and PI3K/mTOR signaling pathways. We demonstrate selectivity and therapeutic efficacy through in vitro screening of kinome inhibition, reduction of cell growth and downstream kinase activation, renormalization of immune cell populations, amelioration of splenomegaly and bone marrow fibrosis, and improved survival in the myeloproliferative leukemia oncogene (MPLW515L) mouse model of myelofibrosis. Furthermore, treatment with LP-182 was well tolerated showing no observable pharmacotoxicity, and combination with the JAK inhibitor Ruxolitinib also showed ~35% reduction in spleen size as compared to either treatment alone with a ~60% spleen volume reduction compared to vehicle treatment, in the JAK2+/V617F mutant mouse model. Overall, these data suggest that in vivo bioavailability and distribution of LP-182 to secondary lymphoid tissues attenuates MAPK and PI3K signaling to alleviate disease phenotypes in animal models of MF. Simultaneous targeting of both MAPK and PI3K pathways with LP-182 alone or in combination with JAK inhibition provides the potential for significant new opportunities to improve the clinical outcome of MF patients. Citation Format: Christopher A. Bonham, Youngsoon Jang, Kevin Heist, Amanda Welton, Tanner Robison, Kathryn E. Luker, Gary D. Luker, Winston Y. Lee, Thomas L. Chenevert, Marcian Van Dort, Brian D. Ross. A novel small molecule inhibitor of MAPK and PI3K ameliorates disease phenotypes in myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6366.

Abstract 3365: Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone

Abstract Overactivation of mitogen-activated protein kinase (MAPK) signaling is a frequently observed driver of many cancers. Targeted MAPK pathway inhibitors have been approved as therapies in patient populations with genomic alterations to MAPK pathway genes (e.g. dabrafenib in BRAF-mutant melanoma), with multiple other MAPK-targeted therapies in development. We have previously demonstrated that cell lines with MAPK pathway genomic alterations show increased sensitivity to ASTX029, our potent and selective dual-mechanism ERK inhibitor which is currently undergoing clinical development. However, MAPK oncogene status alone is not a universal indicator of sensitivity, nor is it clear that genomic markers of sensitivity to single agent inhibition represent the optimal approach to predicting response to MAPK targeted agents in the combination setting (e.g. sotorasib + trametinib). We therefore analysed MAPK pathway related transcriptional signatures to assess predictability for response to ASTX029 in combination with a SHP2 inhibitor. We previously presented data from large-scale viability screens assessing the response to ASTX029 as a single agent treatment (437 cell lines), as well as in combination with an inhibitor of SHP2 (491 cell lines), an upstream adaptor protein in the MAPK pathway. Using these data, several published transcriptional signatures related to the MAPK pathway were assessed for their ability to stratify sensitive vs resistant cell lines when benchmarked against MAPK oncogene genomic status alone. All assessed transcriptomics-based scores had a significant positive correlation with growth inhibition for ERK inhibitor single agent treatment as well as in combination with a SHP2 inhibitor. Transcriptomics-based scores were shown to be more sensitive than the use of mutant MAPK oncogene status alone based on assessment of binomial regression models. The significant increase in sensitivity enables the identification of additional cell lines responsive to MAPK pathway inhibition. The sensitivity was further increased when considering response to the combination of an ERK and SHP2 inhibitor compared to the ERK inhibitor as a single agent. This analysis demonstrates the potential use of MAPK pathway-related transcriptomics-based scores for the stratification of MAPK pathway inhibitor response. ASTX029 is currently undergoing clinical development in advanced solid tumours (NCT03520075) and transcriptomics-based methods may provide utility in refining patient selection in the clinic for single agent or combination approaches. Citation Format: Jessica Brothwood, Andrea Biondo, Kim-Hien Dao, Keisha Hearn, Chris Hindley, Navrohit Kandola, Harold Keer, John Lyons, Yoko Nakatsuru, Marc O'Reilly, Nicola Wallis, Harpreet Saini. Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3365.

Pexmetinib suppresses osteoclast formation and breast cancer induced osteolysis via P38/STAT3 signal pathway

Breast cancer metastases to the bone can lead to a series of bone-related events that seriously affect the quality of life. Pexmetinib, a novel p38 mitogen-activated protein kinase (p38) inhibitor that has been evaluated in phase I clinical trials for myelodysplastic syndrome, but the effects of Pexmetinib on breast cancer induced osteolysis haven’t been explored. Here, we found that Pexmetinib inhibited receptor activator of nuclear factor-κB ligand-induced osteoclast formation and bone resorption in vitro. Pexmetinib suppressed p38-mediated signal transducer and activator of transcription 3 (STAT3), which direct regulated transcription of the nuclear factor of activated T cells 1 (NFATc1), leading to reduced osteoclast formation. Moreover, Pexmetinib exerted anti-tumor effects in breast cancer cells in vitro via suppressing p38-mediated STAT3 activation and matrix metalloproteinases (MMPs) expression. Furthermore, Pexmetinib suppressed breast cancer-associated osteolysis in vivo. These results suggest that Pexmetinib may be a promising drug for the treatment of breast cancer-induced osteolysis.

LGG-27. Molecular implications of mitogen-activated protein kinase pathway inhibition by the MEK inhibitor trametinib in BRAF-fusion-driven pediatric pilocytic astrocytoma

Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by driving alterations in the mitogen-activated protein kinase (MAPK) pathway, leading to its constitutive activation and modulating the balance between cell proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA susceptible to MAPK inhibitor (MAPKi) therapies, which show encouraging results in phase 1/2 clinical trials. However, little is known about the molecular implications of MAPK inhibition in PA. The DKFZ-BT66 cell line, derived from a primary KIAA:BRAF-fusion positive PA, was used as a model system. DKFZ-BT66 were treated with the MEKi trametinib for different durations in both proliferative and senescent states. Gene expression was analyzed by gene expression profiling and protein expression/phospho-regulation by data-dependent mass spectrometry followed by label-free quantitative analysis. A time course analysis based on differentially expressed genes and phosphorylated proteins was performed, followed by a single-sample gene set enrichment analysis (ssGSEA) and kinase substrate enrichment analysis, respectively. Differential gene expression analysis revealed that MEK inhibition led to the inhibition of the OIS/SASP gene programs in senescent DKFZ-BT66, with downregulation of key OIS/SASP partners such as IL1B on the protein level. This functionally translated into a de-sensitization of these cells towards the senolytic agent navitoclax. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. This data indicates that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help increase treatment’s efficacy. Validation of these targets by post-translational modification enrichment analysis of the phospho-proteomics dataset is ongoing.

C/EBPβ mediates anti-proliferative effects of 1,25(OH)2D on differentiated thyroid carcinoma cells.

Differentiated thyroid carcinoma (DTC) is the most common endocrine malignancy and highly expresses the receptor for 1,25-dihydroxyvitamin D (1,25(OH)2D). However, it is unclear whether 1,25(OH)2D regulates DTC proliferation and differentiation. Here, we found that 1,25(OH)2D3 inhibited proliferation but not differentiation of the DTC cells. Notably, CYP27B1was elevated in DTC cells and 25-hydroxyvitamin D3 (25(OH)D3) reduced DTC cell proliferation. Knockdown of VDR did not affect the anti-proliferative effects of 1,25(OH)2D3. However, knockdown of CCAAT enhancer-binding protein β (C/EBPβ)abolished 1,25(OH)2D3-suppressed DTC cell proliferation. In addition, 1,25(OH)2D3 induced phosphorylation and translocation of C/EBPβto the nucleus from the cytoplasm. However, inhibition of p38 mitogen-activated protein kinases (MAPK) abrogated 1,25(OH)2D3-induced phosphorylation and nuclear translocation of C/EBPβas well as 1,25(OH)2D3-suppressed DTC cell proliferation. Knockdown of C/EBPβreduced the expression of Notch3. Knockdown of Notch3 blocked 1,25(OH)2D3-suppressed DTC cell proliferation. In the DTC cell-derived xenograft SCID mouse, knockdown of C/EBPβmarkedly increased tumor growth and proliferation and decreased apoptosis. In DTC patients, C/EBPβwas predominantly located in the cytoplasm of DTC cells in the tumor tissue when compared with adjacent non-cancerous tissue in which C/EBPβis located in the nucleus. In conclusion, C/EBPβstimulated Notch3signaling via the p38 MAPK-dependent pathway mediates the inhibitory effect of 1,25(OH)2D on DTC cell proliferation.

Repurposing HDAC and mTOR inhibitors for pancreatic cancer.

e16234 Background: Pancreatic ductal adenocarcinoma (PDAC) will be the second leading cause of cancer death by 2030 through early onset dedifferentiation and metastasis that results in limited treatments. FDA-approved targeted therapies–including proteasome, mitogen-activated protein kinase (MEK), histone deacetylase (HDAC), and mammalian target of rapamycin (mTOR) inhibitors–have shown preclinical benefit but failed in clinic. However, combination targeted therapy has been largely unexplored in clinical trials. We hypothesized based on prior drug screening efforts in the lab on aggressive subtypes of pancreatic cancer that combinations of these targeted therapies could be synergistically active in preclinical PDAC models. Methods: We optimized combinations of proteasome, MEK, mTOR, and HDAC inhibitors by performing cell viability experiments in human PDAC lines in 2D and 3D cell culture conditions in vitro. Optimized drug combinations from these experiments were then trialed in two different in vivo animal models–human FG xenografts in immunocompromised mice (NSG) and spontaneous primary PDAC tumors from transgenic pancreatic-specific Kras-overexpressing, P53-deficient mice (KPC). RNA-sequencing and Seahorse metabolic assays were utilized to analyze molecular mechanisms of drug synergy. Dedifferentiation was assessed by histology of KPC tumors. Results: The HDAC inhibitor Panobinostat and mTOR inhibitor Everolimus synergistically killed human pancreatic cancer cell lines grown in 2D and 3D cell culture in vitro. Synergy was highest at low concentrations of Panobinostat. In vivo, low-dose Panobinostat and Everolimus synergistically blocked growth of human FG xenografts in NSG mice. In vivo synergy of low-dose Panobinostat and Everolimus was highest in the KPC mouse model, where only combination therapy could significantly reduce tumor growth, stall dedifferentiation, and improve survival. Mechanistically, RNA-sequencing of Panobinostat/Everolimus-treated FG cells revealed that Panobinostat increased tumor suppressor genes opposing dedifferentiation such as p21, EGR1, and CCN2. Everolimus complemented Panobinostat by decreasing expression of enzymes metabolizing acetyl groups such as fatty acid synthase, stearoyl-CoA desaturase, and sterol response element binding factors. Functionally, Everolimus’ activity to reduce oxidative consumption increased histone acetylation specifically in the presence of Panobinostat. Conclusions: A combination of FDA-approved, targeted oral therapies (low-dose Panobinostat/Everolimus) is synergistically active in preclinical models of pancreatic cancer in vitro and in vivo. mTOR inhibitors create drug synergy by specifically increasing histone acetylation in HDAC inhibitor-treated cells by reversing HDAC inhibitor de-repression of acetyl/oxidative metabolism, illustrating a novel connection between cancer epigenetics and metabolism.

Simvastatin Upregulates Lipoxin A4 and Accelerates Neuroinflammation Resolution After Intracerebral Hemorrhage.

Previous studies have demonstrated that statins can relieve inflammatory brain injury after intracerebral hemorrhage (ICH), but the mechanisms remain poorly characterized. This study aims to test whether simvastatin exerts an anti-inflammatory effect by regulating the proresolving mediators. First, male Sprague-Dawley rats had an injection of 200 μL autologous blood. Then, rats were randomly divided into groups treated with simvastatin (i.p. 2 mg/kg) or vehicle. Next, all rats underwent pro-resolving mediator lipoxin A4 (LXA4) level detection, flow cytometric, immunofluorescence, brain edema measurement, neurological scoring and western blot analysis. We found that simvastatin significantly increased the plasma level of LXA4, an endogenous formyl-peptide receptor 2 (FPR2) agonist, in the early stage of ICH. Consistent with the effect of simvastatin, exogenous LXA4 administration also promoted apoptosis of the circulating neutrophils, reduced neutrophils brain infiltration, and ameliorated inflammatory brain injury after ICH. In addition, similar to simvastatin, exogenous LXA4 markedly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) and the apoptosis-related proteins myeloid cell leukemia 1(Mcl-1)/Bax ratio (a decreased ratio represents the induction of apoptosis) in circulating neutrophils isolated from ICH rats. Notably, all of the aforementioned effects of simvastatin on ICH were significantly abolished by Boc-2, a selective antagonist of FPR2. Moreover, simvastatin led to a similar Mcl-1/Bax ratio reduction as SB203580 (a p38 MAPK inhibitor), but it was abolished by P79350 (a p38 MAPK agonist). Collectively, these results suggest that simvastatin ameliorates ICH-mediated inflammatory brain injury, possibly by upregulating the level of pro-resolving mediator LXA4 and further stimulating the FPR2/p38 MAPK signaling pathway.

Inhibitory potential of phytochemicals from Chromolaena odorata L. against apoptosis signal-regulatory kinase 1: A computational model against colorectal cancer

Apoptosis signal kinase 1 (ASK 1) is a member of the mitogen-activated protein kinase (MAPK) family that induces cells apoptosis including colorectal cancer (CRC). CRC is the second most common type of malignancy globally. Hence, ASK 1 plays an essential role in the pathogenesis of CRC and therefore, is an exclusive target in drug design and discovery for CRC. Herein, applied computational approaches including molecular docking, molecular mechanics/generalized born surface area calculation and pharmacokinetic models were performed to propose putative ASK 1 antagonists from natural compounds. Seven (7) ligands were identified as potent inhibitors and two top hit compounds were validated using molecular dynamics (MD) simulation studies. The density function theory (DFT) of the hits were performed at the Becke three Lee Yang Parr/6-31G(d) level of theory to understand their molecular reactivity. Seven compounds identified as ASK 1 antagonists have docking score ranging from −9.10 to −8.14 kcal/mol which is comparable to the reference ligand camptosar (-7.03 kcal/mol). One of the compounds, odoratin has finally emerged as the structurally stable compound with −9.10 kcal/mol and MD simulation over 50 ns indicated that odoratin forms stable interaction with key amino acid residues such as LEU 686, VAL 757 and PRO 758. DFT study showed that the studied compounds have proton donating and accepting ability hence, potent inhibitory and solubility effects. The findings from this study suggest that, odoratin could be considered a potent ASK 1 inhibitor and could be experimentally verified as a lead compound for search of MAPK inhibitors for colorectal cancer.

Antioxidant Activities and Mechanisms of Tomentosin in Human Keratinocytes.

Tomentosin, one of natural sesquiterpene lactones sourced from Inula viscosa L., exerts therapeutic effects in various cell types. Here, we investigated the antioxidant activities and the underlying action mechanisms of tomentosin in HaCaT cells (a human keratinocyte cell line). Specifically, we examined the involvement of tomentosin in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. Treatment with tomentosin for up to 60 min triggered the production of reactive oxygen species (ROS), whereas treatment for 4 h or longer decreased ROS production. Tomentosin treatment also induced the nuclear translocation of Nrf2 and upregulated the expression of Nrf2 and its target genes. These data indicate that tomentosin induces ROS production at an early stage which activates the Nrf2 pathway by disrupting the Nrf2–Keap1 complex. However, at a later stage, ROS levels were reduced by tomentosin-induced upregulation of antioxidant genes. In addition, tomentosin induced the phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 MAPK and c-Jun N-terminal kinase (JNK). SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor) attenuated the tomentosin-induced phosphorylation of Nrf2, suggesting that JNK and p38 MAPK signaling pathways can contribute to the tomentosin-induced Nrf2 activation through phosphorylation of Nrf2. Furthermore, N-acetyl-L-cysteine (NAC) treatment blocked both tomentosin-induced production of ROS and the nuclear translocation of Nrf2. These data suggest that tomentosin-induced Nrf2 signaling is mediated both by tomentosin-induced ROS production and the activation of p38 MAPK and JNK. Moreover, tomentosin inhibited the AhR signaling pathway, as evidenced by the suppression of xenobiotic-response element (XRE) reporter activity and the translocation of AhR into nucleus induced by urban pollutants, especially benzo[a]pyrene. These findings suggest that tomentosin can ameliorate skin damage induced by environmental pollutants.

In Vivo Evaluation of Fibroblast Growth Factor Receptor Inhibition in Mouse Xenograft Models of Gastrointestinal Stromal Tumor.

Advanced gastrointestinal stromal tumors (GIST) are typically treated with tyrosine kinase inhibitors, and imatinib is the most commonly used standard of care in first line treatments. The use of this and other tyrosine kinase inhibitors is associated with objective tumor responses and prolongation of progression-free and overall survival, but the treatment of metastatic disease is non-curative due to the selection or acquisition of secondary mutations and the activation of alternative kinase signaling pathways, leading to resistance and disease progression after an initial response. The present preclinical study evaluated the potential use of the fibroblast growth factor receptor inhibitors infigratinib and dovitinib alone or in combination with the mitogen-activated protein kinase inhibitor binimetinib in mouse models of GIST with different sensitivity or resistance to imatinib. Patient- and cell-line-derived GIST xenografts were established by bilateral, subcutaneous transplantation of human GIST tissue in female adult nu/nu NMRI mice. The mice were treated with dovitinib, infigratinib, or binimetinib, either alone or in combination with imatinib. The safety of treated animals was assessed by well-being inspection and body weight measurement. Antitumor effects were assessed by caliper-based tumor measurement. H&E staining and immunohistochemistry were used for assessing anti-mitotic and pro-apoptotic activity of the experimental treatments. Western blotting was used for assessing effects of the agents on kinase signaling pathways. Anti-angiogenic activity was assessed by measuring tumor vessel density. Dovitinib was found to have antitumor efficacy in GIST xenografts characterized by different imatinib resistance patterns. Dovitinib had better efficacy than imatinib (both at standard and increased dose) and was found to be well tolerated. Dovitinib had better efficacy in a KIT exon 9 mutant model, highlighting a role of patient selection in clinical GIST trials with the agent. In a model with KIT exon 11 and 17 mutations, dovitinib induced tumor necrosis, most likely due to anti-angiogenic effects. Additive effects combining dovitinib with binimetinib were limited.

Retracted: Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis.

This publication has been retracted by the Editor due to the identification of non-original figure images that raise concerns regarding the credibility and originality of the study. Reference: You-Dong Wan, Rui-Xue Zhu, Zhong-Zheng Bian, Xin-Ting Pan. Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitisy. Med Sci Monit, 2019; 25: 4609-4616. DOI: 10.12659/MSM.914538.

Design, Synthesis, and Biological Characterization of Inhaled p38α/β MAPK Inhibitors for the Treatment of Lung Inflammatory Diseases.

The identification of novel inhaled p38α/β mitogen-activated protein kinases (MAPK) (MAPK14/11) inhibitors suitable for the treatment of pulmonary inflammatory conditions has been described. A rational drug design approach started from the identification of a novel tetrahydronaphthalene series, characterized by nanomolar inhibition of p38α with selectivity over p38γ and p38δ isoforms. SAR optimization of 1c is outlined, where improvements in potency against p38α and ligand-enzyme dissociation kinetics led to several compounds showing pronounced anti-inflammatory effects in vitro (inhibition of TNFα release). Targeting of the defined physicochemical properties allowed the identification of compounds 3h, 4e, and 4f, which showed, upon intratracheal instillation, low plasma levels, prolonged lung retention, and anti-inflammatory effects in a rat acute model of a bacterial endotoxin-induced pulmonary inflammation. Compound 4e, in particular, displayed remarkable efficacy and duration of action and was selected for progression in disease models of asthma and chronic obstructive pulmonary disease (COPD).

Vincristine Promotes Transdifferentiation of Fibroblasts Into Myofibroblasts via P38 and ERK Signal Pathways.

Background: Vincristine (VCR) is used in the clinic as an anti-tumor drug. VCR can cause pulmonary fibrosis (PF), leading to respiratory failure. The transformation of fibroblasts into myofibroblasts may play a key role in PF. The present study attempted to reveal the molecular mechanism of VCR-induced PF and the possible involvement of the mitogen-activated protein kinase (MAPK) signaling pathway. Methods: Human embryonic lung fibroblasts (HELFs) were treated with different concentrations of VCR. Inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK were added to HELFs. Cell proliferation state was assessed using cell counting kit-8 and by directly counting the number of cells. The expressions of vimentin and α-smooth muscle actin (α-SMA) were investigated using western blot and immunofluorescence analyses. Activation of ERK and P38 was estimated by the expression of phosphorylated p38 MAPK (p-p38), p38 MAPK, phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 using western blot analysis. Enzyme-linked immunosorbent assay was used to estimate the level of collagen I in cell culture supernatants. Results: Results showed that VCR promoted cellular proliferation, secretion of collagen I and the expression of vimentin and α-SMA. High expression of p-p38 and p-ERK1/2 was associated with the activation of the MAPK signaling pathway. MAPK inhibitors SB203580 and PD98059 suppressed the expression of the above proteins. Conclusion: Our study revealed that VCR could promote the differentiation of fibroblasts into myofibroblasts by regulating the MAPK signal pathway, which may be a promising way to treat VCR-induced PF.

Staphylococcus aureus enhances gelatinase activities in monocytic U937 cells and in human gingival fibroblasts

Background/purposeStaphylococcus aureus (S. aureus) has been suggested to be an initiative pathogen in peri-implantitis because of the solid affinity to titanium. However, the detail pathogenesis for the peri-implantitis initiation by S. aureus is still lacking. This study aimed to in vitro examine the gelatinases’ activities of monocytic U937 cell and human gingival fibroblast after challenges with S. aureus lipoteichoic acid (LTA) and peptidoglycan (PGN). Materials and methodsReleases of gelatinases, including matrix metalloproteinase (MMP)-2 and −9, from cells were measured by zymography. The releases were further examined after being given the S. aureus LTA/PGN. Roles of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways on the enzyme releases were examined by administrating inhibitors. ResultsS. aureus LTA and PGN increased the activities of pro-MMP-9 from U937 cells and pro-MMP-2 and MMP-2 from gingival fibroblasts. By giving the NF-κB inhibitor, the enhanced gelatinase activities in both cells were attenuated. In U937 cells, the enhanced pro-MMP-9 could further be attenuated by MAPK inhibitors, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), P38 MAPK, and c-Jun N-terminal kinase (JNK) inhibitors; however, the attenuation by MAPK inhibitors could not be observed for MMP-2 in gingival fibroblasts. Nevertheless, in gingival fibroblasts, the pro-MMP-2 could be attenuated by JNK inhibitor. ConclusionS. aureus could enhance gelatinase activities of gingival fibroblasts and U937 cells, via NF-κB. The MAPK pathway was also involved in MMP-9 activity of U937 cells; however, the involvement of MAPK in MMP-2 activity of gingival fibroblasts was questioned.

Interleukin‐18 Enhances Contractile Responses to Phenylephrine in Pudendal Artery from Wistar Rats

BackgroundHypertension is a major risk factor for the development of cardiovascular diseases and is a leading cause of mortality. The etiology of essential hypertension remains unclear, and its mechanisms are complex. Inflammation is a complex phenomenon, comprises the first response of the immune system in the presence of harmful stimuli and is considered to be a major cause of increased blood pressure. Indeed, low‐grade inflammation and activation of the immune system have been implicated in hypertension. Cytokine levels are elevated in hypertension and have been reported to be implicated in the associated disturbances in vascular reactivity reported in this disease. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL‐18) in patients with hypertension.HypothesisThis study tests the hypothesis that IL‐18 will enhance contractile responses to phenylephrine in isolated arterial rings from rats.MethodsVascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration‐response curves to phenylephrine (PE; 10‐9‐3x10‐5M), Acetylcholine (ACH; 10‐9 – 3x10‐5M) were performed in the absence or in the presence of IL‐18 (20ng/ml) for 1 hour. 10 minutes prior to the incubation with IL‐18, some arterial rings were incubated separately with the following agents: NADPH oxidase 2 inhibitor, gp91 ds‐tat (10‐5M); NADPH oxidase 1 and 4 inhibitor, GKT137831(10‐6M); and mitogen‐activated protein kinase (MKK / MEK) inhibitor, PD98059 (10‐5M) Concentration‐response curves were analyzed using non‐linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data was analyzed with GraphPad Prism 9.2.0.ResultsIn the Wistar pudendal arterial rings, the potency of PE was increased in the presence of IL‐18 (pEC506.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268) A, IL‐18 plus gp91 ds‐tat (pEC506.643 ± 0.2215 vs 6.264 ± 0.2471 for control; p = 0.0058) B and IL‐18 plus GKT137831 (pEC506.700 ± 0.2937 vs 6.272 ± 0.2963 for control; p = 0.0003) C. Incubation with IL‐18 plus PD98059 restored the contraction to PE to control levels. (pEC506.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120) D. The relaxation response to ACh was not changed by IL‐18 after incubation for 1 hr.ConclusionOur results show that IL‐18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the Mitogen‐Activated Protein Kinase/Extracellular Signal‐Regulated Kinase Pathway.