MAPKAP-K2 Inhibitors Research Articles

Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

ZFP36L1, which is a negative regulator of gene transcripts, has been proven to regulate the progression of several carcinomas. However, its role in sarcoma remains unknown. Here, by using data analyses and in vivo experiments, we found that ZFP36L1 inhibited the lung metastasis of osteosarcoma (OS). Knockdown of ZFP36L1 promoted OS cell migration by activating TGF-β signaling and increasing SDC4 expression. Intriguingly, we observed a positive feedback loop between SDC4 and TGF-β signaling. SDC4 protected TGFBR3 from matrix metalloproteinase (MMP)-mediated cleavage and therefore relieved the inhibition of TGF-β signaling by soluble TGFBR3, while TGF-β signaling positively regulated SDC4 transcription. We also proved that ZFP36L1 regulated SDC4 mRNA decay through adenylate-uridylate (AU)-rich elements (AREs) in its 3'UTR. Furthermore, treatment with SB431542 (a TGF-β receptor kinase inhibitor) and MK2 inhibitor III (a MAPKAPK2 inhibitor that increases the ability of ZFP36L1 to degrade mRNA) dramatically inhibited OS lung metastasis, suggesting a promising therapeutic approach for the treatment of OS lung metastasis.

Investigating the Role of MK2 in Head and Neck Squamous Cell Carcinoma Growth, Metastasis and STING Pathway Activation

Our prior work demonstrated that inhibition of MAPKAPK2 (MK2) can enhance radiation (RT)-mediated in vivo head and neck squamous cell carcinoma (HNSCC) tumor control and survival in preclinical immune incompetent models. The cytosolic DNA sensor cyclic GMP-AMP synthetase (cGAS) and its downstream adaptor protein, stimulator of interferon genes (STING), are conserved proteins within the innate immune signaling pathways and are important for mediating host defense against microbial infection and can play a role in anti-cancer immunity. We hypothesized that loss of MK2 enhances radiation-induced cGAS-STING pathway activation leading to improved tumor control and survival. MK2 shRNA knockdown human (Tu167, CAL27) and MK2 Cas9/CRISPR knockout (KO) syngeneic murine (Ly2, MLM3) HNSCC cell lines were treated with 10 Gy irradiation. Micronuclei were quantitated by DAPI-immunofluorescence (IF). Protein changes in cGAS-STING were evaluated by immunoblot. Inflammatory cytokine production including Type I IFNβ1 were evaluated by RT-qPCR. Ly2 and MLM3 cells were orthotopically or flank engrafted into immune competent mice (Balb/c, C57Bl/6, respectively) for animal tumor control-survival studies. Tumor immune cell infiltrate was examined using FACS and immunohistochemistry. Selected drug studies using the MK2 inhibitor, ATI-450, were performed with RT. Loss of MK2 in HNSCC (Tu167, CAL27, Ly2, MLM3) treated with RT led to a significant increase in micronuclei formation compared to control cells. MK2-enhanced micronuclei generation following RT could be inhibited with the actin filament polymerization inhibitor, cytochalasin B. RT treatment of MK2 shRNA cells led to increased cGAS and phospho-STING levels compared to either treatment alone. IFNβ1 levels were significantly higher in HNSCC cell lines treated with RT and with MK2 inhibited by an MK2 inhibitor (ATI-450) or genetic reduction compared to either treatment alone. In-vivo implantation of MLM3 cells into C57Bl/6 comparing control vs MK2 KO tumors treated with ±8 Gy RT demonstrated improved mouse survival favoring RT+MK2 KO over RT, MK2 KO or parental (63, 58, 58.5, 35 days, respectively). FACS analysis of MLM3 WT v KO tumors 3 days post RT showed an overall increase in the number of CD3/CD8 T-cells infiltrating into the tumor in all groups except for parental tumors. Further analysis demonstrated that loss of MK2 reversed CD8 T-cell exhaustion and when combined with radiotherapy led to increased CD8 T-cell activation. Furthermore, activated CD4 and CD8 T cells were reduced in WT+RT cells compared to WT tumors whereas no reduction was seen in the KO or KO+RT. HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Erythrocyte mitogen-activated protein kinases mediate hemolytic events under osmotic and oxidative stress and in hemolytic diseases

p38 MAPKs are key regulators of cellular adaptation to various stress stimuli, however, their role in mediating erythrocyte cell death and hemolysis is largely unknown. We hypothesized that activation of erythrocyte p38 MAPK is a common event in the stimulation of hemolysis, and that inhibition of p38 MAPK pathways could mitigate hemolysis in hemoglobinopathies. We exposed human erythrocytes to diamide-induced oxidative stress or to hypoosmotic shock in the presence or absence of p38 MAPK inhibitors (SCIO469, SB203580, CMPD1) and used immunoblotting to determine MAPK activity and to identify possible downstream effectors of p38 MAPK. We also evaluated the impact of p38 MAPK inhibitors on stress-induced hemolysis or hypoxia-induced sickling in erythrocytes from mouse models of sickle cell disease. We found that human erythrocytes express conventional MAPKs (MKK3, p38 MAPK, MAPKAPK2) and identified differential MAPK activation pathways in each stress condition. Specifically, p38 MAPK inhibition in diamide-treated erythrocytes was associated with decreased phosphorylation of Src tyrosine kinases and Band 3 protein. Conversely, hypoosmotic shock induced MAPKAPK2 and RSK2 phosphorylation, which was inhibited by SCIO469 or CMPD1. Relevant to hemoglobinopathies, sickle cell disease was associated with increased erythrocyte MKK3, p38 MAPK, and MAPKAPK2 expression and phosphorylation as compared with erythrocytes from healthy individuals. Furthermore, p38 MAPK inhibition was associated with decreased hemolysis in response to diamide treatments or osmotic shock, and with decreased erythrocyte sickling under experimental hypoxia. These findings provided insights into MAPK-mediated signaling pathways that regulate erythrocyte function and hemolysis in response to extracellular stressors or human diseases.

Discovery of CC-99677, a selective targeted covalent MAPKAPK2 (MK2) inhibitor for autoimmune disorders

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.

Allosteric MAPKAPK2 inhibitors improve plaque stability in advanced atherosclerosis.

Atherosclerotic vascular disease resulting from unstable plaques is the leading cause of morbidity and mortality in subjects with type 2 diabetes (T2D), and thus a major therapeutic goal is to discover T2D drugs that can also promote atherosclerotic plaque stability. Genetic or pharmacologic inhibition of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2 or MK2) in obese mice improves glucose homeostasis and enhances insulin sensitivity. We developed two novel orally active small-molecule inhibitors of MK2, TBX-1 and TBX-2, and tested their effects on metabolism and atherosclerosis in high-fat Western diet (WD)-fed Ldlr-/- mice. Ldlr-/- mice were first fed the WD to allow atherosclerotic lesions to become established, and the mice were then treated with TBX-1 or TBX-2. Both compounds improved glucose metabolism and lowered plasma cholesterol and triglyceride, without an effect on body weight. Most importantly, the compounds decreased lesion area, lessened plaque necrosis, and increased fibrous cap thickness in the aortic root lesions of the mice. Thus, in a preclinical model of high-fat feeding and established atherosclerosis, MK2 inhibitors improved metabolism and also enhanced atherosclerotic plaque stability, suggesting potential for further clinical development to address the epidemic of T2D associated with atherosclerotic vascular disease.

Abstract 705: A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4

Abstract c-MYC overexpression plays an important role in tumorigenesis as well as in the progression of tumors. Historically, direct targeting strategies have been challenging due to c-MYC being a transcription factor. Our observation demonstrated that MAPKAPK2 as one of the two major protein kinases involved in binding and phosphorylating OCT4 at serine 111 residue to induce c-MYC activation in progressive disease neuroblastoma. In targeting this novel pathway of c-MYC activation, we considered two options: direct inhibition of MAPKAPK2 or inhibition of MAPKAPK2-OCT4 protein-protein interaction. Given the limitations of direct targeting kinases, namely resistance and off-target effect, we pursued the inhibition of the protein-protein interaction. The purpose of this study is to introduce a novel cell-based assay for compound screening and validation steps to identify compounds that inhibit OCT4-MAPKAPK2 interaction. First, OCT4 (NTD and POUs: the domains critical for c-MYC activation) and MAPKAPK2 were exogenously expressed by a single vector with CMV promoter using a lentiviral vector system in NCI-H82, a small cell lung cancer cell line. Upon the optimization of the assay, we screened approximately 100,000 compounds to identify “hits” by statistically analyzing the reduction in luminescence. Seventy-six compounds were found to inhibit the interaction of MAPKAPK2-OCT4. Subsequently, we have used a two-step hit validation for the 76 compounds: 1) the decrease in pOCT4S111 expression along with the reduction in c-MYC expression by immunoblotting: 2) and the inhibition of binding between MAPKAPK2 and OCT4 by co-immunoprecipitation. The validation step further narrowed down the hits to three compounds that reduced the phosphorylation of OCT4 at S111 and inhibited the MAPKAPK2-OCT4 interaction. To verify the compounds interfering the MAPKAPK2-OCT4 interaction, we used in vitro assay using recombinant human MAPKAPK2 kinase and OCT4 and all three compounds inhibited phosphorylation of OCT4S111. These all compounds also tested minimally affected ATP-dependent MAPKAPK2 kinase relative to the known MAPKAPK2 inhibitor (PF3644022). In conclusion, our screening assay and validation experiments was successfully implemented in identifying hits and the validation step confirmed three novel compounds that demonstrated: 1) impairment of MAPKAPK2-mediated phosphorylation of OCT4; and 2) inhibition of the MAPKAPK2-OCT4 kinase-substrate interaction. These compounds may be further optimized to minimize systemic toxicities and improve the anti-cancer activity for the treatment of neuroblastoma or small cell lung cancer with high c-MYC expression. Citation Format: Inhyoung Yang, Sung-Jen Wei, Ismail Mohiuddin, Gloria M. Martinez, Min H. Kang. A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 705.

MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma.

Radiation therapy (RT) is a cornerstone of treatment in the management of head and neck squamous cell carcinomas (HNSCC), yet treatment failure and disease recurrence are common. The p38/MK2 pathway is activated in response to cellular stressors, including radiation, and promotes tumor inflammation in a variety of cancers. We investigated MK2 pathway activation in HNSCC and the interaction of MK2 and RT in vitro and in vivo. We used a combination of an oropharyngeal SCC tissue microarray, HNSCC cell lines and patient-derived xenograft (PDX) tumor models to study the effect of RT on MK2 pathway activation and to determine how inhibition of MK2 by pharmacologic (PF-3644022) and genetic (siRNA) methods impacts tumor growth. We show that high phosphorylated MK2 (p-MK2) levels are associated with worsened disease specific survival in p16-negative HNSCC patients. RT increased p-MK2 in both p16-positive, HPV-positive and p16-negative, HPV-negative HNSCC cell lines. Pharmacologic inhibition or gene silencing of MK2 in vitro abrogated RT-induced increases in p-MK2; inflammatory cytokine expression and expression of the downstream MK2 target, heat shock protein 27 (HSP27); and markers of epithelial-to-mesenchymal transition. Mouse PDX models treated with a combination of RT and MK2 inhibitor experienced decreased tumor growth and increased survival. Our results suggest that MK2 is a potential prognostic biomarker for head and neck cancer and that MK2 pathway activation can mediate radiation resistance in HNSCC.

Targeting MK2 Is a Novel Approach to Interfere in Multiple Myeloma

MAPKAPK2 (MK2), the direct substrate of p38 MAPK, has been well-acknowledged as an attractive drug target for cancer therapy. However, few studies have assessed the functions of it in multiple myeloma (MM). In the present study, MK2 expression of MM patients was analyzed by gene expression profiling (GEP) and array-based comparative genomic hybridization (aCGH). Several experiments in vitro including MTT assay, Western blot and flow cytometry analysis were performed to identify the function of MK2 in MM. In addition, we conducted mouse survival experiments to explain the effects of MK2 on MM in vivo. mRNA level of MK2 and chromosomal gain of MK2 locus in MM cells significantly increased compared to normal samples. Furthermore, MM patients with high expression of MK2 were associated with a poor outcome. Follow-up studies showed that MK2 exerted a remarkably positive effect on MM cell proliferation and drug-resistance. Further exploration focusing on MK2 inhibitor IV revealed its inhibitory action on MM growth and drug-resistance, as well as improving survival in mouse models. In addition, a combination of MK2 inhibitor IV and the key MM therapeutic agents including bortezomib, doxorubicin, or dexamethasone facilitated curative effects on inhibiting MM cell proliferation. Taken together, our study reveals the clinical relevance of MK2 inhibition in MM and demonstrates that targeting MK2 may afford a new therapeutic approach to MM.

MK2 Is a Therapeutic Target for High-Risk Multiple Myeloma

p38 mitogen-activated protein kinase (MAPK) is constitutively activated in human multiple myeloma (MM). However, a major substrate of p38, MAPKAPK2 (MK2) has received little attention in MM. In this study, we disclosed that elevation of MK2 on DNA, mRNA and protein levels in MM cells compared to normal plasma control cells by array-based comparative genomic hybridization (aCGH), Gene expression profiling (GEP) and immunohistochemistry (IHC) respectively. High MK2 expression prognosticated inferior outcome of newly diagnosed myelomas, particularly in patients with high-risk disease based on GEP. Follow-up study demonstrated that enforced overexpressing MK2 promoted MM cell proliferation, drug-resistance and xenograft tumorigenesis in vivo, while knockdown MK2 expression by shRNA abrogated these characteristics. Further mechanism study showed MK2 directly bound and activated AKT signaling in MM, while specific MK2 inhibitor inhibited MM cell growth and clonogenicity through suppressing AKT phosphorylation. Our findings not only indicate that MK2 is a potential therapeutic target in high-risk MM but also suggest the clinical relevance of MK2 inhibition in MM. DisclosuresNo relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells.

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells.

Applications of 3D-QSAR and structure-based pharmacophore modeling, virtual screening, ADMET, and molecular docking of putative MAPKAP-K2 (MK2) inhibitors

MAPKAP-K2 (MK2) belongs to the Ser/Thr kinase family, and has emerged as a highly favored target in the search for efficacious and safe anti-inflammatory drug. Thus, 3D-QSAR and structure-based pharmacophore models were developed in this study to identify new potential leads inhibiting MK2 as an efficient way to treat rheumatoid arthritis. Furthermore, these two kinds of models were cascaded together to reduce false positives in virtual screening. The selected compounds were further analyzed and refined using drug-like filters and ADMET analysis. Subsequently, 105 recruited hits were docked into MK2-binding site with GOLD. Finally, 12 molecules were selected as potential MK2 inhibitors, and their consensus score and their structural diversity were evaluated.

Discovery of a Potent Dihydrooxadiazole Series of Non-ATP-Competitive MK2 (MAPKAPK2) Inhibitors.

Inhibition of MK2 has been shown to offer advantages over that of p38 MAPK in the development of cures for inflammatory diseases such as arthritis. P38 MAPK knockout in mice was lethal, whereas MK2-null mice demonstrated strong inhibition of disease progression in collagen-induced arthritis and appeared normal and viable. However, it is challenging to develop ATP-competitive MK2 inhibitors due to high ATP binding affinity to the kinase. Non-ATP-competitive MK2 inhibitors interact and bind to the kinase in a mode independent of ATP concentration, which could provide better selectivity and cellular potency. Therefore, it is desirable to identify non-ATP-competitive MK2 inhibitors. Through structure optimization of lead compound 1, a novel series of dihydrooxadiazoles was discovered. Additional structure-activity relationship (SAR) study of this series led to the identification of compound 38 as a non-ATP-competitive MK2 inhibitor with potent enzymatic activity and good cellular potency. The SAR, synthesis, and biological data of dihydrooxadiazole series are discussed.

A three-step protocol for lead optimization: Quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2 (MAPKAPK2) inhibitors

A three-step protocol for SAR development was introduced and applied to the SAR studies of the MK2 inhibitor program. Following this protocol, key conformational features and functional groups for improving MK2 inhibitor activity were quickly identified. Through this effort, the initial gap observed between in vitro binding activity and cellular activity in the lead identification stage was very much reduced. Compound 28 was identified with single digit binding activity (IC(50)=8 nM) and good cellular activity (EC(50)=310 nM). This provides further evidence that non-ATP-competitive binding MK2 inhibitors are feasible by targeting the outside ATP pocket.

Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2 (MAPKAPK2) Inhibitors

A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-throughput screening using the affinity selection-mass spectrometry-based Automated Ligand Identification System platform. Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective inhibitors of MK2 should be feasible by targeting the outside ATP pocket.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part I

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC50 as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38α and JNKs. TNFα release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFα release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D–E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC50<3nM and inhibit the release of TNFα (IC50<0.3μM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC50=0.05–0.23μM), less potent in cells (IC50<1.1μM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

Guttiferones O and P, Prenylated Benzophenone MAPKAPK-2 Inhibitors from Garcinia solomonensis

Two prenylated benzophenones, guttiferones O (1) and P (2), were isolated from the stem bark of the Papua New Guinean plant Garcina solomonensis. The structures of these compounds and their relative configurations were determined by spectroscopic methods. Both compounds inhibited the phosphorylation of the synthetic biotinylated peptide substrate KKLNRTLSVA by the serine/threonine protein kinase MAPKAPK-2 with IC(50) values of 22.0 microM.

Structural Analysis of Carboline Derivatives As Inhibitors of MAPKAP K2 Using 3D QSAR and Docking Studies

MAPKAPK2, a substrate of p38 MAPKs, plays central role in p38-mediated signal transduction, and its inhibitors are promisingly useful in the treatment of inflammatory diseases. The computational approaches comprising both ligand-based drug design and structure-based drug design were used as virtual screening strategies for the discovery of novel MK2 inhibitors. Two quantitative pharmacophore models were generated with a training set of 27 MK2 inhibitors using HypoGen module of CATALYST. The two models suggested that two hydrogen bond acceptors, one hydrogen bond donor, and one hydrophobic feature are essential for ligand binding. Further, 3D QSAR model with comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) was constructed using a training set of 42 inhibitors. The predictive CoMFA and CoMSIA models have cross-validated coefficients (q2) of 0.804 and 0.765 and regression coefficients (r2) of 0.984 and 0.986, respectively. The structure-based studies were carried out by docking 77 known MAPKAPK2 inhibitors into the active site of receptor using Glide and analyzing the "hotspots" of the active site. Docking studies revealed that Met138, Leu141, Asp207, Lys93, Cys140, Leu70, Thr206, and Gly143 are showing interactions with highly active compounds. The structure activity relationships elucidated here for carboline derivatives combined with their binding information will provide an integrated approach to explore the chemical space further for improving the potency of MAPKAPK2 inhibitors.

The discovery of carboline analogs as potent MAPKAP-K2 inhibitors

The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with IC50s as low as 10nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the p-loop and the hinge region of MK2a.