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. 2024 Jul 10;5(7):e634.
doi: 10.1002/mco2.634. eCollection 2024 Jul.

Remodeling tumor-associated macrophage for anti-cancer effects by rational design of irreversible inhibition of mitogen-activated protein kinase-activated protein kinase 2

Affiliations

Remodeling tumor-associated macrophage for anti-cancer effects by rational design of irreversible inhibition of mitogen-activated protein kinase-activated protein kinase 2

Danyi Wang et al. MedComm (2020). .

Abstract

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti-cancer therapies. The limitations of ATP-competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC50 = 2.3 nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor-promoting macrophage M2 phenotype in vitro and in vivo. In macrophage-rich tumor model, compound 11 notably slowed growth in a dose-dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions.

Keywords: anti‐tumor; irreversible inhibitor; kinase; macrophage; mitogen‐activated protein kinase‐activated protein kinase 2 (MK2).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Association of mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) activity with prognosis in cancer. (A) MK2 gene expression distribution in tumor versus adjacent peritumoral tissues across various cancer types, sourced from The Cancer Genome Atlas (TCGA) database. The x‐axis represents distinct cancer types, excluding samples with a size less than 10, while the y‐axis displays gene expression levels, with different colors indicating separate groups. Statistical differences between groups were evaluated using the Wilcoxon test, with significance levels marked as ** p < 0.01, *** p < 0.001. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; COADREAD, colon adenocarcinoma/rectum adenocarcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIPAN, pan‐kidney cohort, KICH + KIRC + KIRP; KIRC, kidney renal clear cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; STAD, stomach adenocarcinoma; STES, stomach and esophageal carcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. (B) Log‐rank survival analysis of patients with LIHC, READ, and LGG (lower grade glioma) patients according to the level of MK2 mRNA (high MK2: top 25%; low MK2: bottom 75%). Data sourced from TCGA database and the software GraphPad Prism 9.0 produced the figures.
FIGURE 2
FIGURE 2
Rational design of novel MK2 inhibitors. (A) The design and optimization process. By analyzing the co‐crystal structure of compound 1 bound to MK2 (PDB: 2P3G), we utilized the docking method to design a covalent inhibitor (compound 2), and compared with the negative control compound 3. This series was optimized to identify the most potent inhibitor, compound 11. (B) Five kinases having a cysteine located at two residues behind the gatekeeper.
FIGURE 3
FIGURE 3
High selectivity and irreversible inhibition on MK2 by compound 11. (A) Compound 11 inhibits MK2 kinase activity irreversibly. The phosphorylation rate of the substrate peptide, expressed as the conversion rate, illustrates the extent of inhibition. The displayed results reflect the phosphorylation outcomes of the peptide by MK2 kinase following preincubation with the specified inhibitor. The group preincubated without MK2 kinase as background group, or preincubated only with MK2 kinase without inhibitor as E control group. (B) An extensive kinase selectivity profile for compound 11, highlighting its specificity for MK2 against 380 other human recombinant kinases.
FIGURE 4
FIGURE 4
Targeting cellular MK2 signaling with irreversible inhibition by compound 11. (A and B) RT‐PCR analysis of IL‐1β, TNF‐α, and IL‐6 expression in Lipopolysaccharides (LPS)‐stimulated THP‐1 (A) and RAW264.7 (B) Cells, with pre‐treatment of compound 11 for 3 h followed by LPS (3 h) stimulation. (C) The inhibitory effects of compound 11 on TNF‐α and IL‐6 production in LPS‐stimulated RAW264.7 macrophages for 24 h, measured by ELISA. (D and E) Compound 11, alongside CC‐99677 and PF‐3644022, suppressed MK2 pathway activation, validated through western blot analysis post‐LPS stimulation. Cells were preincubated with the specified compounds for 1 h prior to LPS stimulation (100 ng/mL, 30 min). (F) Distinguishes the mode of action between compound 11 and CC‐99677. The complex structure of a small molecule and MK2 (3R2Y) with the same parent nucleus as compound 11 is shown in cyans, and the complex structure of a small molecule and MK2 (3FYJ) with the same parent nucleus as CC‐99677 is shown in orange. (G–I) The effect of indicated inhibitor on Hsp27 phosphorylation following inhibitor washout. Cells were pretreated with indicated compounds for 1 h and then washed to remove free compound and incubated for a further indicated time before activation with LPS. In (A–C), LPS (100 ng/mL) served as the stimulation control, while untreated cells were the baseline control. The data represent as mean ± SD from triplicates, with statistical analysis conducted via one‐way analysis of variance (ANOVA) (## p < 0.01, ### p < 0.001 vs. stimulation control; ***p < 0.001 vs. baseline control).
FIGURE 5
FIGURE 5
Inhibition of pro‐tumorigenic M2‐like polarization in macrophages by compound 11. (A–C) RT‐PCR analysis highlights the impact of compound 11 on MRC1 and ARG‐1 mRNA expression in various macrophage cells treated with IL‐4/IL‐13 alone or combined with compound 11 for 12 h or 24 h. (D) CD206 and Arg‐1 expression in bone marrow‐derived macrophages (BMDM) cells via flow cytometry post 48 h treatment of compound 11 and IL‐4/IL‐13. (E) The treatment effects of compound 11 in THP‐1 cells using western blot analysis. Cells were pretreated with compound 11 for 1 h and followed by IL‐4/IL‐13 treatment (30 min). (F) The influence of MK2 siRNA on MRC1 and ARG‐1 mRNA expression in THP‐1 cells by RT‐PCR analysis post‐IL‐4/IL‐13 stimulation (12 h). (G) Flow cytometric analysis of tumor infiltrating tumor‐associated macrophages (TAMs) and CD206+ TAMs in the MC38 tumor model treated with vehicle or compound 11 for 9 days (n = 15 mice per group). Data are shown as the mean ± SD. * p < 0.05; *** p < 0.001 versus the vehicle group, determined by unpaired t test. In A–D and F, IL‐4/IL‐13 (20 ng/mL) served as the stimulation control, while untreated cells were the baseline control. Data represent as mean ± SD from triplicates, with statistical analysis conducted via one‐way analysis of variance (ANOVA) (## p < 0.01, ### p < 0.001 vs. stimulation control; * p < 0.05 *** p < 0.001 vs. baseline control).
FIGURE 6
FIGURE 6
Delayed tumor growth in the MC38 model by compound 11. Over a course of 10 days, immune‐competent mice bearing MC38 tumors were treated intraperitoneally with either compound 11 or a vehicle control (n = 7/group). The tumor growth trend (Panel A) and the body weight data (Panel B) are displayed as the mean ± SEM. Statistical analysis through one‐way analysis of variance (ANOVA) revealed significance as *** p < 0.001.

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