Mitogen-activated protein kinase-interacting kinases (MNK1/2) have emerged as promising therapeutic targets for metabolic dysfunction-associated steatotic liver disease (MASLD). Through scaffold-conservative optimization of an imidazo[2,1-b][1,3,4]thiadiazole core, we identified HD202A as a highly potent MNK1/2 inhibitor (IC50 = 6.1/8.1 nM) with favorable kinome selectivity within the tested kinase panel, oral bioavailability (F = 42.1%), and favorable pharmacokinetic properties. In a high-fat-diet induced MASLD mice, HD202A markedly reduced body-weight gain, hepatic triglyceride accumulation, and serum lipids while improving glucose tolerance, insulin sensitivity, and inflammatory profiles. Mechanistically, HD202A suppressed MNK-eIF4E signaling, downregulated perilipin 2 and stearoyl-coenzyme A desaturase 1, upregulated adipose triglyceride lipase and peroxisome proliferator-activated receptor gamma coactivator 1α, and enhanced mitochondrial fatty-acid oxidation and redox homeostasis. These findings validate MNK inhibition as a viable strategy for MASLD and establish HD202A as a promising lead compound for further development.

Cell-free DNA (cfDNA) biomarkers derived from Arthrobacter luteus (ALU) repeats and long interspersed nuclear elements 1 (LINE1) - including ALU-115, ALU-247, LINE1-97, and LINE1-266 concentrations, as well as the integrity ratios ALU-247/115 and LINE1-266/97 - are commonly utilized to assess cfDNA quantity and integrity. This study examined the impact of delayed blood processing and prolonged plasma storage on these biomarkers using quantitative polymerase chain reaction. Blood samples were collected from twelve healthy individuals (6 males; mean age, 65.8 +/- 4.69 years) into dipotassium ethylenediaminetetraacetic acid tubes. Plasma cfDNA was extracted after various storage durations and temperatures, with aliquots from immediately processed blood subsequently stored at -80 degrees C for different time intervals. Except for LINE1-97, most biomarkers showed significantly higher levels in plasma isolated from whole blood stored at room temperature compared to plasma processed immediately. Storage at 4 degrees C resulted in fragment-specific effects: ALU-247/115 levels remained stable at 3 hours but decreased at 6 hours, while LINE1-266/97 levels increased at both time points. For plasma stored at -80 degrees C, ALU-derived biomarkers remained stable for up to 12 months; however, LINE1-97 levels significantly declined, accompanied by a corresponding increase in LINE1-266/97 as early as one month after freezing. These findings indicate that both storage duration and temperature significantly impact the measured levels of ALU- and LINE1-derived cfDNA biomarkers. Consequently, standardization of pre-analytical handling of blood and plasma is crucial for studies evaluating cfDNA quantity and integrity.

Marginal zinc deficiency (MZD) is a common but often overlooked nutritional condition associated with impaired intestinal barrier integrity and immune dysfunction. Milk lactopeptide (LP) have attracted interest as food-derived bioactive ingredients with potential zinc-binding capacity and immunomodulatory activity. In this study, the physicochemical characteristics and predicted Zn2+-binding potential of LP were evaluated, and their effects on gut-immune disturbances in MZD rats were investigated. LP were characterized by molecular weight distribution, amino acid composition, spectroscopy, and in silico peptide screening. An MZD rat model was then established to assess the effects of graded LP intervention on growth performance, immune-related parameters, intestinal morphology, zinc status, barrier-related proteins and genes, and gut microbiota composition. The results showed that LP were rich in low-molecular-weight fractions and contained peptide features consistent with potential Zn2+ interaction. In vivo, LP improved growth retardation, partially restored immune organ development, increased blood zinc levels and apparent zinc absorption, and ameliorated colonic structural damage in a dose-dependent manner. These effects were associated with the modulation of tight junction-associated proteins and genes, as well as changes in ZIP6 expression and NF-κB-related signaling. and alterations in gut microbiota composition. Collectively, these findings suggest that fermented LP may help alleviate MZD-associated gut-immune disturbances and may serve as a promising food-derived ingredient for nutritional intervention.

Insulin resistance (IR) is a major pathogenic factor in the progression of MASLD. In the liver, insulin suppresses gluconeogenesis and enhances de novo lipogenesis (DNL). During IR, there is a defect in insulin-mediated suppression of gluconeogenesis, but an unrestrained increase in hepatic lipogenesis persists. The mechanism of increased hepatic steatosis in IR is unclear and remains controversial. The key discrepancy is whether insulin retains its ability to directly regulate hepatic lipogenesis. Blocking insulin/IRS/AKT signaling reduces liver lipid deposition in IR, suggesting insulin can still regulate lipid metabolism; hepatic glucose metabolism that bypasses insulin’s action may contribute to lipogenesis; and due to peripheral IR, other tissues are likely to impact liver lipid deposition. We here review the current understanding of insulin’s action in governing different aspects of hepatic lipid metabolism under normal and IR states, with the purpose of highlighting the essential issues that remain unsettled.

Protein synthesis (mRNA translation) plays a crucial role in cell function by shaping the proteome -making all the proteins each cell requires at the right time and in the right quantities and places. Proteins carry out almost every job in the cell. Protein synthesis is also a major component of the cellular economy, using large amounts of metabolic energy and resources, especially amino acids. Accordingly, it is tightly regulated through diverse mechanisms which respond, for example, to nutrients, growth factors, hormones, neurotransmitters and stressful situations.

Under normal conditions, insulin promotes hepatic de novo lipogenesis (DNL). However, during insulin resistance (IR), when insulin signalling is blunted and accompanied by hyperinsulinaemia, the promotion of hepatic DNL continues unabated and hepatic steatosis increases. Here, we show that WD40 repeat-containing protein 6 (WDR6) promotes hepatic DNL during IR. Mechanistically, WDR6 interacts with the beta-type catalytic subunit of serine/threonine-protein phosphatase 1 (PPP1CB) to facilitate PPP1CB dephosphorylation at Thr316, which subsequently enhances fatty acid synthases transcription through DNA-dependent protein kinase and upstream stimulatory factor 1. Using molecular dynamics simulation analysis, we find a small natural compound, XLIX, that inhibits the interaction of WDR6 with PPP1CB, thus reducing DNL in IR states. Together, these results reveal WDR6 as a promising target for the treatment of hepatic steatosis.

[This corrects the article DOI: 10.1021/acsmedchemlett.2c00442.].

Control of protein synthesis (mRNA translation) plays key roles in shaping the proteome and in many physiological, including homeostatic, responses. One long-known translational control mechanism involves phosphorylation of initiation factor, eIF2, which is catalysed by any one of four protein kinases, which are generally activated in response to stresses. They form a key arm of the integrated stress response (ISR). Phosphorylated eIF2 inhibits eIF2B (the protein that promotes exchange of eIF2-bound GDP for GTP) and thus impairs general protein synthesis. However, this mechanism actually promotes translation of certain mRNAs by virtue of specific features they possess. Recent work has uncovered many previously unknown features of this regulatory system. Several studies have yielded crucial insights into the structure and control of eIF2, including that eIF2B is regulated by several metabolites. Recent studies also reveal that control of eIF2 and the ISR helps determine organismal lifespan and surprising roles in sensing mitochondrial stresses and in controlling the mammalian target of rapamycin (mTOR). The latter effect involves an unexpected role for one of the eIF2 kinases, HRI. Phosphoproteomic analysis identified new substrates for another eIF2 kinase, Gcn2, which senses the availability of amino acids. Several genetic disorders arise from mutations in genes for eIF2α kinases or eIF2B (i.e. vanishing white matter disease, VWM and microcephaly, epileptic seizures, microcephaly, hypogenitalism, diabetes and obesity, MEHMO). Furthermore, the eIF2-mediated ISR plays roles in cognitive decline associated with Alzheimer's disease. New findings suggest potential therapeutic value in interfering with the ISR in certain settings, including VWM, for example by using compounds that promote eIF2B activity.

Mitogen-activated protein kinase-interacting protein kinases (MNKs) phosphorylate eukaryotic initiation factor 4E (eIF4E) and regulate the processes of cell proliferation, cell cycle, and migration and invasion of cancer cells. Selectively inhibiting the activity of MNKs could be effective in treating cancers. In this study, we report a series of novel MNK inhibitors with an imidazo[2,1-b][1,3,4]thiadiazol scaffold, from which, compound 18 inhibited the phosphorylation of eIF4E in various cancer cell lines potently. Compound 18 was more potent against MNK2 than MNK1, and decreased the levels of cyclin-B1, cyclin-D3, and MMP-3 in A549 and MDA-MB-231 cells, impaired cell growth and colony formation, arrested the cell cycle in the G0/G1 phase, and inhibited cell migration and the secretion of TNF-α, MCP-1, and IL-8 from A549 cells. It represents a starting compound to design further inhibitors that selectively target MNKs and apply in other diseases.

Cells within solid tumours can become deprived of nutrients; in order to survive, they need to invoke mechanisms to conserve these resources. Using cancer cells in culture in the absence of key nutrients, we have explored the roles of two potential survival mechanisms, autophagy and elongation factor 2 kinase (eEF2K), which, when activated, inhibits the resource-intensive elongation stage of protein synthesis. Both processes are regulated through the nutrient-sensitive AMP-activated protein kinase and mechanistic target of rapamycin complex 1 signalling pathways. We find that disabling both autophagy and eEF2K strongly compromises the survival of nutrient-deprived lung and breast cancer cells, whereas, for example, knocking out eEF2K alone has little effect. Contrary to some earlier reports, we find no evidence that eEF2K regulates autophagy. Unexpectedly, eEF2K does not facilitate survival of prostate cancer PC3 cells. Thus, eEF2K and autophagy enable survival of certain cell-types in a mutually complementary manner. To explore this further, we generated, by selection, cells which were able to survive nutrient starvation even when autophagy and eEF2K were disabled. Proteome profiling using mass spectrometry revealed that these 'resistant' cells showed lower levels of diverse proteins which are required for energy-consuming processes such as protein and fatty acid synthesis, although different clones of 'resistant cells' appear to adapt in dissimilar ways. Our data provide further information of the ways that human cells cope with nutrient limitation and to understanding of the utility of eEF2K as a potential target in oncology.

The MNKs (mitogen-activated protein kinase-interacting protein kinases) phosphorylate eIF4E (eukaryotic initiation factor 4 E) at serine 209; eIF4E plays an important role in the translation of cytoplasmic mRNAs, all of which possess a 5’ ‘cap’ structure to which eIF4E binds. Elevated levels of eIF4E, p-eIF4E and/or the MNK protein kinases have been found in many types of cancer, including solid tumors and leukemia. MNKs also play a role in metabolic disease. Regulation of the activities of MNKs (MNK1 and MNK2), control the phosphorylation of eIF4E, which in turn has a close relationship with the processes of tumor development, cell migration and invasion, and energy metabolism. MNK knock-out mice display no adverse effects on normal cells or phenotypes suggesting that MNK may be a potentially safe targets for the treatment of various cancers. Several MNK inhibitors or ‘degraders’ have been identified. Initially, some of the inhibitors were developed from natural products or based on other protein kinase inhibitors which inhibit multiple kinases. Subsequently, more potent and selective inhibitors for MNK1/2 have been designed and synthesized. Currently, three inhibitors (BAY1143269, eFT508 and ETC-206) are in various stages of clinical trials for the treatment of solid cancers or leukemia, either alone or combined with inhibitors of other protein kinase. In this review, we summarize the diverse MNK inhibitors that have been reported in patents and other literature, including those with activities in vitro and/or in vivo.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

PD-L1 is abnormally regulated in many cancers and is critical for immune escape. Fully understanding the regulation of PD-L1 expression is vital for improving the clinical efficacy of relevant anticancer agents. TGF-β plays an important role in the low reactivity of PD-1/PD-L1 antibody immunotherapy. However, it is not very clear whether and how TGF-β affects PD-L1 expression. In the present study, we show that TGF-β upregulates the expression of the transcriptional coactivator MRTF-A in non-small-cell lung cancer cells, which subsequently interacts with NF-κB/p65 rather than SRF to facilitate the binding of NF-κB/p65 to the PDL1 promoter, thereby activating the transcription and expression of PD-L1. This leads to the immune escape of NSCLC cells. This process is dependent on the activation of the TGF-β signaling pathway. In vivo, inhibition of MRTF-A effectively suppresses the growth of lung tumor syngrafts with enrichment of NK and T cells in tumor tissue. Our study defines a new signaling pathway that regulates the transcription and expression of PD-L1 upon TGF-β treatment, which may have a significant impact on research into the application of immunotherapy in treating lung cancer.

Background Eukaryotic elongation factor-2 kinase (eEF2K) is a Ca 2+ /calmodulin (CaM)-dependent protein kinase that inhibits protein synthesis. However, the role of eEF2K in cancer development was reported paradoxically and remains to be elucidated. Methods Herein, A549 cells with eEF2K depletion or overexpression by stably transfected lentivirus plasmids were used in vitro and in vivo study. MTT and colony assays were used to detect cell proliferation and growth. Extracellular glucose and lactate concentration were measured using test kit. Immunoblot and co-immunoprecipitation assays were used to examine the molecular biology changes and molecular interaction in these cells. LC-MS/MS analysis and [γ- 32 P] ATP kinase assay were used to identify combining protein and phosphorylation site. Nude mice was utilized to study the correlation of eEF2K and tumor growth in vivo. Results We demonstrated that eEF2K inhibited lung cancer cells proliferation and affected the inhibitory effects of EGFR inhibitor gefitinib. Mechanistically, we showed that eEF2K formed a complex with PKM2 and STAT3, thereby phosphorylated PKM2 at T129, leading to reduced dimerization of PKM2. Subsequently, PKM2 impeded STAT3 phosphorylation and STAT3-dependent c-Myc expression. eEF2K depletion promoted the nuclear translocation of PKM2 and increased aerobic glycolysis reflected by increased lactate secretion and glucose. Conclusions Our findings define a novel mechanism underlying the regulation of cancer cell proliferation by eEF2K independent of its role in protein synthesis, disclosing the diverse roles of eEF2K in cell biology, which lays foundation for the development of new anticancer therapeutic strategies.

Objectives: Diet-driven obesity is increasingly widespread. Its consequences pose major challenges to human health and health care systems. There are MAP kinase-interacting kinases (MNKs) in mice, MNK1 and MNK2. Studies have demonstrated that mice lacking either MNK1 or MNK2 were partially protected against high-fat diet (HFD)-induced weight gain and insulin resistance. The aims of this study were to evaluate the phenotype of mice lacking both MNKs when given an HFD, to assess whether pharmacological inhibition of MNK function also protects against diet-induced obesity (DIO) and its consequences and to probe the mechanisms underlying such protection. Methods: Male wild-type (WT) C57Bl6 mice or mice lacking both MNK1 and MNK2 (double knockout, DKO) were fed an HFD or control diet (CD) for up to 16 weeks.In a separate study, WT mice were also given an HFD for 6 weeks, after which half were treated with the recently-developed MNK inhibitor ETC-206 daily for 10 more weeks while continuing an HFD. Metabolites and other parameters were measured, and the expression of selected mRNAs and proteins was assessed. Results: MNK-DKO mice were almost completely protected from HFD-induced obesity. Higher energy expenditure (EE) in MNK-DKO mice was observed, which probably reflects the changes in a number of genes or proteins linked to lipolysis, mitochondrial function/biogenesis, oxidative metabolism, and/or ATP consumption. The MNK inhibitor ETC-206 also prevented HFD-induced weight gain, confirming that the activity of the MNKs facilitates weight gain due to excessive caloric consumption. Conclusions: Disabling MNKs in mice, either genetically or pharmacologically, strongly prevents weight gain on a calorie-rich diet. This finding likely results from increased energy utilisation, involving greater ATP consumption, mitochondrial oxidative metabolism, and other processes.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.