Regulation Of Kinase Activity Research Articles

14-3-3 phosphorylation inhibits 14-3-3θ's ability to regulate LRRK2 kinase activity and toxicity.

LRRK2 mutations are among the most common genetic causes for Parkinson's disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3θ isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3θ phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3θ mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3θ mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3θ mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3θ phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3θ and phosphorylated T2524 LRRK2 was important for 14-3-3θ's ability to regulate kinase activity, as wildtype and S232A 14-3-3θ failed to reduce the kinase activity of G2019S/T2524A LRRK2. Finally, we found that the S232D mutation failed to protect against G2019S LRRK2-induced neurite shortening in primary cultures, while the S232A mutation was protective. We conclude that 14-3-3θ phosphorylation destabilizes the interaction of 14-3-3θ with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity and toxicity.

Adenosine protects cardiomyocytes against acrolein-induced cardiotoxicity by enhancing mitochondrial homeostasis, antioxidant defense, and autophagic flux via ERK-activated FoxO1 upregulation

Acrolein is a ubiquitous gaseous air pollutant and endogenous toxicant, which poses strong risk for oxidative stress-related diseases such as cardiovascular disease. Adenosine has been identified as potential therapeutic agent for age-related cardiovascular disease, while the molecular mechanisms underlying its cardioprotection remain elusive. In the present study, we investigated the myocardial protective effects and the mechanism of adenosine on acrolein-induced toxicity in H9c2 cells and primary neonatal rat cardiomyocytes. We found that acrolein caused apoptosis of cardiomyocytes resulting from oxidative damage, autophagy defect, and mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential, impairment of mitochondrial biogenesis, dynamics, and oxidative phosphorylation, decrease of mitochondrial deoxyribonucleic acid (mtDNA) copy number and adenosine 5’-triphosphate (ATP) production. Adenosine pretreatment protected against acrolein-induced cardiotoxicity by maintaining mitochondrial homeostasis, activating the phase II detoxifying enzyme system, promoting autophagic flux, and alleviating mitochondrial-dependent apoptosis. We further demonstrated that the up-regulation of forkhead box protein O1 (FoxO1) mediated by extracellular regulated protein kinases (ERK) activation contributes to the cardioprotection of adenosine. These results expand the application of adenosine in cardioprotection to preventing myocardial damages induced by environmental pollutant acrolein exposure, and uncover the adenosine-ERK-FoxO1 axis as the underlying mechanism mediating the protection of mitochondrial homeostasis, Nrf2-mediated antioxidant defense and autophagic flux, shedding light on the better understanding about the pathological mechanism of cardiovascular disease caused by environmental pollutants and applications of adenosine in cardioprotection.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

HPK1 citron homology domain regulates phosphorylation of SLP76 and modulates kinase domain interaction dynamics

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed β-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.

Structural insights into the regulation of protein-arginine kinase McsB by McsA

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

Structural determinants for membrane binding of the EGFR juxtamembrane domain.

Overactivation of the epidermal growth factor receptor (EGFR) is critical for the development of multiple cancers. Previous studies have shown that the cell membrane is a key regulator of EGFR kinase activity through its interaction with the EGFR juxtamembrane domain (JM). However, the lipid recognition specificity of EGFR-JM and its interaction details remain unclear. Using lipid strip and liposome pulldown assays, we showed that EGFR-JM could specifically interact with PI(4,5)P2-or phosphatidylserine-containing membranes. We further characterized the JM-membrane interaction using NMR-titration-based chemical shift perturbation and paramagnetic relaxation enhancement analyses, and found that residues I649 - L659 comprised the membrane-binding site. Furthermore, the membrane-binding region contains the predicted dimerization motif of JM, 655LRRLL659, suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity.

Genetic information is transcribed from genomic DNA to mRNA, which is then translated into three-dimensional proteins. mRNAs can undergo various post-transcriptional modifications, including RNA editing that alters mRNA sequences, ultimately affecting protein function. In this study, RNA editing was identified at the 499th base (c.499) of human vaccinia-related kinase 2 (VRK2). This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine (with adenine base) to valine (with guanine base). Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2, which leads to an increase in tumor cell proliferation. Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein (dysbindin) and results in reducing its stability. Herein, we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valine-containing VRK2. Dysbindin interacts with cyclin D and thereby regulates its expression and function. The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression, resulting in increased tumor growth and reduction in survival rates. It has also been observed that in patient samples, VRK2 level was elevated in breast cancer tissue compared to normal breast tissue. Additionally, the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue. Therefore, it is concluded that VRK2, especially dependent on the 167th variant amino acid, can be one of the indexes of tumor progression and proliferation.

N-Feruloylserotonin inhibits lipopolysaccharide-induced inflammation via SIRT1-stimulated FOXO1 and NF-κB signaling pathways in RAW 264.7 cells.

Macrophages become activated by a variety of stimuli such as lipopolysaccharide (LPS) and participate in the process of immune responses. Activated macrophages produce various inflammatory mediators. In the present study, we investigated the anti-inflammatory mechanism of a serotonin derivative, N-feruloylserotonin, isolated from safflower seeds in RAW 264.7 macrophages. N-Feruloylserotonin treatment significantly attenuated these effects on LPS-induced reactive oxygen species, nitric oxide, and prostaglandin E2 production in RAW 264.7 macrophages. Furthermore, N-feruloylserotonin significantly decreased the abnormal expression of mitogen-activated protein kinase, such as phosphor (p)-c-Jun N-terminal kinase and p-extracellular-signal regulated kinase activation. Further research revealed that N-feruloylserotonin could stimulate sirtuin1 (SIRT1), then promote the forkhead box protein O1 (FOXO1), and suppress nuclear factor-kappa B (NF-kB) signaling pathways. The present study suggests that N-feruloylserotonin may be a new anti-inflammatory component and a promising candidate for anti-inflammatory therapeutic agents through the regulation of SIRT1-stimulated FOXO1 and NF-kB signaling pathways.

Allosteric regulation of kinase activity in living cells

The dysregulation of protein kinases is associated with multiple diseases due to the kinases' involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities, making it difficult to specifically target one kinase, and allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss the methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or ‘sensors,’ are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.

Allosteric regulation of kinase activity in living cells.

The dysregulation of protein kinases is associated with multiple diseases due to the kinases' involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities, making it difficult to specifically target one kinase, and allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss the methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or 'sensors,' are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.

From Tea to Health: Exploring Abrus mollis for Liver Protection and Unraveling Its Potential Mechanisms.

Abrus mollis Hance is a characteristic medicinal herb which is used in Guangdong and Guangxi provinces of China for making soup, medicinal meals, and herbal tea to treat dampheat jaundice and rib discomfort. Current phytochemical study on A. mollis led to the isolation of four new flavones, mollisone A-D (1-4), and thirty two known compounds (5-36). Their structures were characterized by an extensive analysis of spectroscopic data including IR, UV, HR-ESI-MS, and 1D and 2D NMR, as well as electronic circular dichroism calculation. In addition, in order to initially understand their biological activities for traditional applications, in vitro antioxidant and hepatoprotective tests were carried out, whose results illustrated that 25 compounds had significant free radical scavenging ability, and compounds 13 and 16 exhibited protective activities on D-GalN-induced LO2 cell damage than the positive control. Moreover, network pharmacological analysis revealed that the hepatoprotective activity of A. mollis involved multitargets and multipathways such as PI3K/Akt, MAPK, and JAK-STAT pathways and various biological processes such as positive regulation of phosphorylation and regulation of kinase activity. These results suggested that this species could serve as a potential hepatoprotective agent for functional food or medicinal use.

Possible molecular exploration of herbal pair Haizao-Kunbu in the treatment of Graves' disease by network pharmacology, molecular docking, and molecular dynamic analysis.

To promote the development and therapeutic application of new medications, it is crucial to conduct a thorough investigation into the mechanism by which the traditional Chinese herb pair of Haizao-Kunbu (HK) treats Graves' disease (GD). Chemical ingredients of HK, putative target genes, and GD-associated genes were retrieved from online public databases. Using Cytoscape 3.9.1, a compound-gene target network was established to explore the association between prosperous ingredients and targets. STRING, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses visualized core targets and disease pathways. Additionally, we conducted a refined analysis of the binding interactions between active ingredients and their respective targets. To visualize these findings, we employed precise molecular docking techniques. Furthermore, we carried out molecular dynamics simulations to gain insights into the formation of more tightly bound complexes. We found that there were nine key active ingredients in HK, which mainly acted on 21 targets. These targets primarily regulated several biological processes such as cell population proliferation, protein phosphorylation, and regulation of kinase activity, and acted on PI3K-AKT and MAPK pathways to treat GD. Analysis of the molecular interaction simulation under computer technology revealed that the key targets exhibited strong binding activity to active ingredients, and Fucosterol-AKT1 and Isofucosterol-AKT1 complexes were highly stable in humans. This study demonstrates that HK exerts therapeutic effects on GD in a multi-component, multi-target, and multi-pathway manner by regulating cell proliferation, differentiation, inflammation, and immunomodulatory-related targets. This study provides a theoretical foundation for further investigation into GD.

Computational analysis of regulatory regions in human protein kinases.

Eukaryotic proteins often feature modular domain structures comprising globular domains that are connected by linker regions and intrinsically disordered regions that may contain important functional motifs. The intramolecular interactions of globular domains and nonglobular regions can play critical roles in different aspects of protein function. However, studying these interactions and their regulatory roles can be challenging due to the flexibility of nonglobular regions, the long insertions separating interacting modules, and the transient nature of some interactions. Obtaining the experimental structures of multiple domains and functional regions is more difficult than determining the structures of individual globular domains. High-quality structural models generated by AlphaFold offer a unique opportunity to study intramolecular interactions in eukaryotic proteins. In this study, we systematically explored intramolecular interactions between human protein kinase domains (KDs) and potential regulatory regions, including globular domains, N- and C-terminal tails, long insertions, and distal nonglobular regions. Our analysis identified intramolecular interactions between human KDs and 35 different types of globular domains, exhibiting a variety of interaction modes that could contribute to orthosteric or allosteric regulation of kinase activity. We also identified prevalent interactions between human KDs and their flanking regions (N- and C-terminal tails). These interactions exhibit group-specific characteristics and can vary within each specific kinase group. Although long-range interactions between KDs and nonglobular regions are relatively rare, structural details of these interactions offer new insights into the regulation mechanisms of several kinases, such as HASPIN, MAPK7, MAPK15, and SIK1B.

Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue.

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phospho-mimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis.

A comprehensive rhythmicity analysis of host proteins and immune factors involved in malaria pathogenesis to decipher the importance of host circadian clock in malaria.

Circadian rhythms broadly impact human health by regulating our daily physiological and metabolic processes. The circadian clocks substantially regulate our immune responses and susceptibility to infections. Malaria parasites have intrinsic molecular oscillations and coordinate their infection cycle with host rhythms. Considering the cyclical nature of malaria, a clear understanding of the circadian regulations in malaria pathogenesis and host responses is of immense importance. We have thoroughly investigated the transcript level rhythmic patterns in blood proteins altered in falciparum and vivax malaria and malaria-related immune factors in mice, baboons, and humans by analyzing datasets from published literature and comprehensive databases. Using the Metascape and DAVID platforms, we analyzed Gene Ontology terms and physiological pathways associated with the rhythmic malaria-associated host immune factors. We observed that almost 50% of the malaria-associated host immune factors are rhythmic in mice and humans. Overlapping rhythmic genes identified in mice, baboons, and humans, exhibited enrichment (Q < 0.05, fold-enrichment > 5) of multiple physiological pathways essential for host immune and defense response, including cytokine production, leukocyte activation, cellular defense, and response, regulation of kinase activity, B-cell receptor signaling pathway, and cellular response to cytokine stimulus. Our analysis indicates a robust circadian regulation on multiple interconnected host response pathways and immunological networks in malaria, evident from numerous rhythmic genes involved in those pathways. Host immune rhythms play a vital role in the temporal regulation of host-parasite interactions and defense machinery in malaria.

A critical evaluation of protein kinase regulation by activation loop autophosphorylation.

Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in trans, mediated by transient dimerization of their kinase domains. However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks.

LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease (PD), with growing importance also for Crohn’s disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.

Identification of Key Genes Involved in Carcinogenesis and Progression of Colon Cancer Based on Bioinformatics

This study aimed to identify key genes associated with colon cancer development. Two datasets (GSE101502 and GSE20916) were obtained from the GEO database and subjected to online analysis. The mirDIP tool predicted target genes based on differentially expressed miRNAs in GSE101502. The DAVID database performed Gene Ontology (GO) and KEGG pathway enrichment analyses on differentially expressed genes (DEGs). The PPI network of DEGs was constructed using the STRING database and visualized with Cytoscape software. From GSE101502, 21 differentially expressed miRNAs were identified, while GSE20916 yielded 921 DEGs. By intersecting the two datasets, 112 common DEGs (co-DEGs) were screened. GO analysis revealed that DEGs were involved in various biological processes, including extracellular matrix organization, kinase activity regulation, and cell-matrix adhesion. KEGG pathway analysis indicated their participation in cancer-related pathways, such as viral carcinogenesis and microRNAs in cancer. Nine hub genes were identified, namely CCNB1, XPO4, KIF1B, PLK4, KMT2A, EP300, ECT2, FBN1, and RB1. These hub genes are closely associated with colon cancer and hold potential as biomarkers for its diagnosis and prognosis.

Analysis on benzothiazole necroptosis inhibitors with chiral substitutions in the solvent-accessible region of RIP kinase domain

Receptor-interacting protein kinase 1 (RIPK1) and RIPK3, two imperative targets of the necroptosis pathway, are associated with various inflammatory-related diseases. Regulating kinase activity with inhibitors has been confirmed as a promising strategy for inflammation treatment. However, most of the reported type I and II kinase inhibitors of RIPK1 and RIPK3, including benzothiazole compounds discovered by our group, have selective limitations due to interaction with ATP-binding pockets. Fortunately, a solvent exposure E0 region of the kinase domain, which extends into the linker region, has been reported to be related to the potency and selectivity of inhibitors. Hence, based on our previous study, a series of benzothiazole necroptosis inhibitors with chiral substitutions in the linker region were developed to investigate RIPK1/3 inhibitory potency. The results showed a 2-to 6-fold increase in anti-necroptotic activity for these chiral compounds. The improved selectivity on RIPK1 or RIPK3 was demonstrated on different derivatives. Predicted binding conformations of enantiomers with RIPK1/3 gave an explanation for their activity differences, guiding further rational design of chiral necroptosis inhibitors.