Human Protein Kinase Research Articles

Assessing Darkness of the Human Kinome from a Medicinal Chemistry Perspective.

In drug discovery, human protein kinases (PKs) represent one of the major target classes due to their central role in cellular signaling, implication in various diseases as a consequence of deregulated signaling, and notable druggability. Individual PKs and their disease biology have been explored to different degrees, giving rise to heterogeneous functional knowledge and disease associations across the human kinome. The U.S. National Institutes of Health previously designated 162 understudied ("dark") human PKs and lipid kinases due to the lack of functional annotations and high-quality molecular probes for functional investigations. Given the large volumes of available PK inhibitors (PKIs) and activity data, we have systematically analyzed the distribution of PKIs and associated data at different confidence levels across the human kinome and distinguished between chemically explored, underexplored, and unexplored PKs. The analysis provides a medicinal chemistry-centric view of PK exploration and further extends prior assessment of the dark kinome.

Prediction of protein-ligand binding sites modulating activity of MAST protein kinases

Aim. Identification of the protein-ligand binding sites, that may be the target of compounds, affecting individual human protein kinases of the MAST family (MAST1, 2, 3, 4 and MASTL / GWL). Methods. Literature and database search. Comparison of protein and ligand structures. Protein structure modeling, structural superimposition, etc. Results. The structural alignment demonstrates significant similarity of catalytic domains in MAST1, 2, 3, 4 and MASTL (GWL). It justifies transferring of reference ligands from PDB structures to human MASTs, discovering potential sites of ligand binding. 13 sites of ligand-binding were specified based on refrence ligands, transferred from RCSB Protein Data Bank structures, and differences in sites amino acid composition of MAST family members were discovered. Сonclusions. Based on the differences in the amino acid composition of the studied pockets in MAST1, 2, 3, 4 and MASTL (GWL), the sites B, C, D, E, F, were selected for further study and virtual screening for new selective inhibitors of individual members of MAST protein kinase family.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

Human DNA-dependent protein kinase catalytic subunit deficiency: A comprehensive review and update

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an essential role in the non-homologous end-joining pathway that repairs DNA double-strand breaks in V(D)J recombination involved in the expression of T- and B-cell receptors. Whereas homozygous mutations in PRKDC define the scid mouse, a model that has been widely used in biology, human mutations in PRKDC are extremely rare and the disease spectrum has not been described so far. To provide an update on the genetics, clinical spectrum, immunological profile, and therapy of DNA-PKcs deficiency in human. The clinical, biological, and treatment data from the 6 cases published to date and from 1 new patient were obtained and analyzed. Rubella PCR was performed on available granuloma material. We report on 7 patients; Six patients displayed the autosomal recessive p.L3062R mutation in PRKDC gene encoding DNA-PKcs. Atypical severe combined immunodeficiency with inflammatory lesions, granulomas, and autoimmunity was the predominant clinical manifestation (n=5/7). Rubella viral strain was detected in the granuloma of 1 patient over the 2 tested. T-cell counts, including naïve CD4+CD45RA+ T cells and T-cell function were low at diagnosis for 6 patients. For most patients with available values naïve CD4+CD45RA+ T cells decreased over time (n=5/6). Hematopoietic stem cell transplantation (HSCT) was performed in 5 patients, of whom 4 are still alive without transplant-related morbidity. Sustained T- and B-cell reconstitution was respectively observed for 4 and 3 patients, after a median follow-up of 8 years (range 3-16 y). DNA-PKcs deficiency mainly manifests as an inflammatory disease with granuloma and autoimmune features, along with severe infections.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Kinase Inhibition via Small Molecule‐Induced Intramolecular Protein Cross‐Linking

AbstractRemarkable progress has been made in the development of cysteine‐targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K–E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D‐targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide‐based small‐molecule inhibitors capable of inducing intramolecular cross‐linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide‐facilitated, ligand‐induced mechanism leading to intramolecular kinase cross‐linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

RedOx regulation of LRRK2 kinase activity by active site cysteines

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson’s disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the “CC” motif. Among all human protein kinases, only LRRK2 contains this “CC” motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S-) in GaMD simulations allowed us to analyse how each of the cysteines in the “CC” motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.

Abstract 7195: Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer

Abstract Background and Aim: Osimertinib is the only 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved for the 1st line therapy of advanced NSCLC patients with EGFR mutations. However, drug resistance is severely limiting its clinical efficacy. Aberrant MET activation is an important mechanism causing osimertinib resistance. There is no effective therapeutic option after osimertinib failure. This study investigated the repurposing of non-oncology MET inhibiting drug to overcome osimertinib resistance. Method: A few clinically-approved drugs with putative MET inhibitory effects were identified by a computational drug repurposing tool called “DRAR-CPI” via analysis of the chemical-protein interactome of known MET inhibitors (crizotinib, cabozantinib, foretinib, and tivantinib). Fexofenadine (FEX), originally indicated for allergic rhinitis and chronic urticaria, was chosen for detailed investigation in osimertinib-resistant NSCLC cell lines that exhibit aberrant MET activation with/without other EGFR-dependent osimertinib resistance mechanisms. The selectivity of kinase inhibition by FEX was evaluated by a KINOME profiling assay against a panel of human protein kinases. Western blot analysis was conducted to examine the activation status of Met and EGFR downstream signaling. RNA sequencing was performed to identify differentially expressed genes (DEGs) triggered by the most promising repurposed drug, presumably contributing to osimertinib resistance circumvention. Sulforhodamine B assay was used to evaluate cell proliferation. The circumvention of osimertinib resistance by FEX was further verified in a patient-derived tumor xenograft (PDX) from an advanced-stage osimertinib-refractory NSCLC patient. Result: FEX was found to inhibit recombinant MET kinase in cell-free assays in a dose- and time-dependent manner. It also inhibited MET phosphorylation and other downstream signaling molecules in osimertinib-resistant NSCLC cell lines harboring EGFR T790M+MET amplification or EGFR T790M loss+MET amplification. KINOME profiling revealed a similar kinase inhibition profile between FEX and cabozantinib using Spearman rank order correlation analysis. Among the tested osimertinib-resistant NSCLC cell lines, FEX showed the highest effectiveness in potentiating the anticancer effect of osimertinib in H820 cells (having MET amplification & EGFR T790M mutation). Transcriptome profiling analysis in H820 cells before and after FEX treatment revealed that the majority of DEGs were enriched in EMT-related biological processes and pathways. FEX was further shown to significantly potentiate the antitumor effect of osimertinib in a drug refractory PDX model, without inducing notable weight loss in the animals. Conclusion: These findings advocate the clinical evaluation of repurposing FEX to overcome osimertinib resistance. Citation Format: Kenneth K.W. To, Kwong Sak Leung, William C.S. Cho. Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7195.

Cellular and clinical impact of protein phosphatase enzyme epigenetic silencing in multiple cancer tissues

BackgroundProtein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signalling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations.MethodsIn this study, we examined promoter-associated DNA methylation profiles in Protein Phosphatase Enzymes and their Interacting Proteins (PPEIP) in a cohort of 705 cancer patients in five tissues (Large intestine, Oesophagus, Lung, Pancreas and Stomach) in three cell models (primary tumours, cancer cell lines and 3D embedded cancer cell cultures). As a subset of PPEIP are known tumour suppressor genes, we analysed the impact of PPEIP promoter hypermethylation marks on gene expression, cellular networks and in a clinical setting.ResultsHere, we report epimutations in PPEIP are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumours have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity.ConclusionsWe propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.

Rapid depletion of target proteins in plants by an inducible protein degradation system.

Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.

Comprehensive Data-Driven Assessment of Non-Kinase Targets of Inhibitors of the Human Kinome.

Protein kinases (PKs) are involved in many intracellular signal transduction pathways through phosphorylation cascades and have become intensely investigated pharmaceutical targets over the past two decades. Inhibition of PKs using small-molecular inhibitors is a premier strategy for the treatment of diseases in different therapeutic areas that are caused by uncontrolled PK-mediated phosphorylation and aberrant signaling. Most PK inhibitors (PKIs) are directed against the ATP cofactor binding site that is largely conserved across the human kinome comprising 518 wild-type PKs (and many mutant forms). Hence, these PKIs often have varying degrees of multi-PK activity (promiscuity) that is also influenced by factors such as single-site mutations in the cofactor binding region, compound binding kinetics, and residence times. The promiscuity of PKIs is often-but not always-critically important for therapeutic efficacy through polypharmacology. Various in vitro and in vivo studies have also indicated that PKIs have the potential of interacting with additional targets other than PKs, and different secondary cellular targets of individual PKIs have been identified on a case-by-case basis. Given the strong interest in PKs as drug targets, a wealth of PKIs from medicinal chemistry and their activity data from many assays and biological screens have become publicly available over the years. On the basis of these data, for the first time, we conducted a systematic search for non-PK targets of PKIs across the human kinome. Starting from a pool of more than 155,000 curated human PKIs, our large-scale analysis confirmed secondary targets from diverse protein classes for 447 PKIs on the basis of high-confidence activity data. These PKIs were active against 390 human PKs, covering all kinase groups of the kinome and 210 non-PK targets, which included other popular pharmaceutical targets as well as currently unclassified proteins. The target distribution and promiscuity of the 447 PKIs were determined, and different interaction profiles with PK and non-PK targets were identified. As a part of our study, the collection of PKIs with activity against non-PK targets and the associated information are made freely available.

Using explainable machine learning to uncover the kinase-substrate interaction landscape.

Phosphorylation, a post-translational modification regulated by protein kinase enzymes, plays an essential role in almost all cellular processes. Understanding how each of the nearly 500 human protein kinases selectively phosphorylates their substrates is a foundational challenge in bioinformatics and cell signaling. Although deep learning models have been a popular means to predict kinase-substrate relationships, existing models often lack interpretability and are trained on datasets skewed toward a subset of well-studied kinases. Here we leverage recent peptide library datasets generated to determine substrate specificity profiles of 300 serine/threonine kinases to develop an explainable Transformer model for kinase-peptide interaction prediction. The model, trained solely on primary sequences, achieved state-of-the-art performance. Its unique multitask learning paradigm built within the model enables predictions on virtually any kinase-peptide pair, including predictions on 139 kinases not used in peptide library screens. Furthermore, we employed explainable machine learning methods to elucidate the model's inner workings. Through analysis of learned embeddings at different training stages, we demonstrate that the model employs a unique strategy of substrate prediction considering both substrate motif patterns and kinase evolutionary features. SHapley Additive exPlanation (SHAP) analysis reveals key specificity determining residues in the peptide sequence. Finally, we provide a web interface for predicting kinase-substrate associations for user-defined sequences and a resource for visualizing the learned kinase-substrate associations. All code and data are available at https://github.com/esbgkannan/Phosformer-ST. Web server is available at https://phosformer.netlify.app.

Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2

Histidine residues contribute to numerous molecular interactions, owing to their structure with the ionizable aromatic side chain with pKa close to the physiological pH. Herein, we studied how the two histidine residues, His115 and His160 of the catalytic subunit of human protein kinase CK2, affect the binding of the halogenated heterocyclic ligands at the ATP-binding site. Thermodynamic studies on the interaction between five variants of hCK2α (WT protein and four histidine mutants) and three ionizable bromo-benzotriazoles and their conditionally non-ionizable benzimidazole counterparts were performed with nanoDSF, MST, and ITC. The results allowed us to identify the contribution of interactions involving the particular histidine residues to ligand binding. We showed that despite the well-documented hydrogen bonding/salt bridge formation dragging the anionic ligands towards Lys68, the protonated His160 also contributes to the binding of such ligands by long-range electrostatic interactions. Simultaneously, His 115 indirectly affects ligand binding, placing the hinge region in open/closed conformations.

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

An Update on Protein Kinases as Therapeutic Targets-Part II: Peptides as Allosteric Protein Kinase C Modulators Targeting Protein-Protein Interactions.

Human protein kinases are highly-sought-after drug targets, historically harnessed for treating cancer, cardiovascular disease, and an increasing number of autoimmune and inflammatory conditions. Most current treatments involve small molecule protein kinase inhibitors that interact orthosterically with the protein kinase ATP-binding pocket. As a result, these compounds are often poorly selective and highly toxic. Part I of this series reviews the role of PKC isoforms in various human diseases, featuring cancer and cardiovascular disease, as well as translational examples of PKC modulation applied to human health and disease. In the present Part II, we discuss alternative allosteric binding mechanisms for targeting PKC, as well as novel drug platforms, such as modified peptides. A major goal is to design protein kinase modulators with enhanced selectivity and improved pharmacological properties. To this end, we use molecular docking analysis to predict the mechanisms of action for inhibitor-kinase interactions that can facilitate the development of next-generation PKC modulators.

Understanding Glycogen Synthase Kinase-3: A Novel Avenue for Alzheimer's Disease.

Alzheimer's Disease (AD) is the most prevalent form of age-related dementia. Even though a century has passed since the discovery of AD, the exact cause of the disease still remains unknown. As a result, this poses a major hindrance in developing effective therapies for treating AD. Glycogen synthase kinase-3 (GSK-3) is one of the kinases that has been investigated recently as a potential therapeutic target for the treatment of AD. It is also known as human tau protein kinase and is a proline-directed serine-threonine kinase. Since dysregulation of this kinase affects all the major characteristic featuresof the disease, such as tau phosphorylation, amyloid formation, memory, and synaptic function, it is thought to be a major playerin the pathogenesis of AD. In this review, we present the most recent information on the role of this kinase in the onset and progression of AD, as well as significant findings that identify GSK-3 as one of the most important targets for AD therapy. We further discuss the potential of treating AD by targeting GSK-3 and give an overview of the ongoing studies aimed at developing GSK-3 inhibitors in preclinical and clinical investigations.

In silico and structure-based evaluation of deleterious mutations identified in human Chk1, Chk2, and Wee1 protein kinase.

Checkpoint kinases Chk1, Chk2, Wee1 are playing a key role in DNA damage response and genomic integrity. Cancer-associated mutations identified in human Chk1, Chk2, and Wee1 were retrieved to understand the function associated with the mutation and also alterations in the folding pattern. Therefore, an attempt has been made to identify deleterious effect of variants using in silico and structure-based approach. Variants of uncertain significance for Chk1, Chk2, and Wee1 were retrieved from different databases and four prediction servers were employed to predict pathogenicity of mutations. Further, Interpro, I-Mutant 3.0, Consurf, TM-align, and have (y)our protein explained were used for comprehensive study of the deleterious effects of variants. The sequences of Chk1, Chk2, and Wee1 were analyzed using Clustal Omega, and the three-dimensional structures of the proteins were aligned using TM-align. The molecular dynamics simulations were performed to explore the differences in folding pattern between Chk1, Chk2, Wee1 wild-type, and mutant protein and also to evaluate the structural integrity. Thirty-six variants in Chk1, 250 Variants in Chk2, and 29 in Wee1 were categorized as pathogenic using in silico prediction tools. Furthermore, 25 mutations in Chk1, 189 in Chk2, and 14 in Wee1 were highly conserved, possessing deleterious effect and also influencing the protein structure and function. These identified mutations may provide underlying genetic intricacies to serve as potential targets for therapeutic inventions and clinical management.

Association of temozolomide with progressive multifocal leukoencephalopathy: a disproportionality analysis integrated with network pharmacology

ABSTRACT Background Temozolomide (TMZ) is an alkylating agent approved for the management of glioblastoma. The TMZ is not known for progressive multifocal leukoencephalopathy (PML). The main objective of the current study is to find out the association of TMZ with PML using disproportionality analysis of FDA Adverse Event Reporting System (FAERS) data integrated with network pharmacological approaches. Research design and methods OpenVigil tool was used to query the FAERS database. The disproportionality measures were calculated. The network has been constructed using Cytoscape. Finally, the possible binding interactions were studied using Glide, Schrödinger Inc. Results A total number of 3502 cases of PML were reported in the FAERS database. Out of these, 10 cases were found with TMZ. The subgroup analysis results have shown a greater number of cases in females. The network has indicated the involvement of human mitogen-activated protein kinase, 3-phosphoinositide-dependent protein kinase 1 protein, human mTOR complex protein, phosphatidylinositol 4,5-bisphosphate 3-kinase protein, and glycogen synthase kinase-3 beta protein. The docking results have indicated good interactions of TMZ with active site of glycogen synthase kinase-3 beta and mitogen-activated protein kinase 1 as compared to other identified targets. Conclusion The PML is identified as novel signal with temozolomide.