Tyrosine Kinase Domain Research Articles

A recurrent NTRK1 tyrosine kinase domain mutation pair is characteristic in a subset of dedifferentiated liposarcomas

IntroductionDedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes. Materials and Methods131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines. ResultsOut of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant. ConclusionsWe detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

Case report: Glycaemic management and pregnancy outcomes in a woman with an insulin receptor mutation, p.Met1180Lys

BackgroundHeterozygous insulin receptor mutations (INSR) are associated with insulin resistance, hyperglycaemia and hyperinsulinaemic hypoglycaemia in addition to hyperandrogenism and oligomenorrhoea in women. Numerous autosomal dominant heterozygous mutations involving the INSR β-subunit tyrosine kinase domain resulting in type A insulin resistance have been previously described. We describe the phenotype, obstetric management and neonatal outcomes in a woman with type A insulin resistance caused by a mutation in the β-subunit of the INSR.Case presentationWe describe a woman with a p.Met1180Lys mutation who presents with hirsutism, oligomenorrhoea and diabetes at age 20. She has autoimmune thyroid disease, Coeliac disease and positive GAD antibodies. She is overweight with no features of acanthosis nigricans and is treated with metformin. She had 11 pregnancies treated with insulin monotherapy (n = 2) or combined metformin and insulin therapy (n = 9). The maximum insulin dose requirement was 134 units/day or 1.68 units/kg/day late in the second pregnancy. Mean birthweight was on the 37th centile in INSR positive offspring (n = 3) and the 94th centile in INSR negative offspring (n = 1).ConclusionThe p.Met1180Lys mutation results in a phenotype of diabetes, hirsutism and oligomenorrhoea. This woman had co-existent autoimmune disease. Her insulin dose requirements during pregnancy were similar to doses observed in women with type 2 diabetes. Metformin may be used to improve insulin sensitivity in women with this mutation. Offspring inheriting the mutation tended to be smaller for gestational age.

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

102TiP A phase III randomised controlled trial of zongertinib (BI 1810631) compared with standard of care (SoC) in patients (pts) with locally advanced or metastatic non-squamous non-small cell lung cancer (NSCLC) harbouring HER2 tyrosine kinase domain (TKD) mutations: Beamion LUNG-2

102TiP A phase III randomised controlled trial of zongertinib (BI 1810631) compared with standard of care (SoC) in patients (pts) with locally advanced or metastatic non-squamous non-small cell lung cancer (NSCLC) harbouring HER2 tyrosine kinase domain (TKD) mutations: Beamion LUNG-2

Docking, Synthesis and Evaluation of 4-hydroxy-1-phenyl-2(1H)-quinolone Derivatives as Anticancer Agents.

The estimated number of cancer cases in India for the year 2022 was found to be 14,61,427. The development of chemotherapeutic agents has reduced the mortality rate, however, they have high toxicity which is a disadvantage. Many researchers have found out that quinolin-2- one possesses anticancer activity, with this background we thought of synthesizing the quinolin-2-one derivatives. This study aimed to carry out docking, synthesis, characterization, and evaluation of 2-(2- (4-Hydroxy-2-oxoquinolin-1(2H)-yl)phenyl/ substituted phenyl)-3-(phenylamino) thiazolidon-4-one derivatives (IVa-g) as an anticancer agent. Diphenylamine and malonic acid treated with phosphoryl chloride gave compound I, which on formylation afforded compound II, which on reaction with various substituted aromatic phenylhydrazine derivatives gave compounds IIIa-g, which on further reaction with thioglycolic acid and anhydrous zinc chloride yielded the compounds IVa-g. Among all the synthesized novel derivatives, compounds IV a-d showed 50% lysis in the IC50 range of 25-50μg for the A549 cell line, and compounds IVa, and IVb showed 50% lysis in the IC50 range of 25-50μg for the MDA-MB cell line. The compound, 3-((4-fluorophenyl)amino)-2-(2-(4- hydroxy-2-oxoquinolin-1(2H)-yl)phenyl)thiazolidin-4-one (IVg) was found to be the most active against both the cell line, A549 and MDA-MB with IC50 value of 0.0298μmol and 0.0338μmol respectively. The docking results revealed that the synthesized compounds exhibited well-conserved hydrogen bonding with one or more amino acid residues in the active pocket of EGFR tyrosine kinase domain with 4-anilinoquinazoline inhibitor erlotinib (PDB ID:1M17). Compound IVg showed the highest MolDock score of -137.813 compared to the standard drug Imatinib having a MolDock score of -119.354. Compound IVg showed the highest MolDock score and was also found to be most potent against both the cell line, A549, and MDA-MB.

Compounds Consisting of Quinazoline, Ibuprofen, and Amino Acids with Cytotoxic and Anti-Inflammatory Effects.

In this research work, a series of 16 quinazoline derivatives bearing ibuprofen and an amino acid were designed as inhibitors of epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) and cyclooxygenase-2 (COX-2) with the intention of presenting dual action in their biological behavior. The designed compounds were synthesized and assessed for cytotoxicity on epithelial cancer cells lines (AGS, A-431, MCF-7, MDA-MB-231) and epithelial non-tumorigenic cell line (HaCaT). From this evaluation, derivative 6 was observed to exhibit higher cytotoxic potency (IC50) than gefitinib (reference drug) on three cancer cell lines (0.034 μM in A-431, 2.67 μM in MCF-7, and 3.64 μM in AGS) without showing activity on the non-tumorigenic cell line (>100 μM). Furthermore, assessment of EGFR-TKD inhibition by 6 showed a discreet difference compared to gefitinib. Additionally, 6 was used to conduct an in vivo anti-inflammatory assay using the 12-O-tetradecanoylphorbol-3-acetate (TPA) method, and it was shown to be 5 times more potent than ibuprofen. Molecular dynamics studies of EGFR-TKD revealed interactions between compound 6 and M793. On the other hand, one significant interaction was observed for COX-2, involving S531. The RMSD graph indicated that the ligand remained stable in 50 ns.

Exploring In-Silico, In-Vitro Antioxidant, and Cytotoxic Potential of Valerian wallichii by On Cervical Epithelial Carcinoma (HeLa) Cell Lines.

Traditional medicinal practices often utilize herbal remedies for treating various diseases. This study focuses on exploring the phytochemical constituents, in-silico, in-vitro antioxidant, and anticancer activities of Valerian wallichii root extracts on human cervical epithelial carcinoma (HeLa) cell lines. The molecular docking approach was employed to predict the ligand molecule's orientation within the receptor like Epidermal growth factor receptor tyrosine kinase domain (PDB ID: 1M17) using Schrodinger's GLIDE model. Among the selected phytocompounds, hesperidin exhibited promising inhibitory activity against EGFR (Epidermal Growth Factor Receptor) domain with -8.701 kcal/mol docking score and interactions with MET 769, ASP 831, ASP776, LEU694 and ASN818 residues as compared to standard doxorubicin with -7.6 kcal/mol docking score and interactions with ASP 831, ASN818 and ASP776 residues and further, various antioxidant activity was assessed and In-vitro anticancer activity against HeLa cell lines was evaluated by hydroalcoholic root extracts demonstrated antioxidant capacities, and selective cytotoxicity was observed, with IC50 : 45.759±0.42 μg/mL for the overall extract and IC50 : 30.245±0.58 μg/mL for the EAF fraction as compared to standard doxorubicin with IC50 : 25.9891±0.25 μg/mL, respectively. The present study concluded that Valerian wallichii L possesses potential human cervical epithelial carcinoma cell line inhibition properties based on the computer aided drug design models and in-vitro activity.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

BRCC36 associates with FLT3-ITD to regulate its protein stability and intracellular signaling in acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.

Secondary metabolites of Trichoderma spp. as EGFR tyrosine kinase inhibitors: Evaluation of anticancer efficacy through computational approach.

The present study explores the epidermal growth factor receptor (EGFR) tyrosine kinase inhibition efficacy of secondary metabolites in Trichoderma spp. through molecular docking, molecular dynamics (MD) simulation and MM-PBSA approach. The result of molecular docking confirmed that out of 200 metabolites screened, three metabolites such as Harzianelactone A, Pretrichodermamide G and Aspochalasin M, potentially bound with the active binding site of EGFR tyrosine kinase domain(PDB ID: 1M17) with a threshold docking score of ≤- 9.0 kcal/mol when compared with the standard EGFR inhibitor (Erlotinib). The MD simulation was run to investigate the potential for stable complex formation in EGFR tyrosine kinase domain-unbound/lead metabolite (Aspochalasin M)-bound/standard inhibitor (Erlotinib)-bound complex. The MD simulation analysis at 100 ns revealed that Aspochalasin M formed the stable complex with EGFR. Besides, the in silico predication of pharmacokinetic properties further confirmed that Aspochalasin M qualified the drug-likeness rules with no harmful side effects (viz., hERG toxicity, hepatotoxicity and skin sensitization), non-mutagenicity and favourable logBB value. Moreover, the BOILED-Egg model predicted that Aspochalasin M showed a higher gastrointestinal absorption with improved bioavailability when administered orally and removed from the central nervous system (CNS). The results of the computational studies concluded that Aspochalasin M possessed significant efficacy in binding EGFR's active sites compared to the known standard inhibitor (Erlotinib). Therefore, Aspochalasin M can be used as a possible anticancer drug candidate and further in vitro and in vivo experimental validation of Aspochalasin M of Trichoderma spp. are required to determine its anticancer potential.

Expansion of the complex genotypic and phenotypic spectrum of FGFR2-associated neurocutaneous syndromes

The fibroblast growth factor receptors comprise a family of related but individually distinct tyrosine kinase receptors. Within this family, FGFR2 is a key regulator in many biological processes, e.g., cell proliferation, tumorigenesis, metastasis, and angiogenesis. Heterozygous activating non-mosaic germline variants in FGFR2 have been linked to numerous autosomal dominantly inherited disorders including several craniosynostoses and skeletal dysplasia syndromes. We report on a girl with cutaneous nevi, ocular malformations, macrocephaly, mild developmental delay, and the initial clinical diagnosis of Schimmelpenning–Feuerstein–Mims syndrome, a very rare mosaic neurocutaneous disorder caused by postzygotic missense variants in HRAS, KRAS, and NRAS. Exome sequencing of blood and affected skin tissue identified the mosaic variant c.1647=/T > G p.(Asn549=/Lys) in FGFR2, upstream of the RAS signaling pathway. The variant is located in the tyrosine kinase domain of FGFR2 in a region that regulates the activity of the receptor and structural mapping and functional characterization revealed that it results in constitutive receptor activation. Overall, our findings indicate FGFR2-associated neurocutaneous syndrome as the accurate clinical-molecular diagnosis for the reported individual, and thereby expand the complex genotypic and phenotypic spectrum of FGFR-associated disorders. We conclude that molecular analysis of FGFR2 should be considered in the genetic workup of individuals with the clinical suspicion of a mosaic neurocutaneous condition, as the knowledge of the molecular cause might have relevant implications for genetic counseling, prognosis, tumor surveillance and potential treatment options.

A novel approach to study multi-domain motions in JAK1's activation mechanism based on energy landscape.

The family of Janus Kinases (JAKs) associated with the JAK-signal transducers and activators of transcription signaling pathway plays a vital role in the regulation of various cellular processes. The conformational change of JAKs is the fundamental steps for activation, affecting multiple intracellular signaling pathways. However, the transitional process from inactive to active kinase is still a mystery. This study is aimed at investigating the electrostatic properties and transitional states of JAK1 to a fully activation to a catalytically active enzyme. To achieve this goal, structures of the inhibited/activated full-length JAK1 were modelled and the energies of JAK1 with Tyrosine Kinase (TK) domain at different positions were calculated, and Dijkstra's method was applied to find the energetically smoothest path. Through a comparison of the energetically smoothest paths of kinase inactivating P733L and S703I mutations, an evaluation of the reasons why these mutations lead to negative or positive regulation of JAK1 are provided. Our energy analysis suggests that activation of JAK1 is thermodynamically spontaneous, with the inhibition resulting from an energy barrier at the initial steps of activation, specifically the release of the TK domain from the inhibited Four-point-one, Ezrin, Radixin, Moesin-PK cavity. Overall, this work provides insights into the potential pathway for TK translocation and the activation mechanism of JAK1.

Gefitinib: Combination Therapy and Complex Delivery Systems (Review)

Introduction. The search for new methods of therapy for non-small cell lung cancer (NSCLC) is an urgent task of modern science. Gefitinib is a targeted drug widely used in the treatment of NSCLC in patients with a mutation in the epidermal growth factor receptor tyrosine kinase domain. However, using of gefitinib and other drugs from the group of tyrosine kinase inhibitors is to limited by rapidly developing resistance, for this reason finding of a ways overcome drug resistance is actual part of research interests.Text. The review is devoted to the use of gefitinib in modern developments: introduction to various targeted delivery systems (liposomes, micelles, microspheres, etc.), studying it in combination with other chemotherapeutic agents, as well as in combination with photo- or thermosensitive compounds in various micro- and nanostructured complexes.Conclusion. As a result of the analysis of literature data, it was shown that, despite the fact that gefitinib is a first-generation drug, foreign and Russian researchers consider it quite promising for further use in the treatment of NSCLC. At the same time, developments are being carried out both in the field of expanding combination therapy and in the field of creating complex structures of targeted action, into which, in addition to gefitinib, photosensitizers or other compounds with photo- or thermosensitive effects are introduced.

Comprehensive structural and functional analysis of hVEGFR1: Insights into phosphorylation, molecular interactions, and potential inhibitors through docking and dynamics simulations

Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), is an enzyme with tyrosine kinase activity that plays a pivotal role in angiogenesis, the process of new blood vessel formation. This receptor is of significant clinical importance as it is implicated in various cancers, particularly non-small cell lung cancer (NSCLC), where its dysregulation leads to uncontrolled cell growth through ligand-induced phosphorylation. While commercially available drugs target VEGFR1, their prolonged use often leads to drug resistance and the emergence of mutations in cancer patients. To address these challenges, researchers have identified the human tyrosine kinase (hTK) domain of VEGFR1 as a potential therapeutic marker for lung malignancies. The 3D crystal structure of the hTK domain, obtained from Protein Data Bank (PDB ID: 3HNG), has provided vital structural insights of hVEGFR1. This study has revealed variations within the hVEGFR1 tyrosine kinase domain, distinguishing between regions associated with phosphorylase kinase and transferase activities. We identified numerous potential phosphorylation sites within the TK domain, shedding light on the protein's regulation and signaling possible. Detailed molecular interaction analyses have elucidated the binding forces between lead molecules and hVEGFR1, including hydrogen bonds, electrostatic, hydrophobic, and π-sigma interactions. The stability observed during molecular dynamics simulations further underscores the biological relevance of these interactions. Furthermore, docked complexes has highlighted localized structural fluctuations, offering insight into potential allosteric effects and dynamic conformational changes induced by lead molecules. These findings not only provide a comprehensive characterization of hVEGFR1 but also pave the way for the development of targeted therapies. Eventually, this study has the potential in identifying drug to combat diseases associated with hVEGFR1 dysregulation, including cancer and angiogenesis-related disorders, contributing to effective treatment strategies.

Schizandrin A induces non-small cell lung cancer apoptosis by suppressing the epidermal growth factor receptor activation.

The purpose of this study is to explore the biological mechanism of Schizandrin A (SchA) inducing non-small cell lung cancer (NSCLC) apoptosis. The reverse molecular docking tool "Swiss Target Prediction" was used to predict the targets of SchA. Protein-protein interaction analysis was performed on potential targets using the String database. Functional enrichment analyses of potential targets were performed with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The conformation of SchA binding to target was simulated by chemical-protein interactomics and molecular docking. The effect of SchA on the expression and phosphorylation level of EGFR was detected by Western blot. Lipofectamine 3000 and EGFR plasmids were used to overexpress EGFR. Apoptosis was tested with Annexin V-FITC and propidium iodide staining, and cell cycle was detected by propidium iodide staining. The "Swiss Target Prediction" database predicted 112 and 111 targets based on the 2D and 3D structures of SchA, respectively, of which kinases accounted for the most, accounting for 24%. Protein interaction network analyses showed that molecular targets such as ERBB family and SRC were at the center of the network. Functional enrichment analyses indicated that ERBB-related signaling pathways were enriched. Compound-protein interactomics and molecular docking revealed that SchA could bind to the ATP-active pocket of the EGFR tyrosine kinase domain. Laboratory results showed that SchA inhibited the phosphorylation of EGFR. Insulin could counteract the cytotoxic effect of SchA. EGFR overexpression and excess EGF or IGF-1 had limited impacts on the cytotoxicity of SchA. Network pharmacology analyses suggested that ERBB family members may be the targets of SchA. SchA can inhibit NSCLC at least in part by inhibiting EGFR phosphorylation, and activating the EGFR bypass can neutralize the cytotoxicity of SchA.

Critical domains for NACC2-NTRK2 fusion protein activation.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

In Silico Screening of Multi-Domain Targeted Inhibitors for PTK6: A Strategy Integrating Drug Repurposing and Consensus Docking.

Protein tyrosine kinase 6 (PTK6), also known as breast tumor kinase (BRK), serves as a non-receptor intracellular tyrosine kinase within the Src kinases family. Structurally resembling other Src kinases, PTK6 possesses an Src homology 3 (SH3) domain, an Src homology 2 (SH2) domain, and a tyrosine kinase domain (SH1). While considerable efforts have been dedicated to designing PTK6 inhibitors targeting the SH1 domain, which is responsible for kinase activity in various pathways, it has been observed that solely inhibiting the SH1 domain does not effectively suppress PTK6 activity. Subsequent investigations have revealed the involvement of SH2 and SH3 domains in intramolecular and substrate binding interactions, which are crucial for PTK6 function. Consequently, the identification of PTK6 inhibitors targeting not only the SH1 domain but also the SH2 and SH3 domains becomes imperative. Through an in silico structural-based virtual screening approach, incorporating drug repurposing and a consensus docking approach, we have successfully identified four potential ligands capable of concurrently inhibiting the tyrosine kinase domain and SH2/SH3 domains of PT6K simultaneously. This finding suggests potential pathways for therapeutic interventions in PTK6 inhibition.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRβ) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRβ. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.

Synergistic effects of resveratrol with gemcitabine in pancreatic cancer chemotherapy by inhibiting the c-Met/PARP1 axis

Objective: Pancreatic cancer is a highly malignant tumor of the digestive tract with a dismal prognosis. A key challenge of pancreatic cancer is its resistance to chemotherapy. The c-Met/PARP1 axis plays an important role in the therapeutic resistance of breast cancer and hepatocellular carcinoma. Therefore, this study aims to explore potential therapeutic targets for improving chemotherapy for pancreatic cancer and the underlying mechanisms. Methods: Gemcitabine-resistant pancreatic cancer cell lines were constructed by our laboratory using a continuous low-concentration gemcitabine induction method. The proliferation and apoptosis of combination therapy were examined using flow cytometry and comet assay. Synergistic effects of two drugs were determined by Chou-Talalay's combination index (CI). The interactions between proteins were predicted using the AutoDock model and detected through Coimmunoprecipitation assays. Results: The combination of resveratrol and gemcitabine inhibited proliferation and promoted apoptosis of pancreatic cancer cells. We found that c-Met and PARP1 were highly expressed in gemcitabine-resistant pancreatic cancer cells. However, resveratrol inhibited their expression and improved the effectiveness of gemcitabine-induced DNA damage. In addition, our data demonstrated that resveratrol and gemcitabine had synergistic effects (CI < 1). Furthermore, the protein interactions between c-Met and PARP1 were attenuated after the early stage of resveratrol intervention. Using AutoDock models, we predicted the potential binding sites where resveratrol could impact the protein interaction between c-Met (tyrosine kinase domain) and PARP1 (CAT domain). Conclusion: Our results suggested that the synergistic effects of resveratrol with gemcitabine depend on the c-Met/PAPR1 axis. Using resveratrol as a combined chemotherapy agent may have clinical benefits for patients with refractory pancreatic cancer.