Receptor Tyrosine Kinase Domain Research Articles

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain

The oncogenic fusion protein EML4-NTRK3 requires three salt bridges for stability and biological activity

Aim of studyChromosomal translocations involving neurotrophic receptor tyrosine kinases (NTRKs) have been identified in 20 % of soft tissue sarcomas. This work focuses on the EML4-NTRK3 translocation identified in cases of Infantile Fibrosarcoma, which contains the coiled-coil multimerization domain of Echinoderm Microtubule-like protein 4 (EML4) fused with the tyrosine kinase domain of Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3). The aim of the study was to test the importance of tyrosine kinase activity and multimerization for the oncogenic activity of EML4-NTRK3. MethodsThese studies examined EML4-NTRK3 proteins containing a kinase-dead or WT kinase domain, together with mutations in specific salt bridge residues within the coiled-coil domain. Biological activity was assayed using focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT pathways were analyzed for downstream activation of signaling pathways. Localization of EML4-NTRK3 proteins was examined by immunofluorescence microscopy, and the ability of the EML4 coiled-coil domain to drive protein multimerization was examined by biochemical assays. ResultsActivation of EML4-NTRK3 relies on both the tyrosine kinase activity of NTRK3 and salt-bridge stabilization within the coiled-coil domain of EML4. The tyrosine kinase activity of NTRK3 is essential for the biological activation of EML4-NTRK3. Furthermore, EML4-NTRK3 activates downstream signaling pathways MAPK/ERK, JAK/STAT3 and PKC/PLCγ. The disruption of three specific salt bridge interactions within the EML4 coiled-coil domain of EML4-NTRK3 blocks downstream activation, biological activity, and the ability to hetero-multimerize with EML4. We also demonstrate that EML4-NTRK3 is localized in the cytoplasm and fails to associate with microtubules. Concluding statementThese data suggest potential therapeutic strategies for Infantile Fibrosarcoma cases bearing EML4-NTRK3 fusion through inhibition of salt bridge interactions and disruption of multimerization.

Effects of heterologous kinase domains on growth factor receptor specificity

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.

Small molecule inhibitors of the VEGF and tyrosine kinase for the treatment of cervical cancer.

Cervical cancer accounts for most deaths due to cancer in women, majorly in developing nations. The culprit behind this disease is the human papillomavirus(HPV) which accounts for more than 90% of cervical cancer cases. The viral strains produce proteins that favor the knocking down of the apoptosis process and continuous growth of cells in the cervix leading to tumor growth. Proangiogenic growth factors, such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), angiopoietins, and other endothelial growth factors (EGF), are secreted by tumor cells and the surrounding microenvironment, which further advances the development of cancer. The extracellular domain of receptor tyrosine kinases is employed by ligands (like VEGF and EGF) to engage and activate them by inducing receptor dimerization, which facilitates the cascade impact of these factors. The tyrosine kinase domains of each receptor autophosphorylate each other, activating the receptor and initiating signaling cascades that promote angiogenesis, migration, proliferation, and survival of endothelial cells. Cancer cells benefit from its modified signaling pathways, which cause oncogenic activation. Upon early cervical cancer detection, the second-line therapy strategy involves blocking the signaling pathways with VEGF and small molecule tyrosine kinase inhibitors (TKIs). This review paper highlights the genesis of cervical cancer and combating it using VEGF and tyrosine kinase inhibitors by delving into the details of the currently available inhibitors. Further, we have discussed the inhibitor molecules that are currently in various phases of clinical trials. This paper will surely enhance the understanding of cervical cancer and its treatment approaches and what further interventions can be done to alleviate the disease currently serving as a major health burden in the developing world.

Catalytic green synthesis of Tin(IV) oxide nanoparticles for phenolic compounds removal and molecular docking with EGFR tyrosine kinase

In this study, tin dioxide nanoparticles (SnO2 NPs) were successfully synthesized through an eco-friendly method using basil leaves extract. The fabricated SnO2 NPs demonstrated significant adsorption capabilities for phenol (PHE), p-nitrophenol (P-NP), and p-methoxyphenol (P-MP) from water matrices. Optimal conditions for maximum removal efficiency was determined for each phenolic compound, with PHE showing a remarkable 95% removal at a 3 ppm, 0.20 g of SnO2 NPs, pH 8, and 30 min of agitation at 35 °C. Molecular docking studies unveiled a potential anticancer mechanism, indicating the ability of SnO2 NPs to interact with the epidermal growth factor receptor tyrosine kinase domain and inhibit its activity. The adsorption processes followed pseudo-second order kinetics and Temkin isotherm model, revealing spontaneous, exothermic, and chemisorption-controlled mechanisms. This eco-friendly approach utilizing plant extracts was considered as a valuable tool for nano-sorbent production. The SnO2 NPs not only exhibit promise in water treatment and also demonstrate potential applications in cancer therapy. Characterization techniques including scanning electron microscopy, UV–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy (XRD), and energy-dispersive X-ray spectroscopy (EDAX) provided comprehensive insights into the results.

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.

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.

Clinical advances in EGFR-TKI combination therapy for EGFR-mutated NSCLC: a narrative review.

Mutations located in epidermal growth factor receptor (EGFR) tyrosine kinase domains have been described as the 'Achilles heel' of non-small cell lung cancer (NSCLC) and can be targeted by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). However, the clinical benefits of EGFR-TKIs are limited, and drug resistance inevitably occurs in NSCLC patients after long-term exposure to certain drugs. EGFR-TKI combination therapies, including combined targeted therapy, radiotherapy, chemotherapy, and immunotherapy, have shown promise in addressing this issue. This literature review analyzed the rationale and controversies of clinical research related to various EGFR-TKI combination therapies. The PubMed database was searched to retrieve articles published from January 1, 2001 to April 15, 2023 using the following Medical Subject Headings (MeSH) terms: "EGFR-mutated non-small cell lung cancer" and "clinical trial". Google Scholar was also reviewed to retrieve additional articles. The search was limited to articles published in English. In this review, we summarized EGFR-TKI combination therapies, including combined targeted therapy, radiotherapy, chemotherapy, and immunotherapy, most of which have shown efficacy and safety in patients with EGFR-mutated NSCLC. A number of clinical studies with large sample sizes have analyzed the activity and toxicity of combined therapies and explored potential and well-tolerated treatment options. EGFR mutations have been detected in many NSCLC patients and can be targeted by EGFR-TKIs. However, drug resistance after long-term exposure remains a significant challenge for this type of treatment. Most clinical trials have shown that the combination of EGFR-TKIs and targeted therapy, chemotherapy, radiotherapy or immunotherapy is efficacious and safe in the treatment of EGFR-mutated NSCLC. It should be noted that in some instances, serious adverse events have led to the termination of trials. However, EGFR-TKI combination therapy is indeed an effective approach for the treatment of patients with EGFR-mutated NSCLC and deserves further development.

Synthesis, Biological Evaluation, and Molecular Docking of Novel Azolylhydrazonothiazoles as Potential Anticancer Agents.

A novel set of thiazolylhydrazonothiazoles bearing an indole moiety were synthesized by subjection reactions of carbothioamide derivative and hydrazonoyl chlorides (or α-haloketones). The cytotoxicity of the synthesized compounds was evaluated against the colon carcinoma cell line (HCT-116), liver carcinoma cell line (HepG2), and breast carcinoma cell line (MDA-MB-231), and demonstrated encouraging activity. Furthermore, when representative products were assessed for toxicity against normal cells, minimal toxic effects were observed, indicating their potential safety for use in pharmacological studies. The mechanism of action of the tested products, as inhibitors of the epidermal growth factor receptor tyrosine kinase domain (EGFR TK) protein, was suggested through docking studies that assessed their binding scores and modes, in comparison to a reference standard (W19), thus endorsing their anticancer activity.

Synthesis and study of some properties of new tetrazole-containing derivatives of morpholin-4-yl-1,3,5-triazine and 4-methylpiperidin-1-yl-1,3,5-triazine

New tetrazole-containing derivatives of morpholin-4-yl-1,3,5-triazine and 4-methylpiperidin-1-yl-1,3,5-triazine were synthesized. Cytotoxic activity of the compounds obtained against human liver tumor cell lines Huh-7 and human lung A549 was studied by the MTT test. It was shown that these substances do not exhibit a pronounced cytotoxic effect. The most significant antitumor activity was shown by 1,3,5-triazine containing 5-phenyltetrazol-2-ylacetohydrazide fragment and 4-methylpiperidine ring as substituents, as well as 1,3,5-triazine containing 5-methyl-1 H -tetrazol-1-ylacetohydrazide fragment and two morpholine rings. For these compounds, the interaction with DNA was studied by UV spectroscopy. For N ’-(4,6-dimorpholino-1,3,5-triazin-2-yl)-2-(5-methyl-1 H -tetrazol-1-yl)acetohydrazide, the DNA binding constant was determined ( K bin 9.02× 104 M.-1) and studied the ability to inhibit the tyrosine kinase domain of surface receptors. It was shown that the studied tetrazole-containing derivatives of 1,3,5-triazine do not exhibit antioxidant properties with respect to NO-radicals and do not cause photoinduced hemolysis.

Antioxidant properties and cytotoxic effects of Oxalis corniculata on human Hepatocarcinoma (Hep-G2) cell line: an in vitro and in silico evaluation

BackgroundOxalis corniculata is a well-known medicinal plant used in folk medicine for the management of many diseases. The aim is to determine the physico-chemical properties, microscopic study, phytochemical properties, in-silico, in-vitro antioxidant and anticancer activity on human Hepatocarcinoma (Hep-G2) cell line of O. corniculata plant extract.ResultsMicroscopical study reaveals that presence of pericyclic fibres, starch gains, trichomes etc, and phytochemical screening is carried to find out secondary metabolites. The molecular docking study concluded that some of the phytocompounds showed inhibition of epidermal growth factor receptor tyrosine kinase domain (PDB ID: 1M17) inhibitor. Furthermore, ADMET and drug likeness study hints some of phytocompounds may act as lead for anticancer drug discovery and development. Among selected phytocompounds, compound apigenin possesses − 7.90 kcal/mol as compared to standard drug doxorubicin possesses − 7.63 kcal/mol against the epidermal growth factor receptor tyrosine kinase. The plant extract shown antioxidant activities based on the different tests were performed. The hydroalcoholic plant extracts were found to be selectively cytotoxic in vitro to human Hepatocarcinoma (Hep-G2) cell line with IC50values 34.494 ± 0.42 µg/ml and EAF showed at IC50 value 30.245 ± 0.58 µg/ml of the cells were inhibited at the concentration of 50 µg/ml as compared with standard doxorubicin at IC50 value 24.8939 ± 0.25 µg/ml, respectively.ConclusionThe present study concluded that O. corniculata possesses potential antioxidant and cytotoxic properties based upon the computer aided drug design models and in-vitro activity.

Gatekeeper mutations activate FGF receptor tyrosine kinases by destabilizing the autoinhibited state

Many types of human cancers are being treated with small molecule ATP-competitive inhibitors targeting the kinase domain of receptor tyrosine kinases. Despite initial successful remission, long-term treatment almost inevitably leads to the emergence of drug resistance mutations at the gatekeeper residue hindering the access of the inhibitor to a hydrophobic pocket at the back of the ATP-binding cleft. In addition to reducing drug efficacy, gatekeeper mutations elevate the intrinsic activity of the tyrosine kinase domain leading to more aggressive types of cancer. However, the mechanism of gain-of-function by gatekeeper mutations is poorly understood. Here, we characterized fibroblast growth factor receptor (FGFR) tyrosine kinases harboring two distinct gatekeeper mutations using kinase activity assays, NMR spectroscopy, bioinformatic analyses, and MD simulations. Our data show that gatekeeper mutations destabilize the autoinhibitory conformation of the DFG motif locally and of the kinase globally, suggesting they impart gain-of-function by facilitating the kinase's ability to populate the active state.

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as β1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

Structural insights into conformational stability and binding of thiazolo-[2,3-b] quinazolinone derivatives with EGFR-TKD and in-vitro study

Heterocyclic molecules are well-known drugs against various diseases including cancer. Many tyrosine kinase inhibitors including erlotinib, osimertinib, and sunitinib were developed and approved but caused adverse effects among treated patients. Which prevents them from being used as cancer therapeutics. In this study, we strategically developed heterocyclic thiazolo-[2,3-b]quinazolinone derivatives by an organic synthesis approach. These synthesized molecules were assessed against the epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) by in silico methods. Molecular docking simulations unravel derivative 17 showed better binding energy scores and followed Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties. The binding affinity displayed by synthetic congener and reference molecule erlotinib was found to be −8.26 ± 0.0033 kcal/mol and −7.54 ± 0.1411 kcal/mol with the kinase domain. Further, molecular dynamic simulations were conducted thrice to validate the molecular docking study and achieved significant results. Both synthetic derivative and reference molecule attained stability in the active site of the TKD. The synthetic congener and erlotinib showed free energy binding (ΔGbind) −102.975 ± 3.714 kJ/mol and −130.378 ± 0.355 kJ/mol computed by Molecular Mechanics Poison Boltzmann Surface Area (MM-PBSA) method. In addition, the motions of each sampled system including the Apo complex were determined by the principal component analysis and Gibbs energy landscape analysis. The in-vitro apoptosis study was performed using MCF-7 and H-1299 cancer cell lines. However, thiazolo-[2,3-]-quinazoline derivative 17 showed fair anti-proliferative activity against MCF-7 and H-1299. Further, the in-vivo study is necessary to determine the effectivity of the potent anti-proliferative, non-toxic molecule against TKD.

Mulberroside A could serve as a pan inhibitor for the tyrosine kinase domains of the HER family

Numerous medicines were authorized for their targeting of the tyrosine kinase domain (TKD) of human epidermal receptors (HER). However, it has been demonstrated that these TKDs exhibit persistent resistance, necessitating the development of additional inhibitors with different modes of action. A single pharmacophore can be manipulated to create a pan-inhibitor where the binding site of these receptors has been highly conserved. The development of anti-cancer agents and the introduction of synergistic action to increase the efficacy of current medications have both recently benefited from the use of medicinal plants and herbal extracts as a reliable source. The binding affinity to the TKDs was predicted by molecular docking, followed by molecular dynamic simulation to examine the changes in the motion of the enzymes, with a focus on the components responsible for catalytic activity (C α-helix), activation (activation loop), and autophosphorylation (C-terminal loop). According to the ∆G value provided by Autodock Vina (-40.54, -40.12, -37.20, -37.62 kJ/mol for HER1, HER2, HER3, and HER4, respectively) and MM/PBSA, which indicates a strong affinity for the TKDs, the outcomes are undeniably positive. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and the principal component analysis (PCA) detected significant dynamic changes in the TKDs, particularly in the C α-helix and the activation loop for all TKDs, suggesting that mulberroside A may alter the function of these enzymes. This study will expand our understanding of the dynamics of the TKDs and shed light on the anticancer potential of the polyphenolic compounds.

Profiling of gene fusion involving targetable genes in Chinese gastric cancer.

BACKGROUNDApproximately half of all new cases of gastric cancer (GC) and related deaths occur in China. More than 80% of patients with GC are diagnosed at an advanced stage, which results in poor prognosis. Although HER2-directed therapy and immune checkpoint inhibitors have been somewhat successful, new drugs are still needed for the treatment of GC. Notably, several gene fusion-targeted drugs have been approved by the United States Food and Drug Administration for solid tumors, including GC, such as larotrectinib for NTRK fusion-positive cancers and zenocutuzumab for NRG1 fusion-positive cancers. However, gene fusions involving targetable genes have not been well characterized in Chinese patients with GC.AIMTo identify the profile of fusions involving targetable genes in Chinese patients with GC using clinical specimens and determine the distribution of patients with gene fusion variants among the molecular subtypes of GC.METHODSWe retrospectively analyzed gene fusion events in tumor tissue samples from 954 Chinese patients with GC. Clinicopathological characteristics were obtained from their medical records. Genetic alterations, such as single nucleotide variants, indels, amplifications, and gene fusions, were identified using a targeted sequencing panel containing 825 genes. Fusions were validated by fluorescence in situ hybridization (FISH) using break-apart probes. The microsatellite instability (MSI) status was evaluated using MSIsensor from the targeted sequencing panel data. Tumor mutational burden (TMB) was calculated using the total number of nonsynonymous mutations divided by the total genomic targeted region. Chi-square analysis was used to determine the enrichment of gene fusions associated with the molecular subtypes of GC.RESULTSWe found that 1.68% (16/954) of patients harbored 20 fusion events involving targetable genes. RARA fusions (n = 5) were the most common, followed by FGFR2, BRAF, MET, FGFR3, RET, ALK, EGFR, NTRK2, and NRG1 fusions. Two of the RARA fusions, EML4-ALK (E6:E20) and EGFR-SEPTIN14 (E7:E10), have been identified in other tumors but not in GC. Surprisingly, 18 gene fusion events were previously not reported in any cancer types. Twelve of the eighteen novel gene fusions included complete exons encoding functional domains of targetable genes, such as the tyrosine kinase domain of receptor tyrosine kinases and the DNA- and ligand-binding domains of RARA. Consistent with the results of detection using the targeted sequencing fusion panel, the results of FISH (fluorescence in situ hybridization) confirmed the rearrangement of FGFR2 and BRAF in tumors from patients 04 and 09, respectively. Genetic analysis indicated that the fusion genes were significantly enriched in patients with ERBB2 amplification (P = 0.02); however, there were no significant differences between fusion-positive and fusion-negative patients in age, sex, MSI status, and TMB.CONCLUSIONWe characterized the landscape of fusions involving targetable genes in a Chinese GC cohort and found that 1.68% of patients with GC harbor potential targetable gene fusions, which were enriched in patients with ERBB2 amplification. Gene fusion detection may provide a potential treatment strategy for patients with GC with disease progression following standard therapy.

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids.

The cytoplasmic domain of receptor tyrosine kinases (RTKs) plays roles as a kinase and a protein scaffold; however, the allocation of these two functions is not fully understood. Here, we analyzed the assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied members of RTKs, by combining single-pair fluorescence resonance energy transfer (FRET) imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (T654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias toward oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of T654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which T654 phosphorylation causes a switch of the major function of EGFR from kinase-activating dimers to scaffolding oligomers.

Synthesis, Molecular Docking and Antiproliferative Activity Studies of a Thiazole‐Based Compound Linked to Hydrazone Moiety

Abstract(A new 4‐oxothiazolidin‐2‐ylidene derivative bearing hydrazone moiety was synthesized via Michael addition between the reaction of 4‐(4‐nitrophenyl)‐3‐thiosemicarbazide and dimethyl acetylenedicarboxylate (DMAD). The structure of synthesized compound was elucidated using various spectral techniques such as FTIR, UV‐spec, 1H NMR and 13C NMR. The structure of the related compound was confirmed by single‐crystal X‐ray analysis. Antiproliferative activity of the synthesized compound was evaluated in two human cancer cell lines, HepG2 (liver hepatocellular carcinoma cell line) and DLD‐1 (human colon cancer cell line). In addition, molecular docking of synthesized compound was investigated to give an insight of its activity against Epidermal Growth Factor Receptor tyrosine kinase domain proteins (EGFR) (lung cancer) (PDB ID: 1 M17), deleted in Liver Cancer 2 proteins (DLC2) (liver cancer) (PDB ID: 2H80), and MLK4 kinase proteins (colon cancer) (PDB ID: 4UYA) were investigated. Furthermore, the ability of the molecule to be a drug was examined by ADME/T analysis.)