Measurement of downstream kinase activity modulation as indicator of epidermal growth factor receptor inhibitor efficacy

The acoustic membrane micro particle (AMMP) technology has been used to quantify single analytes out of multiple sample types. In this study the technology is used to reveal molecular interactions of components of kinase pathways. Specifically, the downstream kinase activity of the EGFR receptor in the presence or absence of EGFR inhibitors is investigated. These experiments substantiate that EGFR stimulation predominantly activates the MEK/ERK pathway. The EGFR inhibitors tested had varying effectiveness at preventing phosphorylation at the EGFR or downstream kinase activity. These experiments reveal the use of the AMMP technology for observing multiple signaling pathways in a single experiment.     

Click chemistry for improvement in selectivity of quinazoline-based kinase inhibitors for mutant epidermal growth factor receptors

Discovery of mutant-selective kinase inhibitors is one of the challenges in medicinal chemistry and is a main issue for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. We tried to improve the selectivity of pan-HER inhibitors for mutant EGFRs. Utilizing click chemistry, triazole-tethered quinazoline derivatives were synthesized, based on a quinazoline scaffold showing pan-HER inhibition. The representative compound 5j exhibited 17- and 52-fold improved selectivity for EGFR L858R/T790M over wild-type EGFR and HER2, respectively, and demonstrated 6.7-fold more potent antiproliferative activity against PC9 cells harboring EGFR-activating mutation than gefitinib. Although the described quinazolines did not surpass pyrimidines as 3rd generation EGFR inhibitors in terms of selectivity for mutant EGFRs, our approach might provide information that would help in the identification of mutant-selective compounds among pan-HER inhibitors using the quinazoline scaffold.     

Mapping inhibitor response to the in-frame deletions,insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase’s active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation–π interactions and hydrophobic contacts.     

A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes

Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.     

Discovery of 5-(methylthio)pyrimidine derivatives as L858R/T790M mutant selective epidermal growth factor receptor (EGFR) inhibitors

To overcome the drug-resistance of first generation EGFR inhibitors and the nonselective toxicities of second generation inhibitors among NSCLC patients, a series of 5-(methylthio)pyrimidine derivatives were discovered as novel EGFR inhibitors, which harbored not only potent enzymatic and antiproliferative activities against EGFR^L858R/T790M mutants, but good selectivity over wide-type form of the receptor. This goal was achieved by employing structure-based drug design and traditional optimization strategies, based on WZ4002 and CO1686. These derivatives inhibited the enzymatic activity of EGFR^L858R/T790M mutants with IC₅₀ values in subnanomolar ranges, while exhibiting hundreds of fold less potency on EGFR^WT. These compounds also strongly inhibited the proliferation of H1975 non-small cell lung cancer cells bearing EGFR^L858R/T790M, while being significantly less toxic to A431 human epithelial carcinoma cells with overexpressed EGFR^WT. The EGFR kinase inhibitory and antiproliferative activities were further validated by Western blot analysis for activation of EGFR and the downstream signaling in cancer cells.     

A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer

Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776^YVMA) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2^YVMA mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776^YVMA insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2^YVMA mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776^YVMA insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2^YVMA mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC₅₀?>?1000 and =27?nM, respectively, suggesting that the bosutinib might be exploited as a selective inhibitor for mutant over wild-type HER2. Structural examination revealed that formation of additional non-bonded interactions such as hydrogen bonds and hydrophobic contacts with HER2 A-loop region due to G776^YVMA insertion is the primary factor to improve bosutinib affinity upon the mutation.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Targeting epidermal growth factor receptors inhibition in non-small-cell lung cancer: a computational approach

Epidermal growth factor receptors (EGFRs) are transmembrane receptors present on cell membranes, play an important role in controlling cell growth, apoptosis and other cellular functions. They have an extracellular binding moiety, a transmembrane component and an intracellular tyrosine kinase unit. Mutations of EGFRs can lead to continual or abnormal activation of the receptors causing unregulated cell division, causing cancer such as non-small-cell lung cancer (NSCLC). Hence, the objective is to recognise the potential drug targets through generating pharmacophoric pattern, identifying and building suitable ligands and docking studies with dynamics applications. The pharmacophore of these compounds explains about physicochemical properties required for designing new compounds which provides the design to develop desired targeted drug therapy. The simulation uncovers changes in the width of the essential channel to the active site, extensive to concede substrates. This study concludes the interaction of EGFRs with its inhibitors through computational modelling which can be important initial steps toward the development of novel pharmaceuticals in the fight against NSCLC.     

Activation of epidermal growth factor receptor via CCR3 in bronchial epithelial cells

We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

EGFR基因在非小细胞肺癌、乳腺癌中突变的研究

表皮生长因子受体(EGFR)基因酪氨酸激酶域体细胞突变与非小细胞肺癌(NSCLC)患者对酪氨酸激酶抑制剂吉非替尼敏感性密切相关。文章分析和检测本院75例非小细胞肺癌、10例乳腺癌患者石蜡包埋标本EGFR基因突变状况。采用PCR技术进行EGFR基因19和21外显子突变分析。结果显示:75例NSCLC患者中有13例(13/75,17.33%)酪氨酸激酶域存在体细胞突变。其中7例(7/75,9.33%)为19外显子缺失突变,6例(6/75,8%)为21外显子替代突变(2573T>G,L858R)。病理分型显示,腺癌突变率高于其他几种类型NSCLC。乳腺癌患者均为免疫组化HER-2阳性女性,EGFR基因的19、21外显子中未见突变发生。中国非小细胞肺癌患者总突变率高于高加索人种,女性患者较男性患者突变率高,提示肺腺癌的患者突变率高可能在吉非替尼的治疗中获益。     

Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma and various other solid tumors

SU11248 sunitinib malate sutent^® is a selective inhibitor of certain protein tyrosine kinases including VEGF-R types 1–3 PDGF-R-a and -b, c-kit, and RET. Its antitumor activity may result from both inhibition of angiogenesis and direct antiprofilerative effects on certain tumor types. In several phase I/II/III studies, sutent^® was found to be effective as second and first line treatment in metastatic renal cell carcinoma (RCC). In fact, with a 37% response rate and an additional 48% stable disease sutent became the drug of choice for first line treatment in RCC. Sutent^® was also effective as second line treatment in patients with gastrointestinal stromal tumors (GIST) with 8% response rate, 70% stable disease and a 20-month median survival. Prolonged stable disease was also documented in neuroendocrine tumors. In addition, a phase II study in multitreated women with breast cancer, sutent^® demonstrated a moderate activity with 16% clinical benefit. Finally, in non-small cell lung cancer (NSCLC) in patients’ progressing on chemotherapy sutent^® was able to achieve a 10% response rate, a level of activity similar to those documented by other agents approved for lung cancer. The agent is being tested in other tumors with a modified schedule of dosage.     

Motility enhancement by tumor-derived mutant E-cadherin is sensitive to treatment with epidermal growth factor receptor and phosphatidylinositol 3-kinase inhibitors

Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell-matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.     

In silico evaluation of the resistance of the T790M variant of epidermal growth factor receptor kinase to cancer drug Erlotinib

Epidermal growth factor receptor kinase is implicated in cancer development due to either overexpression or activation variants in its functional intracellular kinase domain. Threonine to methionine (Thr 790 Met) is one such variant observed commonly in patients showing resistance to kinase inhibitor drug Erlotinib. Two mechanisms for resistance have been proposed (1) steric hindrance and (2) enhanced binding to ATP. In this study, we employed molecular dynamics simulations and studied both the mechanisms. Extensive simulations and free energy of binding analyses has shown that steric hindrance does not explain appropriately the mechanism for resistance against Erlotinib therapy for this variant. It has been observed that conformational switching from an intermediate intrinsically disordered C-helix conformation is required for completion of the kinase’s catalytic cycle. Our study substantiates that T790M variant has greater tendency for early transition to this intrinsically disordered C-helix intermediate state. We propose that enhanced catalytic efficiency in addition to enhanced ATP binding explains mechanism of T790M resistance to drug Erlotinib.     

A quinazoline-derivative DOTA-type gallium(III) complex for targeting epidermal growth factor receptors: synthesis, characterisation and biological studies

The novel DOTA-like chelator 1,4,7,10-tetraazacyclododecane-1-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}propionamide-4,7,10-triacetic acid (H₃L) was synthesised by alkylation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate) with N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-bromopropionamide, followed by hydrolysis of the ester groups with trifluoracetic acid. H₃L has been fully characterised by multinuclear NMR spectroscopy, mass spectrometry and high-performance liquid chromatography (HPLC). Five protonation constants, log K _Hi , of H₃L were determined by potentiometry and UV–vis spectrophotometry and the values found are 10.47, 9.18, 5.24, 4.00 and 2.23. These methods, complemented with variable-pH ⁷¹Ga NMR studies, allowed us to ascertain the stability constant of the Ga(III) complex of L. GaL has a remarkably high thermodynamic stability constant (log K _ML = 24.5). The radioactive complex ⁶⁷GaL was prepared in high yield and high radiochemical purity. Its HPLC chromatogram is identical to that obtained for the GaL complex prepared at the macroscopic level. At pH 7.4, ⁶⁷GaL has an overall neutral charge, is highly hydrophilic (log D = −1.02 ± 0.03) and presents high in vitro stability in physiological media and in the presence of an excess of diethylenetriaminepentaethanoic acid . In vitro studies indicated that H₃L and GaL do not inhibit the cell growth of epidermal growth factor receptor expressing cell lines, such as A431 cervical carcinoma cells, a result which agrees with the very low cell internalisation found for ⁶⁷GaL in the same cell line. Biodistribution studies in mice indicated high in vivo stability for ⁶⁷GaL, a high total excretion rate and a relatively slow blood clearance, in full accordance with its hydrophilic character and the relatively important protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Staurosporine scaffold–based rational discovery of the wild‐type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog‐sensitive kinase technology

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first‐line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small‐molecule kinase inhibitors in EGFR‐targeted chemotherapy. Previously, the reversible pan‐kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR^T790M) over wild‐type kinase (EGFR^WT), suggesting that the staurosporine scaffold is potentially to develop the wild‐type sparing reversible inhibitors of EGFR^T790M. Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold–based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico–in vitro analog‐sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild‐type sparing inhibitors UCN‐01, UCN‐02, AFN941, and SB‐218078 with high or moderate selectivity of 30‐, 45‐, 5‐, and 8‐fold for EGFR^T790M over EGFR^WT, respectively, which are comparable with or even better than that of the parent compound staurosporine (24‐fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN‐02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase‐inhibitor binding. A hydroxyl group of UCN‐02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN‐02 than staurosporine for mutant over wild‐type kinase.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.