Comparison of Elisa-Based Tyrosine Kinase Assays for Screening EGFR Inhibitors

Abstract

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu, Tyr)_1:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.     

S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors

Increased activity of the src family of oncogenic tyrosine kinases is seen in many human tumors and pharmacologic inhibitors of these kinases are investigated as potential anti-tumor agents. A family of pyrido [2, 3-d] pyrimidine compounds (PD) has been characterized as selective inhibitors of Src kinases. We studied the effects of this class of compounds on cancer cell lines and found that they were highly specific inhibitors of cell cycle progression. These compounds inhibit cells either in the mitotic phase or in mid S-phase; these two activities are mutually exclusive: no compound exerts both activities. We undertook experiments to determine the mechanistic basis for these differences and found additional biochemical activities associated with the S-phase inhibitors. Treatment of cells with the S-phase blocker PD179483 causes abnormal and persistent hyperactivation of Cdk2 and Cdc2 due to Tyr-15 dephosphorylation. These effects were associated with hyperphosphorylation of the upstream regulatory kinase Myt1 and Wee1. They were not observed with the anti-mitotic compounds. Furthermore, the S-phase inhibitors PD179483 and PD166326, but not the anti-mitotic compounds, inhibit Wee1 in vitro at concentrations that cause S-phase block in vivo. These data identify a novel subset of pyridopyrimidine compounds which are inhibitors of src and Wee1 kinases and which inhibit tumor cell growth through cell cycle arrest in mid S-phase.     

Selecting Tyrosine Kinase Inhibitors for Gastrointestinal Stromal Tumor with Secondary KIT Activation-Loop Domain Mutations

Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation.     

Cloning and Expression of EGFR Tyrosine Kinase and Construction of the Screening Model for the Enzymatic Inhibitors

表皮生长因子受体(Epidermal growth factor receptor,EGFR)属受体酪氨酸激酶(Tyrosine kinase,TK)家族,其胞内的酪氨酸激酶在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。本研究从人脐静脉内皮细胞(HUVEC) 提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶催化域的编码基因。将其克隆至载体pET-30a,在E.coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化。通过对酶的活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,拟对微生物代谢产物进行筛选。     

表皮生长因子受体酪氨酸激酶的克隆表达和酶抑制剂筛选模型的构建

表皮生长因子受体（epidermal growth factor receptor,EGFR）属受体酪氨酸激酶（tyrosine kinase,TK）家族,其胞内的酪氨酸激酶在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。从人脐静脉内皮细胞（HUVEC）提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶催化域的编码基因。将其克隆至载体pET-30a,在E．coliBL21（DE3）中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化。通过对酶的活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,拟对微生物代谢产物进行筛选。     

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

Objectives

Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells.

Methods

We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy.

Results and Conclusions

Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment.     

Synthesis,Molecular Docking,and Cytotoxicity Evaluation of New Khellinone Derivatives as Tyrosine Kinase Inhibitors

Russian Journal of Bioorganic Chemistry - A series of new benzofuran derivatives were synthesized using khellinone. The acetyl function of khellinone was converted into its corresponding enaminone,...     

Computer Modeling and Synthesis of Potential Inhibitors of Tyrosine Kinase BCR-ABL with the T315I Mutation

Russian Journal of Bioorganic Chemistry - A comparative analysis of the interaction of the chimeric protein BCR-ABL, of the normal type and with the T315I mutation, with known inhibitors as well as...     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.     

Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation

Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.     

Combination of mTOR and EGFR Kinase Inhibitors Blocks mTORC1 and mTORC2 Kinase Activity and Suppresses the Progression of Colorectal Carcinoma

Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT^S473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR^T1068). The parallel increase of AKT^S473 and EGFR^T1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.     

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

KDR酪氨酸激酶的克隆表达及纯化

VEGF作为一种血管内皮细胞有丝分裂原,通过与内皮细胞表面特定受体结合,从而导致新生血管的形成,其中VEGFR-2(KDR/FIk-1)在肿瘤血管形成中起重要作用,因此其抑制剂有可能发展成为治疗肿瘤的新药.采用大肠杆菌成功表达了具有酪氨酸激酶活性的KDR酪氨酸激酶催化域(KDR-CD).采用RT-PCR从人脐静脉内皮细胞中提取总RNA,获得KDR-CD的编码基因,将其克隆至pET-30a载体,在E. coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化,Western blotting结果显示表迭的KDR-CD蛋白自身磷酸化,重组KDR-CD蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性.     

Multi-Targeted Antiangiogenic Tyrosine Kinase Inhibitors in Advanced Non-Small Cell Lung Cancer: Meta-Analyses of 20 Randomized Controlled Trials and Subgroup Analyses

Background

Multi-targeted antiangiogenic tyrosine kinase inhibitors (MATKIs) have been studied in many randomized controlled trials (RCTs) for treatment of advanced non-small cell lung cancer (NSCLC). We seek to summarize the most up-to-date evidences and perform a timely meta-analysis.

Methods

Electronic databases were searched for eligible studies. We defined the experimental arm as MATKI-containing group and the control arm as MATKI-free group. The extracted data on objective response rates (ORR), disease control rates (DCR), progression-free survival (PFS) and overall survival (OS) were pooled. Subgroup and sensitivity analyses were conducted.

Results

Twenty phase II/III RCTs that involved a total of 10834 participants were included. Overall, MATKI-containing group was associated with significant superior ORR (OR 1.29, 95% CI 1.08 to 1.55, P = 0.006) and prolonged PFS (HR 0.83, 0.78 to 0.90, P = 0.005) compared to the MATKI-free group. However, no significant improvements in DCR (OR 1.08, 1.00 to 1.17, P = 0.054) or OS (HR 0.97, 0.93 to 1.01, P = 0.106) were observed. Subgroup analyses showed that the benefits were predominantly presented in pooled results of studies enrolling previously-treated patients, studies not limiting to enroll non-squamous NSCLC, and studies using MATKIs in combination with the control regimens as experimental therapies.

Conclusions

This up-to-date meta-analysis showed that MATKIs did increase ORR and prolong PFS, with no significant improvement in DCR and OS. The advantages of MATKIs were most prominent in patients who received a MATKI in combination with standard treatments and in patients who had previously experienced chemotherapy. We suggest further discussion as to the inclusion criteria of future studies on MATKIs regarding histology.     

Evaluation of Indole Esters As Inhibitors of p60c-Src Receptor Tyrosine Kinase and Investigation of the Inhibition Using Receptor Docking Studies

Several indole esters were tested as inhibitors of tyrosine kinase p60c-Src. Compound (4) was found fairly active against the enzyme with IC50=1.34?μM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60c-Src tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.