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Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

ABSTRACT: Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

SUBMITTER: Antczak C

PROVIDER: S-EPMC3277729 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

Antczak Christophe C Bermingham Alun A Calder Paul P Malkov Dmitry D Song Keming K Fetter John J Djaballah Hakim H

Assay and drug development technologies 20120126 1

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal g ...[more]

PMID: 22280060

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Project description:This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with 64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh (n = 7) and EGFRlow (n = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD = 7 ± 5 nM) and murine (KD = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. 64Cu-Gp2-EGFR tracer effectively localized to EGFRhigh tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlow tumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhigh tumors (0.5 ± 0.3%ID/g, p < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFRhigh tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial in vivo characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents.

Project description:PurposeSubset analyses from phase III evaluation of epidermal growth factor receptor inhibition (EGFRi) suggest improved outcomes in patients with EGFR-amplified gastroesophageal adenocarcinoma (GEA), but large-scale analyses are lacking. This multi-institutional analysis sought to determine the role of EGFRi in the largest cohort of patients with EGFR-amplified GEA to date.Patients and methodsA total of 60 patients from 15 tertiary cancer centers in six countries met the inclusion criteria. These criteria required histologically confirmed GEA in the metastatic or unresectable setting with EGFR amplification identified by using a Clinical Laboratory Improvement Amendments-approved assay, and who received on- or off-protocol EGFRi. Testing could be by tissue next-generation sequencing, plasma circulating tumor DNA next-generation sequencing, and/or fluorescence in situ hybridization performed by a Clinical Laboratory Improvement Amendments approved laboratory. Treatment patterns and outcomes analysis was also performed using a deidentified clinicogenomic database (CGDB).ResultsSixty patients with EGFR-amplified GEA received EGFRi, including 31 of 60 patients (52%) with concurrent chemotherapy. Across treatment lines, patients achieved a 43% objective response rate with a median progression-free survival of 4.6 months (95% CI, 3.5 to 6.4). Patients receiving EGFRi in first-, second-, and third-line therapy achieved a median overall survival of 20.6 months (95% CI, 13.5 to not reached [NR]), 9 months (95% CI, 7.9 to NR), and 8.4 months (7.6 to NR), respectively. This survival far exceeded the 11.2-month (95% CI, 8.7 to 14.2) median overall survival from first-line initiation of non-EGFRi therapy in patients with EGFR-amplified GEA in the CGDB. Despite this benefit, analysis of the CGDB (January 2011-December 2020) suggests that only 5% of patients with EGFR-amplified GEA received EGFRi.ConclusionPatients with EGFR-amplified GEA derive significant benefit from EGFRi. Further prospective investigation of EGFRi in a well-selected patient population is ongoing in an upcoming trial of amivantamab in EGFR and/or MET amplified GEA.

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Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

Publications

Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

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