Development of a multiparametric cell imaging assay to assess inverse regulation of cancer associated biomarkers

Programme : Back to Dr Thomas Jones

Poster

Development of a multiparametric cell imaging assay to assess inverse regulation of cancer associated biomarkers

Authors

T Jones¹; D Kostomiris²; M Bilyard¹; A Ahmed³; A Madin⁴; G Kauffman⁵; S Mlynarski⁵; R Ziegler⁵; H Liu⁵; N Umbreit⁵; D Zindel¹;
¹ AstraZeneca Mechanistic and Structural Biology, Cambridge, UK; ² Astrazeneca Mechanistic and Structural Biology, Waltham, United States; ³ AstraZeneca Cellular Assay Development, Cambridge, UK; ⁴ AstraZeneca Hit Discovery, Cambridge, UK; ⁵ AstraZeneca Oncology, Waltham, United States

Discussion

Authors

Discussion

Background: Our protein of interest (POI) is commonly overexpressed in cancers, associates with a poorer prognosis and negatively regulates elements of the DNA damage response and thus is an important therapeutic target. Inhibitors of the POI have been previously identified to drive a antiproliferative response.

Objectives: To develop and validate a multiparametric cellular high content screening (HCS) assay that allows co-investigation of POI proximal and POI distal effects following treatment with small molecules targeting the POI. To generate cell-based data to drive structure-activity-relationship (SAR) understanding for the small molecule project.

Methods: Co-assessment of distal biomarker upregulation and POI expression was performed using the MCF-7 cell line, 28 hours post-treatment with tool inhibitor compound, using the Cell Insight imaging platform. Fluorescence channel specificity was assessed by fluorophore switching.

Results: Here we demonstrate concentration-dependent inverse biomarker regulation, observing distal biomarker upregulation and POI downregulation in MCF-7 cells treated with tool compound. Both biomarkers are robustly detected over a range of potencies and fluorophore switching confirms fluorescence channel specificity. In addition, we have identified internal equity out of lead generation potentially co-modulating the biomarkers of interest, complementing biochemical binding and catalytic assay data.

Conclusions: We have developed a novel, HTS applicable, multiparametric cell imaging assay to assess compound induced co-regulation of both POI and distal biomarker. Using a tool we can demonstrate that our HCS assay is suitable to generate cellular SAR-enabling data and have identified compounds co-modulating the POI and a distal biomarker in MCF-7 cells.