Category: Assay Development and Screening

1100-A - A biochemical and cell-based screening approach to identify new RET kinase inhibitors

Monday, February 5, 2018
2:00 PM - 3:00 PM

RET (Rearranged during transfection) is a cell surface receptor of the receptor tyrosine kinase (RTK) family. RET kinase plays a key role in various cell signalling pathways and RET mutations have been linked to various diseases. RET gene fusions or single point RET mutations lead to constitutive activation of RET, which is linked to increased cell proliferation and cell survival as well as cell invasion. These mutations have been identified in different types of cancer, such as thyroid cancer, pancreatic cancer and breast cancer. Targeting RET presents a promising therapeutic strategy for cancer patients, carrying these mutations. However, up to now only kinase inhibitors with low RET selectivity are available.
To identify new RET inhibitors with increased RET selectivity, we used a 384-well plate format luminescence-based biochemical RET kinase assay to screen compounds for their activity against wild type RET. Hits confirmed against wild type RET were then tested for activity against single point mutated RET (G691S), a mutation present in a subset of pancreatic cancer, using the same biochemical assay. Vandetanib, a non-selective RET inhibitor was used as the positive control. This assay format uses low reaction volumes and is compatible with the use of automated equipment and high throughput screening. For further biological profiling, compounds were then tested in a target-driven cell-based assay, using BA/F3 cells, a murine interleukin-3 dependent cell line. This model system was used to assess the cell permeability and, general cytotoxicity of prioritised compounds, as well as RET selectivity, determining off-target activity on other kinases, such as KDR. For this assay format, BA/F3 cells were seeded into 384-well plates, incubated with compounds at different doses for 48 h and cell viability was assessed using a resazurin based metabolic assay.
We tested a library of 742 FDA approved drugs and identified 8 compounds with RET inhibitory effect which we determined IC50 values for in the biochemical assay against single point mutated RET (G691S) and wild type RET. No difference in the inhibitory effect between the different types of RET kinase were observed. These compounds were subsequently tested in the cell-based assay and 2 compounds were identified with activity against BA/F3 RET cells in the low to sub-micromolar range and showed a lower effect in BA/F3 control cells. These compounds were similar/more active than the control, vandetanib.
To further explore the effects of RET inhibition in tumorigenesis, cell based assays using a panel of cancer cells carrying wildtype RET or RET fusion/single point mutations are being used to assess the effect of RET inhibition on cell invasion in these cell lines.

Elke Kaemmerer

Postdoctoral Researcher
Discovery Biology, Griffith University & CRC for Cancer Therapeutics
Nathan, Queensland, Australia

Elke studied biology at Technische Universit├Ąt (TU) Darmstadt (Darmstadt, Germany) and graduated in 2010 with a Diploma in Biology (equivalent to M.Sc.). She then commenced a PhD project in 2010 focusing on bio-engineered 3D cell culture systems for ovarian cancer and high resolution microscopy. For her PhD project she conducted research at TU Darmstadt and Queensland University of Technology (Brisbane, Australia). In 2014 she was awarded a Doctor of Natural Sciences (equivalent to PhD degree) from TU Darmstadt. She then worked as a postdoctoral researcher at University of Queensland (Brisbane, Australia) focusing on the role of calcium signalling in mediating drug resistance in breast cancer. Since 2016 she is part of the Discovery Biology and Cancer Therapeutics CRC team at the Griffith Institute for Drug Discovery, Griffith University (Brisbane, Australia) developing and performing biochemical and cell-based high throughput assays to identify new molecules for cancer therapy.