Artios is developing novel anti-cancer medicines targeting the DNA damage response. To enable this, we developed a range of novel assays to measure cellular DNA repair activity. These assays had a major impact on our programs leading to clinical stage assets being tested in patients with high unmet medical needs.
Accurately measuring on-target cellular activity is essential for progressing novel targeted therapeutics. Generating assays for DNA repair factors with the required properties for fueling an efficient drug discovery campaign has been challenging, whether it's a need for high throughput, robust signal-to-noise, rapid turnaround, and ideally, portability to different biological systems. We have developed a suite of robust quantitative and titratable luminescence-based reporter assays to measure the proficiency of four major double-strand break repair pathways, homologous recombination, non-homologous end joining, microhomology-mediated end joining, and single-strand annealing.
These reporter assays are validated as reading out pathway-dependent repair, are sensitive to pharmacological modulation, and have a fast turnaround time. The repair substrates we have developed are extrachromosomal so they can be transiently transfected into models of interest and easily scaled into a high-throughput format. We believe our assay systems have an important place in enabling DDR drug discovery, but also in furthering our understanding of DDR biology more broadly.
In this respect, we hope the methods help accelerate the identification and prosecution of new drug targets, leading to medicines for diseases, such as cancer, where there is high unmet medical need. To begin, generate each reporter substrate and check their concentration using a spectrophotometer. Obtain 200 nanograms of reporter substrate.
Electrophorese each reporter substrate alongside a high molecular weight DNA ladder in a 0.7%agarose TAE gel containing a fluorescent double-stranded DNA stain. Verify that defined bands of the correct size are observed for each reporter substrate. If assessing the impact of compounds on the reporter assay, dispense compounds dissolved in the vehicle into the wells of a 96-well plate.
To prepare the transfection mix for a 96-well plate, dilute the Firefly control plasmid and NanoLuc double-strand break repair reporter substrate in 500 microliters of transfection buffer in a 1.5-milliliter tube. After adding the lipid-based transfection reagent, vortex the DNA mixture briefly and incubate for 10 minutes at room temperature. To harvest the cells, trypsinize and resuspend them in fresh medium containing 10%fetal bovine serum or FBS.
Then, determine the cell count and resuspend three times 10 to the power of six cells in 8.5 milliliters of medium with FBS in a fresh 15-milliliter tube. Add the DNA transfection mix into the cell suspension and invert the tube several times to mix. Plate 80 microliters of cell suspension per well and incubate the 96-well plate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
Prepare the control luciferase and the reporter luciferase reagent according to the manufacturer's instructions. Add substrate into an appropriate volume of assay buffer at a 1:100 ratio and mix. After the plate and reagents reach room temperature, add 80 microliters of control Firefly luciferase reagent per well.
Shake the plate for three minutes on an orbital shaker at 450 revolutions per minute. Place the plate in the luminescence plate reader and measure the control luciferase luminescence signal. To measure the reporter luciferase signal, add 80 microliters of NanoLuc reporter luciferase reagent per well.
After a three-minute incubation in an orbital shaker at 450 revolutions per minute, leave the plate to rest at room temperature for seven minutes. Finally, measure the reporter luciferase luminescence signal in a luminescence plate reader. Evaluation of the component NanoLuc and Firefly luminescence signals showed that the observed repair defect was driven by a reduction in the NanoLuc signal while the Firefly control signal was unperturbed.
