Testing on mutagenic strains
Please go to Researchers involved for a list of researchers who contributed to this work
One way to confirm the binding mode of the hit compounds is to perform Green site saturation mutagenesis under targeting compound selection experiments. To do this, the conditions for testing were explored by applying the hits RA-3272, RA-3301, and RA-3318 to three human cell lines:
- U937-DCSIGN cells = human monocyte cell line
- Huh7.5 = human hepatocyte cell line
- C6/36 = insect cell line
Below are the results for RA-3301 and RA-3318; only RA-3301 is showing activity (in line with other data), but this is accompanied by cytotoxicity in the human cell lines (in line with other data). The original saturation mutagenesis experiments that helped confirm the Green site as a suitable pocket was done on the insect cell line C6/36, which is also showing the best activity/cytotoxicity for RA-3301.
The concern is the relatively high IC50 (~6uM) of RA-3301, along with the steep Hillslope (~6) which doesn't make the compound ideal for continued testing in the mutagenic studies. Possible hits from the Second expansion could provide more suitable compounds to explore.