Lirafugratinib

Potency assay (off target): Within target family: RLY-4008 has >250-fold selectivity over FGFR1, and >80- and >5,000-fold selectivity over FGFR3 and FGFR4, respectively. Outside Target family: TREEspot depicting selectivity of RLY-4008 screened against 468 kinases via KINOMEscan (DiscoverX, Eurofins). At the test concentration of 500 nmol/L, three kinases showed greater than 75% inhibition: FGFR2 (94.1%), MEK5 (92.4%), and MKNK2 (89%). Neither kinase has a cysteine residue in the same position on the P-loop as the FGFRs and is thus not expected to be inhibited irreversibly by RLY-4008

Potency assay, off target (cells): Selectivity of RLY-4008 in cells was evaluated on the proliferation of FGFR2-dependent (KATO III, NCI-H716, SNU-16, ICC13-7, JHUEM-2, MFE-296, AN3CA), FGFR1-dependent (JMSU-1, Li-7), FGFR3-dependent (RT-112, RT-4), and FGFR4-dependent (MDA-MB-453) cell lines. RLY-4008 inhibited cellular proliferation with IC50 <14 nmol/L in FGFR2-dependent cell lines including those derived from FGFR2-amplified gastric carcinoma (KATO III, SNU-16) and colorectal adenocarcinoma (NCI-H716), FGFR2 fusion–positive iCCA (ICC13–7), and FGFR2-mutant endometrial adenocarcinoma (MFE-296, FGFR2N549K and AN3CA, FGFR2K310R;N549K) and breast carcinoma (JHUEM-2, FGFR2C383R). In contrast to pan-FGFRi, RLY-4008 did not have strong inhibitory activity in FGFR1-, FGFR3-, or FGFR4-dependent cell lines. The potency (expressed as IC50) of RLY-4008 on these cell lines was 212 nmol/L to >10 mmol/L, demonstrating a high degree of selectivity for RLY-4008 on FGFR2 relative to other FGFR family members in cellular assays. RLY-4008 did not inhibit the proliferation of ICC13-7–FGFR2C491S, confirming that the antiproliferative activity of RLY-4008 is on-target.