Figure 3. Resistant variants from the active site act by steric hindrance or disrupting active-site integrity.

(a) A cartoon depiction of JAK2 active site bound with ruxolitinib (blue sticks) showing its interaction with Glu 930 and Asn 981 by hydrogen bonding (dashed line). Residue Tyr 931 interacts with Gln 853, Leu 855 and Gly 935 by hydrogen bonds with 5 water molecules (red circles). Residue Asp 994 (orange stick) from the DFG motif directly interacts with ruxolitinib and catalytic Lys 882. (b) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition of Y931C by ruxolitinib (L983F is completely resistant). Upper panel showing increasing phospho-JAK2 levels with increasing drug concentration, possibly due to preferential binding and stabilization of the active conformation⁷³. Lower panel showing phospho-STAT5 level. Blots were stripped and reprobed for total JAK2 and STAT5. (c) Structural model of ruxolitinib, showing steric clash with phenylalanine substituted for Leu 983.