Structural analysis of JAK inhibitors and PROTACs bound to JAK2. (A) The JAK2 JH1 (kinase) domain assumes a “DFG-in” conformation upon binding ruxolitinib (pink sticks). Protein color is graded from blue at the N terminus (N-ter) to red at the C terminus (C-ter), with ALL mutations shown as red dots, the mobile P-loop in dark blue, and the ruxolitinib binding site shown as a pink surface close to the activation loop containing the DFG motif (green dots); 2 phosphorylated tyrosines (p-Tyr) are shown as yellow dots. The black arrow near ruxolitinib indicates the direction of linker extension for the design of PROTACs as shown in (B). (B) The orientation of ruxolitinib bound to human JAK2 (pink) is flipped with respect to its position in complex with SRC (4U5J, from chicken)⁶³ shown in gray as conformation A and B for both molecules in the asymmetric unit, guiding the linker attachment point (red circle) for the PROTAC design toward solvent. Polder OMIT electron density maps are shown as green mesh at 3σ and hydrogen bonds are shown as yellow dotted lines for all structures in panels B-E. (C) Baricitinib bound to JAK2 shows a different placement of ligand moieties, such as the nitrile group, compared with BMP2-inducible kinase (4W9X).⁶⁴ PROTAC precursor compounds (cmpd) 3 (D) and 4 (E) possess an additional H-bond interaction between the linker and the backbone of the hinge region and extend into solvent.