Extended Data Fig. 2. HRO761 and related WRN inhibitors are selective WRN inhibitors mixed competitive with ATP and uncompetitive with DNA.

a. Biochemical ATPase IC₅₀ activity and concentration response curves of the WRN inhibitors HRO761 (4), and analogues 2 and 3 on WRN as well as RecQ helicases BLM, RecQ1 and RecQ5. Data represent mean of quadruplicates expressed as %inhibition ± SD. b. Comparison of biochemical ATP binding, ATPase and helicase IC₅₀ across different WRN constructs and mutants (reported IC₅₀ values in a and b are the geometrical means of at least 2 independent experiments., structure of 5 shown on the left.). c. Michaelis-Menten plots of the WRN ATPase assay (0.5 nM WRN D1D2RH, 3 nM ssDNA FLAP26, 50 mM NaCl) at varying concentrations of 4 (for legend, see inset) revealed a non-competitive or mixed mode of action (Lineweaver-Burk plot on the right), interpreted as mixed ATP-competition due to competition in ATP binding assay (b.) d. K_DNA determination in the ATPase assay (0.5 nM WRN D1D2RH, 100 µM ATP, 150 mM NaCl, ssDNA: FLAP26) at varying concentrations of 5 (for legend, see inset) revealed a non-competitive or mixed mode of action (double reciprocal plot on the right), interpreted as a non-competitive mode of action due lack of DNA competition in a radioactive binding assay (e.). For c, d, Data represent initial Kobs determined by following the ATPase reaction for 20 min. e. Saturation binding experiments of radiolabeled 6 (structure shown in f.) performed with 10 nM WRN (D1D2RH) in absence (left) or in presence of 50 nM dsDNA (right) using a Scintillation Proximity Assay resulted in a similar K_d (10/14 nM). Data points represent the mean of triplicate ±SD (each experiment was repeated at least twice).