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. 2022 Jun 3;8(22):eabm5563. doi: 10.1126/sciadv.abm5563

Fig. 4. KK174 is a SMO agonist that functions at the TMD.

Fig. 4.

(A) Structures of cholesterol and KK174. (B) The interaction between hSMOΔC and Nb8 was assessed using a pull-down assay in the presence of the indicated SMO ligands (100 μM each for 16 hours). Immunoblot shows the amount of hSMOΔC that coprecipitates with Nb8 captured on beads. Figure S5A shows the abundance of SMO in flow-through samples from this pull-down. (C, D, and F) Fold increase in Gli1 mRNA induced by the addition of the indicated SMO agonists (100 nM SAG, 300 μM MβCD:KK174, and 50 nM SHH for 20 hours) in Smo−/− cells stably expressing mSMO-WT, mSMO-D99A, mSMO-D477G, or mSMO-V333F. (E) A ligand-affinity assay was used to measure the amount of hSMOΔC-BRIL-V329F (7) captured on 20(S)-yne–coupled beads in the presence of 50 μM MβCD:20(S)-OHC or MβCD:KK174. The immunoblot shows 1% of the protein added to each binding reaction (input) and 50% of the protein captured on beads. Exact P values for comparisons: (C) WT untreated versus KK174 = 0.0003, D99A untreated versus KK174 = 0.0009, and WT versus D99A (+KK174) = 0.9194. (D) WT untreated versus SHH < 0.0001, WT untreated versus SAG < 0.0001, WT untreated versus KK174 = 0.0003, D477G untreated versus SHH < 0.0001, D477G untreated versus SAG > 0.9999, D477G untreated versus KK174 = 0.0002, and WT versus D477G (+KK174) > 0.9999. (F) WT untreated versus SAG < 0.0001, WT untreated versus KK174 < 0.0001, V333F untreated versus SAG > 0.9999, and V333F untreated versus KK174 > 0.9999.