Figure 1. Thalidezine binds and activates AMPK in vitro.

(A) Chemical structure of thalidezine. (B) Kinetic analysis of the interaction between AMPK and thalidezine by BLI. The Ni-NTA biosensor tips coated with HIS-tagged AMPK were dipped in increasing concentrations of thalidezine (12.5, 25, 50, and 100 μM) to measure binding affinity of thalidezine to AMPK (K_on 1.17±0.231×104 Ms⁻¹) and subsequently moved to wells containing buffer to measure dissociation rates (K_off 2.22±0.407 s⁻¹). The affinity constant was calculated as the ratio of the K_off to the K_on (K_D 189±50.9 μM). (C) Thalidezine directly activates AMPK kinase. AMPK protein was incubated without (control) or with increasing concentrations of thalidezine (Tha) (1, 2.5, 5, and 10 μM) or AMP (10 μM, positive control) for 20 min. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (D) Thalidezine activates the AMPK-mTOR signaling pathway. HeLa cells were treated with 10 μM of thalidezine for 0-24 h, rapamycin (Rapa, 200 nM) was used as the positive control. Immunoblots indicated p-AMPK, total AMPK, p-p70S6K, total p70S6K, and β-actin detection. Uncropped blots images were shown in Supplementary Figure 4A. Data were representative of three to five independent experiments.