EX229 (C991) is an allosteric activator of AMPK, with Kds of 0.06 μM, 0.06 μM and 0.51 μM for α1β1γ1, α2β1γ1, and α1β2γ1, respectively.

AMPK α1β1γ1:0.06 μM (Kd), AMPK α1β2γ1:0.51 μM (Kd), AMPK α2β1γ1:0.06 μM (Kd)

Treatment of hepatocytes with EX229 (991) alone results in a slight increase in the phosphorylation of AMPK and RAPTOR only at 0.3 μM, whereas a robust increase in ACC phosphorylation is readily observed and saturated at a concentration of 0.03 μM EX229. AICAR or C13 alone robustly increases T172 phosphorylation of AMPKα, and when 991 is coincubated, there is a modest additional dose-dependent increase in AMPKα phosphorylation. RAPTOR phosphorylation is modestly increased by AICAR or C13 alone, and it is dose-dependently increased when coincubation is carried out with EX229. EX229 also dose-dependently (0.01 and 0.1 μM) inhibits lipogenesis (34% and 63%, respectively), which is further reduced when it is coincubated with a low dose of AICAR (0.03 mM) or C13 (1 μM). Treatment with EX229 promotes dose-dependent increases in ACC and RAPTOR phosphorylation. Similar to the observations in hepatocytes [2].