Fig. 3: Functional validation and structural basis of dual AKT and PRKG1 inhibition by ipatasertib.

A Binding pose of ipatasertib on AKT binding site extracted from PDB ID 4ekl. B Superposition of ATP binding pocket in AKT (gray) and PRKG1 (blue) showing a different 3D conformation for the equivalent glutamic acid residue (278 in Akt, 488 in PRKG1). C Binding pose of ipatasertib docked to PRKG1 after induced fit (pink). Original PRKG1 conformation in blue. D 3D structures of AKT (PDB ID 5kcv, gray and orange) and PRKG1-Iβ (7lv3, blue). E Western blot showing PRKG1 and AKT levels in whole cell extracts upon PRKG1 knockdown (shPRKG1#1 and shPRKG1#2) and AKT knockdown (shAKT#1 and shAKT#2) in RH4 cells. Tubulin as loading control. Blots are representative of two independent biological replicates. F Cell viability curves at 72 h in shControl (shCtrl#LKO, purple), shAKT (shAKT#1, orange), and shPRKG1 (shPRKG1#2, blue) upon treatment with ipatasertib or miransertib, respectively. Mean ± SEM from independent biological replicates (n = 2 for ipatasertib, n = 3 for miransertib). Dose-response curves fitted using a four-parameter logistic model with variable slope. Source Data is available. G Western blot analysis of PRKG1 levels in protein extracts from SCR (control) and KO_PRKG1 cells in RH4. Tubulin was used as loading control. Blot is representative of four independent biological replicates. H Cell viability curves at 72 h for AKT inhibitors, showing the percentage of viable cells in control (SCR) versus PRKG1_KO cells treated with ATP-competitive inhibitors (ipatasertib and GSK690693) and allosteric inhibitors (MK-2206 and miransertib). Data represent mean ± SEM; n = 3 independent biological replicates for ipatasertib, miransertib, and MK-2206, and n = 2 for GSK. Dose-response curves were fitted using a four-parameter logistic model to calculate IC50 values (see table on the right). Source Data is available.