Structure‐Based Design of AG‐946, a Pyruvate Kinase Activator

Abstract Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure‐based design of AG‐946, an activator of PK isoforms, including red blood cell‐specific forms of PK (PKR). This was designed to have a pseudo‐C2‐symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off‐targets observed from the original scaffold. AG‐946 (1) demonstrated activation of human wild‐type PK (half‐maximal activation concentration [AC 50 ]=0.005 μM) and a panel of mutated PK proteins (K410E [AC 50 =0.0043 μM] and R510Q [AC 50 =0.0069 μM]), (2) displayed a significantly longer half‐time of activation (>150‐fold) compared with 6‐(3‐methoxybenzyl)‐4‐methyl‐2‐(methylsulfinyl)‐4,6‐dihydro‐5H‐thieno[2′,3′:4,5]pyrrolo[2,3‐d]pyridazin‐5‐one, and (3) stabilized PKR R510Q, an unstable mutant PKR enzyme, and preserved its catalytic activity under increasingly denaturing conditions. As a potent, oral, small‐molecule allosteric activator of wild‐type and mutant PKR, AG‐946 was advanced to human clinical trials.