Characterization of 3‑[(Carboxymethyl)thio]picolinic Acid: A Novel Inhibitor of Phosphoenolpyruvate Carboxykinase

Phosphoenolpyruvate
carboxykinase (PEPCK) has traditionally been
characterized for its role in the first committed step of gluconeogenesis.
The current understanding of PEPCK’s metabolic role has recently
expanded to include it serving as a general mediator of tricarboxylic
acid cycle flux. Selective inhibition of PEPCK in vivo and in vitro has been achieved with 3-mercaptopicolinic
acid (MPA) (Ki ∼ 8 μM), whose
mechanism of inhibition has been elucidated only recently. On the
basis of crystallographic and mechanistic data of various inhibitors
of PEPCK, MPA was used as the initial chemical scaffold to create
a potentially more selective inhibitor, 3-[(carboxymethyl)­thio]­picolinic
acid (CMP), which has been characterized both structurally and kinetically
here. These data demonstrate that CMP acts as a competitive inhibitor
at the OAA/PEP binding site, with its picolinic acid moiety coordinating
directly with the M1 metal in the active site (Ki ∼ 29–55 μM). The extended carboxy tail
occupies a secondary binding cleft that was previously shown could
be occupied by sulfoacetate (Ki ∼
82 μM) and for the first time demonstrates the simultaneous
occupation of both OAA/PEP subsites by a single molecular structure.
By occupying both the OAA/PEP binding subsites simultaneously, CMP
and similar molecules can potentially be used as a starting point
for the creation of additional selective inhibitors of PEPCK.