Cancer cells frequently rewire central carbon metabolism to sustain rapid proliferation, and one recurring feature of this metabolic shift is an increased reliance on glutamine catabolism. A key node in this pathway is mitochondrial malic enzyme 2 (ME2), which converts malate to pyruvate while generating NAD(P)H. ME2 is markedly upregulated in several aggressive cancers, including pancreatic, melanoma, and lung tumors, yet its structural tractability and potential for selective pharmacological inhibition remain insufficiently explored. To address this gap, we undertook a comprehensive structural and biochemical analysis of all three human malic enzyme isoforms with the goal of defining inhibitor binding modes, identifying cryptic ligandable pockets, and establishing a framework for isoform‑selective drug discovery.
Recombinant ME1, ME2, and ME3 were expressed and kinetically characterized in the presence of known and newly identified inhibitors. We determined a complete set of X‑ray crystal structures for each isoform bound to the potent small‑molecule inhibitor NPD‑389, capturing a metal‑coordinated binding mode that is conserved across isoforms. These structures revealed an unexpected conformational state of the malic enzyme scaffold, providing a previously unappreciated template for structure‑based ligand discovery. Leveraging this conformation, we performed virtual screening of a curated library of approximately 14 million drug‑like and fragment‑sized molecules. Multiple scaffolds emerged with measurable biochemical activity, validating the structural model as a productive search space.
Among these hits, one chemotype—FLA—displayed a distinctive selectivity profile and bound ME2 in a cryptic allosteric pocket adjacent to the NAD⁺ cofactor and malate‑binding site. Structural analysis showed that this pocket is not apparent in ligand‑free structures but forms through local rearrangements upon inhibitor engagement, highlighting a previously unrecognized opportunity for isoform‑specific targeting. Cellular assays in melanoma and triple‑negative breast cancer models demonstrated that both NPD‑389 and FLA reduce proliferation, supporting their potential as chemical leads for further optimization.
Together, these findings establish mitochondrial malic enzymes, particularly ME2 and ME3, as structurally tractable and therapeutically relevant metabolic targets. The elucidation of both a conserved metal‑binding inhibitory mode and a newly discovered cryptic allosteric pocket provides a robust foundation for rational design of next‑generation inhibitors with improved potency, isoform selectivity, and mitochondrial targeting. Such compounds hold promise as adjuncts to existing cancer therapies by exploiting metabolic vulnerabilities that underlie tumor growth and survival.