Unveiling the enzymatic activity of a dimeric <scp>LDH</scp> isoform and its implications for allosteric inhibition strategies

Lactate dehydrogenase (LDH) is a key enzyme in cancer metabolism, with isoforms LDH5 and LDH1 supporting glycolysis and oxidative lactate metabolism, respectively. While the development of competitive LDH inhibitors has faced diverse challenges, allosteric strategies targeting LDH tetramerization have recently attracted increasing attention. To further explore this alternative, we investigated the factors influencing LDH tetramerization and enzymatic activity using a truncated form of human LDH-B (LDHBtr), which was reported to exist predominantly as a dimer. Unexpectedly, LDHBtr exhibited measurable activity at high concentrations, correlating with increased protein stability and a structural transition to the tetrameric form. Preincubation with NADH further enhanced LDHBtr activity, stability, and self-association, consistent with cofactor-promoted tetramer assembly. Crystallographic studies confirmed the tetrameric structure of LDHBtr bound to NADH. Furthermore, reported LDH allosteric inhibitors, including cGmC9 and fluoxetine, preferentially inhibited LDHBtr compared to the native LDHB, by preventing tetramer formation. Overall, this work highlights the central role of tetramerization in regulating LDH activity, and the therapeutic potential of targeting this process. It also establishes LDHBtr as a valuable tool for screening tetramerization disruptors, paving the way for next-generation LDH inhibitors to target cancer metabolism.