The assembly of tetrameric human L-lactate dehydrogenase is regulated by ionic strength, β-NADH binding, cyclic...

Lactate dehydrogenases catalyze the reduction of pyruvate to lactate, the generation of which is of importance for the energetic metabolism of malignant cells. In particular, human cancer cells overexpress lactate dehydrogenase A (hLDH-A), whose catalytic activity depends on its assembly into the corresponding homotetramer (denoted as hLDH-5), suggesting that compounds interfering with the protein-protein interactions responsible for the generation of hLDH-5 should represent inhibitors of high selectivity. Not surprisingly, quite a number of competitive inhibitors were designed, synthesized, and tested against hLDH-5. However, only a few representatives of the repertoire accordingly obtained feature an appropriate selectivity. Here we show how the ionic strength and β-NADH binding affect the monomers-to-tetramer hLDH-A transition. Moreover, by taking advantage of the isolation of monomeric hLDH-A at neutral pH, we tested the effectiveness of cyclic peptides and long-chain dicarboxylates in hampering the assembly of hLDH-5. Interestingly, the cyclic peptide LCO15 and the long-chain dicarboxylate crocetin were found to be very effective inhibitors of hLDH-A: the catalytic activity of the enzyme was inhibited by 97% and 86% when these compounds were administered at 40 and 20 μM, respectively. Furthermore, the amount of lactate produced by MCF7 human cancer cells was found to decrease by 30% in the presence of 80 μM crocin (a diester of crocetin). Overall, our observations indicate that the assembly of hLDH-A into catalytically competent hLDH-5 can be appropriately inhibited by means of cyclic peptides and long-chain dicarboxylates.