Exploring the Key Amino Acid Residues Surrounding the Active Center of Lactate Dehydrogenase A for the Development of Ideal Inhibitors.

Lactate dehydrogenase A (LDHA) primarily catalyzes the conversion between lactic acid and pyruvate, serving as a key enzyme in the aerobic glycolysis pathway of sugar in tumor cells. LDHA plays a crucial role in the occurrence, development, progression, invasion, metastasis, angiogenesis, and immune escape of tumors. Consequently, LDHA not only serves as a biomarker for tumor diagnosis and prognosis but also represents an ideal target for tumor therapy. Although LDHA inhibitors show great therapeutic potential, their development has proven to be challenging. In the development of LDHA inhibitors, the key active sites of LDHA are emphasized. Nevertheless, there is a relative lack of research on the amino acid residues around the active center of LDHA. Therefore, in this study, we investigated the amino acid residues around the active center of LDHA. Through structure comparison analysis, five key amino acid residues (Ala30, Met41, Lys131, Gln233, and Ala259) were identified. Subsequently, the effects of these five residues on the enzymatic properties of LDHA were investigated using site-directed mutagenesis. The results revealed that the catalytic activities of the five mutants varied to different degrees in both the reaction from lactic acid to pyruvate and pyruvate to lactic acid. Notably, the catalytic activities of LDHAM41G and LDHAK131I were improved, particularly in the case of LDHAK131I. The results of the molecular dynamics analysis of LDHAK131I explained the reasons for this phenomenon. Additionally, the optimum temperature of LDHAM41G and LDHAQ233M increased from 35 °C to 40 °C, whereas in the reverse reaction, the optimum temperature of LDHAM41G and LDHAK131I decreased from 70 °C to 60 °C. These findings indicate that Ala30, Met41, Lys131, Gln233, and Ala259 exert diverse effects on the catalytic activity and optimum temperature of LHDA. Therefore, these amino acid residues, in addition to the key catalytic site of the active center, play a crucial role. Considering these residues in the design and screening of LDHA inhibitors may lead to the development of more effective inhibitors.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Designed ß-Hairpins Inhibit LDH5 Oligomerization and Enzymatic Activity.

Lactate dehydrogenase 5 (LDH5) is overexpressed in metastatic tumors and is an attractive target for anticancer therapy. Small-molecule drugs have been developed to target the substrate/cofactor sites of LDH5, but none has reached the clinic to date, and alternative strategies remain almost unexplored. Combining rational and computer-based approaches, we identified peptidic sequences with high affinity toward a ß-sheet region that is involved in protein-protein interactions (PPIs) required for the activity of LDH5. To improve stability and potency, these sequences were grafted into a cyclic cell-penetrating ß-hairpin peptide scaffold. The lead grafted peptide, cGmC9, inhibited LDH5 activity in vitro in low micromolar range and more efficiently than the small-molecule inhibitor GNE-140. cGmC9 inhibits LDH5 by targeting an interface unlikely to be inhibited by small-molecule drugs. This lead will guide the development of new LDH5 inhibitors and challenges the landscape of drug discovery programs exclusively dedicated to small molecules.

Polyunsaturated fatty acid-containing phosphatidic acids selectively interact with L-lactate dehydrogenase A and induce its secondary structural change and inactivation.

Phosphatidic acid (PA) consists of various molecular species that have different fatty acyl chains at the sn-1 and sn-2 positions; and consequently, mammalian cells contain at least 50 structurally distinct PA molecular species. However, the different roles of each PA species are poorly understood. In the present study, we attempted to identify dipalmitoyl (16:0/16:0)-PA-binding proteins from mouse skeletal muscle using liposome precipitation and tandem mass spectrometry analysis. We identified L-lactate dehydrogenase (LDH) A, which catalyzes conversion of pyruvate to lactate and is a key checkpoint of anaerobic glycolysis critical for tumor growth, as a 16:0/16:0-PA-binding protein. LDHA did not substantially associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphoinositides and cardiolipin at physiological pH (7.4), indicating that LDHA specifically bound to PA. Interestingly, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA also interacted with LDHA, and their binding activities were stronger than 16:0/16:0-PA at pH 7.4. Moreover, circular dichroism spectrometry showed that 18:0/20:4- and 18:0/22:6-PA, but not 16:0/16:0- or 18:0/18:0-PA, significantly reduced the α-helical structure of LDHA. Furthermore, 18:0/20:4- and 18:0/22:6-PA attenuated LDH activity. Taken together, we demonstrated for the first time that LDHA is a PA-binding protein and is a unique PA-binding protein that is structurally and functionally controlled by associating with 18:0/20:4- and 18:0/22:6-PA.