Kinetic and Structural Characterization of a Novel Allosteric Inhibitor Targeting Human Lactate Dehydrogenase A in Cancer Metabolism
DOI:
https://doi.org/10.70749/ijbr.v3i7.1941Keywords:
Lactate dehydrogenase A (LDHA), allosteric inhibitor, cancer metabolism, enzyme kinetics, X-ray crystallographyAbstract
Lactate dehydrogenase A (LDHA) is a critical metabolic enzyme upregulated in cancers that drives glycolytic flux and supports tumor growth. While LDHA inhibition represents a promising therapeutic strategy, existing active-site inhibitors face challenges including poor selectivity and competition with endogenous substrates. Here, we characterize LADX-21, a novel allosteric inhibitor exhibiting potent (IC50 = 4.3 ± 0.6 μM) and selective inhibition of LDHA. Kinetic studies revealed non-competitive inhibition with respect to pyruvate (Km unchanged, Vmax reduced by 67%), confirming an allosteric mechanism distinct from traditional substrate mimics. Thermal shift assays demonstrated strong binding (+5.4°C ΔTm) and X-ray crystallography (2.1 Å resolution) identified a unique allosteric pocket near the αC-helix/NADH domain, explaining its isoform specificity. Notably, LADX-21 showed >50-fold selectivity for LDHA over LDHB and reduced lactate production by 47% in A549 lung cancer cells at 10 μM, while sparing normal fibroblasts. Structural analysis revealed key interactions with Tyr239 and Arg168 that induce conformational changes destabilizing the catalytic loop. Unlike NADH-competitive inhibitors, LADX-21 maintained efficacy at physiological pyruvate concentrations (0.5 mM). The inhibitor's reversible binding mode and favorable physicochemical properties suggest improved drug-like characteristics compared to earlier LDHA-targeting compounds. These findings establish LADX-21 as both a valuable chemical probe for studying LDHA biology and a promising lead compound for anticancer drug development. Its novel mechanism bypasses limitations of active-site inhibition and provides a framework for designing next-generation allosteric modulators of cancer metabolism.
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1. Angulo-Elizari, E., Gaviria-Soteras, L., Zubiri, I., Ramos-Inza, S., Sanmartin, C., & Plano, D. (2023). Unmasking the Warburg effect: unleashing the power of enzyme inhibitors for cancer therapy. Drugs and Drug Candidates, 2(3), 728-769.
https://doi.org/10.3390/ddc2030037
2. Cheng, A., Zhang, P., Wang, B., Yang, D., Duan, X., Jiang, Y., . . . Ding, C. (2019). Aurora-A mediated phosphorylation of LDHB promotes glycolysis and tumor progression by relieving the substrate-inhibition effect. Nature communications, 10(1), 5566.
https://doi.org/10.1038/s41467-019-13485-8
3. Di Ianni, L. (2018). Exploring the role of LDH in cancer cells through the use of small-molecule inhibitors.
4. Di Stefano, G., Manerba, M., Di Ianni, L., & Fiume, L. (2016). Lactate dehydrogenase inhibition: exploring possible applications beyond cancer treatment. Future medicinal chemistry, 8(6), 713-725.
https://doi.org/10.4155/fmc.16.10
5. El Khoury, A., & Papaneophytou, C. (2025). Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation. Molecules, 30(14), 2923.
https://doi.org/10.3390/molecules30142923
6. Farhana, A., & Lappin, S. L. (2023). Biochemistry, lactate dehydrogenase. In StatPearls [internet]: StatPearls Publishing.
7. Friberg, A., Rehwinkel, H., Nguyen, D., Pütter, V., Quanz, M., Weiske, J. r., . . . Langer, G. (2020). Structural evidence for isoform-selective allosteric inhibition of lactate dehydrogenase A. ACS omega, 5(22), 13034-13041.
https://doi.org/10.1021/acsomega.0c00715
8. Fukushi, A., Kim, H.-D., Chang, Y.-C., & Kim, C.-H. (2022). Revisited metabolic control and reprogramming cancers by means of the Warburg effect in tumor cells. International journal of molecular sciences, 23(17), 10037.
https://doi.org/10.3390/ijms231710037
9. Garcia, I., Cornely, K., Peterson, C. N., & Berkmen, M. B. (2024). Roles of the oncometabolite enantiomers of 2-hydroxyglutarate and their metabolism by diverse dehydrogenases. Essays in Biochemistry, 68(2), 161-171.
https://doi.org/10.1042/ebc20230077
10. Jiang, X., Yan, N., Deng, D., & Yan, C. (2022). Structural aspects of the glucose and monocarboxylate transporters involved in the Warburg effect. IUBMB life, 74(12), 1180-1199.
https://doi.org/10.1002/iub.2668
11. Laganà, G., Barreca, D., Calderaro, A., & Bellocco, E. (2019). Lactate dehydrogenase inhibition: biochemical relevance and therapeutical potential. Current medicinal chemistry, 26(18), 3242-3252.
https://doi.org/10.2174/0929867324666170209103444
12. Li, C., Zhang, G., Zhao, L., Ma, Z., & Chen, H. (2015). Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer. World journal of surgical oncology, 14(1), 15.
https://doi.org/10.1186/s12957-016-0769-9
13. Li, W., Liao, L.-p., Song, N., Liu, Y.-j., Ding, Y.-l., Zhang, Y.-y., . . . Wang, H.-b. (2022). Natural product 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase. Acta Pharmacologica Sinica, 43(2), 470-482.
14. Lin, L.-C., Chang, H.-Y., Lin, T. E., Lin, J.-R., Hsia, S.-M., Hsu, K.-C., & Huang, T.-C. (2022). Structure-based virtual screening discovers novel kidney-type glutaminase inhibitors. Biomedicine & pharmacotherapy, 154, 113585.
https://doi.org/10.1016/j.biopha.2022.113585
15. Liu, G.-M., & Zhang, Y.-M. (2018). Targeting FBPase is an emerging novel approach for cancer therapy. Cancer cell international, 18(1), 36.
https://doi.org/10.1186/s12935-018-0533-z
16. Malla, A., Gupta, S., & Sur, R. (2023). Inhibition of Lactate Dehydrogenase A (LDH-A) by Diclofenac Sodium Induces Apoptosis in HeLa Cells by Activation of AMPK. bioRxiv, 2023.2010. 2002.560620.
https://doi.org/10.1101/2023.10.02.560620
17. Martinez-Vaz, B. M., Howard, A. L., Jamburuthugoda, V. K., & Callahan, K. P. (2024). Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules. Essays in Biochemistry, 68(2), 173-181.
18. Pedley, A. M., & Benkovic, S. J. (2017). A new view into the regulation of purine metabolism: the purinosome. Trends in biochemical sciences, 42(2), 141-154.
https://doi.org/10.1016/j.tibs.2016.09.009
19. Petrassi, M., Barber, R., Be, C., Beach, S., Cox, B., D’Souza, A.-M., . . . Hunt, P. (2017). Identification of a novel allosteric inhibitory site on tryptophan hydroxylase 1 enabling unprecedented selectivity over all related hydroxylases. Frontiers in Pharmacology, 8, 240.
20. Rai, G., Urban, D. J., Mott, B. T., Hu, X., Yang, S.-M., Benavides, G. A., . . . Lee, T. D. (2020). Pyrazole-based lactate dehydrogenase inhibitors with optimized cell activity and pharmacokinetic properties. Journal of medicinal chemistry, 63(19), 10984-11011.
https://doi.org/10.1021/acs.jmedchem.0c00916
21. Reyes Romero, A., Lunev, S., Popowicz, G. M., Calderone, V., Gentili, M., Sattler, M., . . . Holak, T. A. (2021). A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity. Communications biology, 4(1), 949.
22. Schneider, N. O. (2024). Characterizing Substituted Imidazolidinetriones as a Novel Class of Pyruvate Carboxylase Inhibitors: Marquette University.
23. Storey, K. B. (2016). Comparative enzymology—new insights from studies of an “old” enzyme, lactate dehydrogenase. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 199, 13-20.
https://doi.org/10.1016/j.cbpb.2015.12.004
24. Tang, P., Xu, J., Oliveira, C. L., Li, Z. J., & Liu, S. (2017). A mechanistic kinetic description of lactate dehydrogenase elucidating cancer diagnosis and inhibitor evaluation. Journal of enzyme inhibition and medicinal chemistry, 32(1), 564-571.
25. Tanner, J. J., Fendt, S.-M., & Becker, D. F. (2018). The proline cycle as a potential cancer therapy target. Biochemistry, 57(25), 3433-3444.
https://doi.org/10.1021/acs.biochem.8b00215
26. Woodford, M. R., Baker-Williams, A. J., Sager, R. A., Backe, S. J., Blanden, A. R., Hashmi, F., . . . Castelli, M. (2021). The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect. Nature structural & molecular biology, 28(8), 662-670.
https://doi.org/10.1038/s41594-021-00633-2
27. Xu, X., Wang, J., Wang, M., Yuan, X., Li, L., Zhang, C., . . . Tong, C. (2021). Structure-enabled discovery of novel macrocyclic inhibitors targeting glutaminase 1 allosteric binding site. Journal of medicinal chemistry, 64(8), 4588-4611.
https://doi.org/10.1021/acs.jmedchem.0c02044
28. Yang, W.-H., Qiu, Y., Stamatatos, O., Janowitz, T., & Lukey, M. J. (2021). Enhancing the efficacy of glutamine metabolism inhibitors in cancer therapy. Trends in cancer, 7(8), 790-804.
https://doi.org/10.1016/j.trecan.2021.04.003
29. Yizhak, K., Chaneton, B., Gottlieb, E., & Ruppin, E. (2015). Modeling cancer metabolism on a genome scale. Molecular systems biology, 11(6), 817.
https://doi.org/10.15252/msb.20145307
30. Zeng, S., Wang, Y., Ying, M., Jin, C., Ying, C., Wang, D., . . . Hu, X. (2024). Elucidating the kinetic and thermodynamic insight into regulation of glycolysis by lactate dehydrogenase and its impact on tricarboxylic acid cycle and oxidative phosphorylation in cancer cells. bioRxiv, 2024.2006. 2026.600909.
https://doi.org/10.7554/elife.99576
31. Zhang, W., Zhang, S.-L., Hu, X., & Tam, K. Y. (2015). Targeting tumor metabolism for cancer treatment: is pyruvate dehydrogenase kinases (PDKs) a viable anticancer target? International journal of biological sciences, 11(12), 1390.
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