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Abstract

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.