Structure-based design of stapled peptides targeting the oligomeric state of lactate dehydrogenase.

Lactate dehydrogenase (LDH) catalyzes the reversible conversion of pyruvate, NADH, and H+ into lactate and NAD+, maintaining the energy metabolism and the cellular redox balance. It also plays a key role in metabolic reprogramming processes observed in cancer and other proliferative conditions. Consequently, LDH has long been considered as an attractive therapeutic target, and numerous inhibitors have been developed, albeit with limited clinical success. Most of these efforts have focused on the highly conserved active site, a strategy often hampered by poor selectivity and off-target effects. Here, we explored an alternative approach based on targeting LDH oligomerization, which may help circumvent some limitations associated with active-site inhibition. Our research focuses on optimizing a perfluoroaryl cysteine-stapled octapeptide named macrocycle 7, which was previously identified to bind the LDH tetramerization site with micromolar affinity. Through systematic structure-activity relationship studies and the incorporation of unnatural amino acids, we report the design and synthesis of improved analogs with enhanced binding affinity, stability, and inhibitory efficacy. Biophysical and enzymatic assays confirmed that these optimized peptides effectively destabilize LDH and inhibit its catalytic activity. In cellular models, the most potent analogs significantly altered LDH thermal stability, confirming target engagement, and 72-h treatments in HCT116 and MDA-MB-231 human cancer cells further revealed significant cytotoxic anticancer effects. These optimized peptides demonstrate the potential of targeting protein oligomerization as a general strategy to regulate LDH activity through structural destabilization rather than active site competition.