Optimized Stapled Peptides Targeting LDH Oligomerization Induce Cytotoxicity in Human Cancer Cells

Lactate dehydrogenase (LDH) is a critical enzyme in energy metabolism and redox balance, playing a pivotal role in the metabolic reprogramming observed in cancer and other proliferative conditions. Current therapeutic strategies often target the highly conserved LDH active site, but these approaches frequently encounter limitations due to poor selectivity and off-target effects, hindering clinical success. This research explores an alternative, novel strategy: targeting LDH oligomerization to induce structural destabilization, aiming to circumvent the challenges associated with active-site inhibition and provide a more specific therapeutic avenue.

Researchers optimized macrocycle 7, a perfluoroaryl cysteine-stapled octapeptide previously identified to bind the LDH tetramerization site with micromolar affinity. Through systematic structure-activity relationship studies and the incorporation of unnatural amino acids, improved analogs were designed and synthesized to enhance binding affinity, stability, and inhibitory efficacy. Biophysical and enzymatic assays were employed to confirm LDH destabilization and inhibition of its catalytic activity. In cellular models, the most potent analogs were tested for their ability to alter LDH thermal stability, confirming target engagement. Finally, 72-h treatments were conducted in HCT116 and MDA-MB-231 human cancer cell lines to assess cytotoxic anticancer effects.

The systematic optimization efforts yielded stapled peptide analogs with significantly enhanced binding affinity, stability, and inhibitory efficacy compared to the original macrocycle 7. Biophysical and enzymatic assays consistently 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, providing clear evidence of successful target engagement. This structural destabilization mechanism represents a distinct approach from traditional active-site competition.

72-h treatments with these optimized peptides in HCT116 and MDA-MB-231 human cancer cells further revealed significant cytotoxic anticancer effects, demonstrating their therapeutic potential.