Design of Inhibitory Peptides Based on TRPM4-KCTD5 Interaction

High expression of TRPM4 has been reported to be associated with pathologies that alter cytoskeletal rearrangement and cell migration, including fibrosis and metastatic cancer, across different cell lines. The KCTD5 protein was identified as a positive regulator of TRPM4, thereby increasing Ca²⁺ sensitivity. KCTD5 is involved in cell migration by regulating TRPM4. TRPM4 and KCTD5 expression levels are elevated in breast cancer with poor prognosis. Therefore, the TRPM4-KCTD5 interaction represents an attractive target for the development of peptides that modulate TRPM4 activity with therapeutic potential. Based on this premise, two peptides derived from the TRPM4-KCTD5 interface were designed in silico and evaluated using in vitro assays in HEK293, HEK293^KCTD5-/-, and MDA-MB231 cells, including intracellular sodium recordings, patch clamp, cell invasion, and bimolecular fluorescent complementation (BiFC). In BiFC assays, a decrease in the fluorescence of TRPM4-KCTD5 complexes was observed upon treatment with the peptides TAT-TRPM4-HA or TAT-KCTD5-HA, indicating a loss of the TRPM4-KCTD5 interaction. A significant decrease in TRPM4-dependent Na⁺ influx and currents was observed in HEK293 cells treated with the peptides TAT-TRPM4-HA and TAT-KCTD5-HA compared to untreated controls. Furthermore, a significant decrease in cell invasion was observed in MDA-MB-231 cells treated with the TAT-TRPM4-HA peptide compared with controls. Overall, these experiments demonstrate that the in silico-designed peptides can inhibit the TRPM4-KCTD5 interaction and reduce MDA-MB-231 cell invasion, highlighting this protein interface as a promising therapeutic target for metastatic breast cancer.