Development of terfenadine-derived small-molecule inhibitors of TFE3-O dimerization for translocation renal cell carcinoma

Translocation renal cell carcinoma (tRCC) is an aggressive kidney cancer subtype with poor clinical outcome and no effective standard therapies. It is characterized by gene fusions involving members of the MiT transcription factor family, most commonly TFE3 fused to various partner genes. Work from our laboratory has previously identified the antihistamine terfenadine as a promising compound capable of inhibiting TFE3 dimerization, highlighting dimerization as a targetable mechanism in tRCC. These findings suggest that disrupting TFE3 fusion protein dimerization may enhance therapeutic response and could be leveraged in combination with existing treatment options, including tyrosine kinase inhibitors (TKIs). The purpose of this study was to optimize terfenadine-derived tool compounds and identify novel small molecules that enhance its antiproliferative activity while reducing terfenadine well-known long QT associated cardiotoxicity. We aimed to determine whether these derivatives selectively inhibit dimerization of TFE3 fusion proteins and improve activity against tRCC diminishing hERG interaction and cardiotoxicity. Two series of terfenadine analogs based on chemical structure modifications were synthesized to evaluate groups specific functional contribution. All compounds were purified and structurally verified by NMR, LTQ mass spectrometry, and HPLC. Their antiproliferative activity was assessed in tRCC cell lines harboring distinct TFE3 fusions (R07: SPFQ-TFE3; UOK-109: NONO-TFE3; UOK-146: PRCC-TFE3). FRET-based assays are being established to measure inhibition of TFE3/TFE3-O dimerization, and hERG activity monitored. Several newly synthesized compounds, in particular two leading candidates exhibited improved antiproliferative effects (IC 50 s ~2 µM) as compared to terfenadine (IC 50 s ~7 µM). These results provide a foundation for further structural refinement and assessment of pharmacological properties with modifications also aiming to reduce its cardiotoxic effect. Combination studies with TKIs demonstrated that co-treatment with terfenadine significantly reduced IC50 values relative to monotherapy, as confirmed by Combination Index analysis. This supports the hypothesis that inhibiting TFE3 fusion protein dimerization enhances drug response to TKIs, potentially by reducing drug sequestration in the lysosome. In conclusion, this work identifies terfenadine derivatives with improved antiproliferative potential and reduced predicted cardiotoxicity, and highlights TFE3 dimerization as a therapeutically targetable mechanism that may help overcome drug resistance in tRCC.