Structural and cellular insights into the inhibition of the drug efflux activity of the Hedgehog receptor PTCH1

Abstract PTCH1, the receptor for the Sonic Hedgehog morphogen, mediates cholesterol transport across the plasma membrane by harnessing the proton motive force. In cancer, PTCH1 is frequently overexpressed and promotes chemoresistance by transporting drugs such as doxorubicin (dxr) out of cells. Among the inhibitors identified, PAH stands out for its ability to significantly enhance the efficacy of several chemotherapeutic drugs on melanoma and breast cancer cells. To investigate PTCH1’s structure in complex with its inhibitor PAH, we overexpressed a construct spanning residues 1-619 and 720-1305 in HEK293 cells. The protein localized to the membrane, and transfected cells exhibited reduced sensitivity to dxr compared to control cells. Additionally, we observed a pH-dependent efflux of dxr, which was reversed by PAH, confirming that the PTCH1 construct used in this study functions as an active drug-efflux pump. In the structure of PTCH1 bound to PAH determined using cryo-electron microscopy, PAH occupies a hydrophobic cavity in an extracellular domain which is normally occupied by cholesterol in other PTCH1 structures, and engages in a key hydrogen bond via one of its hydroxyl groups, a feature previously established as essential for its inhibitory function. These findings not only clarify the molecular basis of PAH’s action but also provide a structural roadmap for rational drug design, enabling the development of next-generation inhibitors with enhanced potency.