Folate-ClipTAC: Design and Proof of Concept Study

Proteolysis-targeting chimaeras (PROTAC) technology is a rapidly emerging and intensively investigated strategy in the field of new drug research and development. However, due to the tripartite structure of PROTAC molecules, their molecular weights are generally high, which results in poor cell membrane permeability and targeting. A folate-CLIPTAC system was proposed to overcome these limitations.

For proof of concept, the estrogen receptor (ER) was selected as the model target protein. Two clickable fragments (Part A and B) were synthesized first. Then, the stability, targeting, and membrane permeability were examined in vitro. Finally, the ER degradation effect was evaluated by Western blot (WB).

The structures of the two precursors were confirmed by NMR and MS. Both Part A and Part B were able to stably exist within 48 hours in artificial plasma, and they showed distinct targeting and membrane permeability properties when incubated with breast cancer cell line MCF-7 for a certain period. When administered at intervals, WB analysis revealed a pronounced reduction in target protein levels.

The folate-CLIPTAC system was designed to overcome poor permeability by using two small precursor molecular fragments that undergo in situ click reactions and achieve tissue targeting by introducing folate groups on both small molecular fragments. Subsequently, when the two precursor fragments encounter each other within the cell, a click reaction occurs, generating the PROTAC macromolecule in situ, thereby achieving degradation of the POI.

Based on targeted degradation of ER as an example, this study provides primary proof-of-concept evidence for the folate-CLIPTAC system and lays the groundwork for future investigations.