Development of In-Cell Self-Assembly Proteolysis Targeting Chimeras Exploiting the Biorthogonal Reaction Between Vinyl Boronic Acids and 1,2,4,5-Tetrazines

Background Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules composed of a ligand of the protein of interest (POI) and a ligand of an E3-ligase, connected by a suitable linker. PROTACs induce POI ubiquitination and degradation via the 26S proteasome. Despite their potential to target undruggable proteins, PROTACs often violate the Lipinski’s rule of 5, potentially leading to bioavailability issues. [1] Aims A possible solution is represented by in-cell self-assembly PROTACs: the degrader is split into two fragments bearing biorthogonal groups that react inside the cell to form the active molecule. In this work, the reaction between vinyl boronic acids and 1,2,4,5-tetrazines[2] was used to develop self-assembling PROTACs targeting BRD4, an epigenetic reader that binds acetylated histones and drives oncogenic transcription. Methods The BRD4 inhibitor (+)-JQ1, originally reported in 2010[3], and lenalidomide, a recruiter of the E3 ligase CRBN, were selected as the binding moieties of the PROTAC. Based on feasible exit vectors and synthetic accessibility, routes to obtain the functionalized fragments were developed. The formation of the full PROTAC through the selected biorthogonal reaction is intended to be monitored through HPLC and NMR[4]. Computational studies are being performed using molecular docking based on the available crystal structures of BRD4 in complex with JQ1 and CRBN in complex with lenalidomide. Linker lengths were designed by comparison with reported active PROTACs. The stability of both the binary (PROTAC–BRD4 and PROTAC–CRBN), and ternary complex (BRD4–PROTAC–CRBN) was evaluated through molecular dynamics simulations. Results An efficient route was developed for the synthesis of a (+)-JQ1 derivative bearing a vinyl boronic acid (VBA) moiety, as well as for a lenalidomide-based fragment functionalized with a tetrazine group (Tz). The selected biorthogonal reaction was validated on model compounds, supporting its applicability to the full self-assembling PROTAC. Computational studies indicated favorable stability of both binary and ternary complexes. Ongoing work includes biophysical evaluation of the binding affinity of each fragment and of the assembled PROTAC, as well as assessment of intracellular PROTAC formation and evaluation of its degradation activity through cellular essays. Conclusions This work demonstrates the feasibility of designing self-assembling PROTACs based on biorthogonal chemistry. Synthetic and computational results support the stability and potential functionality of the system. Ongoing studies will validate intracellular assembly and biological activity, providing insight into this strategy as a solution to overcome pharmacokinetic limitations of conventional PROTACs. References [1] He, S.; Dong, G.; Cheng, J.; Wu, Y.; Sheng, C. Strategies for Designing Proteolysis Targeting Chimaeras (PROTACs). Med. Res. Rev., 2022, 42 (3), 1280–1342. https://doi.org/10.1002/med.21877. [2] Eising, S.; van der Linden, N. G. A.; Kleinpenning, F.; Bonger, K. M. Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells. Bioconjugate Chem., 2018, 29 (4), 982–986. https://doi.org/10.1021/acs.bioconjchem.7b00796. [3] Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; et al. Selective Inhibition of BET Bromodomains. Nature, 2010, 468 (7327), 1067–1073. https://doi.org/10.1038/nature09504. [4] Guo, Z.; Sun, Y.; Liang, L.; Lu, W.; Luo, B.; Wu, Z.; Huo, B.; Hu, Y.; Huang, P.; Wu, Q.; et al. Design and Synthesis of Dual EZH2/BRD4 Inhibitors to Target Solid Tumors. J. Med. Chem., 2022, 65 (9), 6573–6592. https://doi.org/10.1021/acs.jmedchem.1c01876.