Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized by structural modeling with molecular dynamics.

Proteasome substrate receptor hRpn13 is a promising target for cancer therapy. hRpn13 proteolysis-targeting chimera (PROTACs) induce apoptosis by targeting the hRpn13 proteolytic product hRpn13(Pru), which contains an intact ubiquitin- and proteasome-binding Pru domain. We generated a PROTAC series based on hRpn13(Pru)-targeting XL5 by varying the linker that connects it to a warhead against the VHL-based ubiquitin E3 ligase machinery. Among eight tested derivatives, XL5-VHL-7 with a -(CH(2))(5)- alkyl linker promoted hRpn13(Pru) degradation and induced cellular apoptosis with 2-fold improved potency compared to the original PROTAC. By using this PROTAC series with slight chemical modifications in the linker region, we were able to evaluate the efficacy of structural modeling with molecular dynamics for refining PROTACs. Overall, we found that the experimental data correlated with efficacy predictions based on molecular dynamics and structural modeling. Moreover, we could observe hRpn13:PROTAC:VHL complexes by 2D NMR that support the structural modeling and stronger affinity of XL5-VHL-7 compared to the original hRpn13 PROTAC. Our NMR data further indicate that hRpn13 Pru affinity for XL5-VHL-7 is higher within the VHL complex present than with XL5-VHL-7 alone. Altogether, we develop an hRpn13 PROTAC with 2-fold increased potency by optimizing the linker and demonstrate the current benefit and limitations for including modeling with molecular dynamics to aid PROTAC optimization.