Abstract
Macrocyclization is a compelling strategy for conventional drug design for improving biological activity, target specificity, and metabolic stability, but it was rarely applied to the design of PROTACs possibly due to the mechanism and structural complexity. Herein, we report the rational design of the first series of "Head-to-Tail" macrocyclic PROTACs. The resulting molecule SHD913 exhibited pronounced Brd4 protein degradation with low nM DC50 values while almost totally dismissing the "hook effect", which is a general character and common concern of a PROTAC, in multiple cancer cell lines. Further biological evaluation revealed that the compound exhibited positive cooperativity and induced de novo protein-protein interactions (PPIs) in both biophysical and cellular NanoBRET assays and outperformed macroPROTAC-1 that is the first reported macrocyclic Brd4 PROTAC, in cellular assays. In vitro liver microsomal stability evaluation suggested that SHD913 demonstrated improved metabolic stability in different species compared with the linear counterpart. The co-crystal structure of Brd4BD2: SHD913: VCB (VHL, Elongin C and Elongin B) complex determination and molecular dynamics (MD) simulation also elucidated details of the chemical-induced PPIs and highlighted the crucial contribution of restricted conformation of SHD913 to the ternary complex formation. These results collectively support that macrocyclization could be an attractive and feasible strategy for a new PROTAC design.