We reasoned that another strategy for overcoming this tight binding limit would involve employing spy molecules with higher binding affinity to VBC. In this way higher IC50 values would be expected from the PROTAC, allowing the titrations to be performed at VBC concentrations suitable with a good assay performance. Based on our previous work exploring different spy molecules probing VBC, spy molecules 7 (Kd VBC = 67 ± 14 µM) and 18 (Kd VBC = 25 ± 3 µM) were selected with this goal in mind (Scheme 3), because they are both tighter VBC binders than 19 (Kd VBC = 145 ± 29 µM). However, fast transverse relaxation times (T2) were observed for both compounds solely in presence of Brd4(2), especially spy molecule 7 (Figure ). This observation indicates that spy molecules 7 and 18 can adventitiously weakly bind to Brd4(2), making them unsuitable for measuring α of BET targeting PROTACs.