Overall, the PAMPA permeabilities of our new PROTAC series followed the same trends shown by our model compounds. In three of the four amide-to-ester matched pairs (MZ1 (21) and OMZ1 (25), ARV-771 (22) and OARV-771 (26), and AB2 (24) and OAB2 (28)), the amide-toester substitution led to an increase in permeability by 10-, 1.5-and 7.5-fold, respectively (Table , Figure , SI Table ). As expected, substituting the amide in AB1 (23) for an ester in OAB1 (27) caused a 2.5-fold reduction in permeability in the last matched pair. This decrease in permeability is due to the increased lipophilicity of 27, ALogP = 5.5, pushing the ester into the insoluble ALogP regime. Compound 28 had the highest PAMPA permeability (0.6 x 10 -6 cm/s), with an ALogP of 4.4. PROTACs with ALogP values >4.4 started to show a decrease in PAMPA permeability (Figure ). Compounds 22 and 26 contained an extra chiral methyl group at the benzyl position of the VH032 ligand. The effects of this additional methyl group on permeability can be seen when comparing two alternative matched pairs within the amide series, 22 vs 24, and within the ester series, 26 vs 28. In the amide pairing, an additional methyl group increases PAMPA permeability by 2.5-fold, whereas, in the case of the ester pairing, permeability decreases by 2-fold. Again, these trends are likely the result of the "inverted-parabola" relationship between ALogP and permeability. As MDCK permeability measurements are less sensitive to poor compound solubility than PAMPA permeability at high lipophilicities, we attempted to collect MDCK cell permeabilities for these PROTAC compounds starting with 21 and 25. However, both compounds were below the limit of detection in the apical to basal permeation (SI Table ). Thus, we did not pursue MDCK permeabilities on the remaining PROTACs. Taken together, our data are consistent with increasing lipophilicity and reducing PEG-like character of the PROTAC linker, producing similar trends in permeability as described for the model compounds above. Furthermore, all eight PROTACs were stable in plasma after 90 minutes with no detectable reduction in PROTAC levels (Figure ). This suggests that a rigid and sterically bulky POI-ligand, like JQ1, provides sufficient protection from ester hydrolysis, as also suggested by the model compounds. Next, we evaluated the cellular activities of all eight PROTACs in HEK293 cells to obtain a degradation (DC50) profile for BET proteins, Brd4, Brd3, and Brd2 (Figure , SI Figure ). Notably, the improvements in permeability seen when substituting the amide in the known degraders 21 and 22 for an ester in 25 and 26 translated into meaningful improvements in bioactivity. Compound 25 showed a 1.5-to 2-fold increase in degradation potency over 21 for both Brd4 (DC50 = 44 nM vs 60 nM, respectively) and Brd2 (DC50 = 133 nM vs 230 nM, respectively), while 25 showed near equipotent degradation compared to 21 for Brd3 (DC50 = 221 nM vs 239 nM respectively). Strikingly, 26 showed to be the most potent degrader out of this series, with a 5.5-fold more potent degradation of Brd4 compared to its amide counterpart, 22 (DC50 = 6 nM vs 33 nM, respectively), a 42-fold increase for Brd2 (DC50 = 1 nM vs 42 nM, respectively) and a 12fold increase for Brd3 (DC50 = 4 nM vs 47 nM, respectively). Similarly, 27 gave a 2-fold increase in degradation potency over its amide counterpart, 23 for both Brd4 (DC50 = 133 nM vs 57 nM, respectively) and Brd2 (DC50 = 87 nM vs 166 nM, respectively), and also, a 1.5-fold increase with Brd3 (DC50 = 107 nM vs 158 nM, respectively). Finally, 28 showed a 4-fold increase in degradation potency against Brd4 when compared to its amide counterpart, 24 (DC50 31 nM vs 125 nM, respectively), a 4-fold increase with Brd2 (DC50 = 68 nM vs 273 nM, respectively) and a 3.2-fold increase with Brd3 (DC50 = 68 nM vs 273 nM, respectively) (Figure , SI Figure ). Together, the cellular degradation data demonstrate that the amide-to-ester substitution has a profound beneficial effect on PROTAC activity.