Amide-to-ester substitution as a strategy for optimizing PROTAC permeability and cellular activity

Abstract

Bifunctional PROTAC degraders belong to "beyond Rule of 5" chemical space, and criteria for predicting their drug-like properties are underdeveloped. PROTAC components are often combined via late-stage amide couplings, due to the reliability and robustness of amide bond formation. Amides, however, can give rise to low cellular permeability and poor ADME properties. We hypothesized that a bioisosteric replacement of an amide with a less polar ester could lead to improvements in both physicochemical properties and bioactivity. Using a library of model compounds, bearing either amides or esters at various linker-warhead junctions, we identify parameters for optimal compound lipophilicity and permeability. We next applied these learnings to design a set of novel amide-to-ester substituted, VHL-based BET degraders with increased permeability. Our ester-PROTACs remarkably retained intracellular stability, were overall more potent degraders than their amide counterparts and showed an earlier onset of the hook effect. These enhanced cellular features were found to be driven by greater cell permeability rather than improvements in ternary complex formation. This largely unexplored amide-to-ester substitution therefore provides a simple and practical strategy to enhance PROTAC permeability and degradation performance. Such approach could prove equally beneficial to other classes of beyond Ro5 molecules. Introduction:Targeted protein degraders, also known as Proteolysis Targeting Chimeras (PROTACs), are becoming a widespread source of chemical probes and lead compounds that degrade rather than inhibit target proteins, providing a different drug modality with potential to expand the "druggable" proteome. [1][2][3][4][5][6][7] These chimeric molecules typically contain a protein-of-interest (POI)targeting ligand (or warhead) and a ligand which binds to an E3 ligase, connected by a linker. 8-10 PROTAC-induced ternary complexes between the POI and E3 ligase are required for polyubiquitination and targeted degradation of the POI. 11 PROTACs do not require full target occupancy, because a single PROTAC molecule can induce degradation of more than one target protein molecule over time, thereby acting catalytically at sub-stoichiometric target occupancy. These distinct features of PROTACs mode of action have been shown to result in increased target selectivity, higher potencies, and fewer off-target effects compared to small molecule inhibitors. 10, 12-14 Furthermore, unlike small molecule inhibitors, PROTACs can bind the target at any position, including non-functional binding sites. 10,15 Notably, PROTACs have shown to be developable for use in humans, with several compounds reaching the clinic, including ARV-110 and ARV-471 that have recently progressed into Phase II clinical trials for prostate and breast cancer, respectively, demonstrating both safety and efficacy in patients. [16][17][18] While PROTACs harbor several advantages as a new modality within drug discovery, their bifunctional nature and chemical composition mean th...

Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

“…Based on the presented DMS data, for instance exemplified by VHL P71I and VHL H110L , it is conceivable that ARV-771 induces a ternary complex of a different geometry than the ones previously resolved for MZ1 10 or macroPROTAC-1 37 , or the one we here resolve for AT2. In support of these predictions, are the observations that (i) ARV-771-induced ternary complex assemblies that involve VCB and BET bromodomains have thus far proven to be unsuccessful to crystallization efforts; (ii) ARV-771 and MZ1 displayed distinct intra-BET bromodomain cooperativity profiles in FP ternary complex assays 51 . Hence, this and related observations emerged from this study together underscore that nuanced, differentiated mutational profiles and sensitivities can arise even with degraders which share otherwise highly similar chemical structures, mechanisms, and cellular activities.…”

Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

“…It is interesting to note that all esters ( 22–25 ) are more potent than their respective amide counterparts ( 18–21 ) by between 2- and 126-fold in DC 50 (Table ). Recently, we have shown that the amide-to-ester substitution can provide a simple strategy to increase the PROTAC degradation activity due to increased lipophilicity and cellular permeability while maintaining remarkable intracellular stability . This trend is well reflected in this compound set, as all esters ( 22–25 ) were cell-active and showed more potent DC 50 and a prominent hook effect at the higher concentrations compared to their amide counterparts.…”

Bioisostere Effects on the EPSA of Common Permeability-Limiting Groups

“…29 In accordance with this observation, the lesser dipole embedded within 1,2,4-oxadiazoles led to reductions in EPSA on the order of Δavg = -12±9 when compared to matched amide counterparts. We found that N-methylation (Δavg = -12±5) 30 , 31 and ester substitution (Δavg = -15±9) 32,33 were among the simplest and most effective tactics to reduce the EPSA of amide-containing compounds, a finding presaged by the abundance of these groups in naturally occurring, permeable macrocyclic peptides. 34 Thus, among common amide bioisosteres that preserve the parent group's HBA capacity while removing its donor function, only 1,2,3-triazoles failed to reliably reduce EPSA.…”