Abstract
Criteria for predicting the druglike properties of “beyond Rule of 5” Proteolysis Targeting Chimeras (PROTAC) degraders are underdeveloped. PROTAC components are often combined via amide couplings due to their reliability. Amides, however, can give rise to poor absorption, distribution, metabolism, and excretion (ADME) properties. We hypothesized that a bioisosteric amide-to-ester substitution could lead to improvements in both physicochemical properties and bioactivity. Using model compounds, bearing either amides or esters, we identify parameters for optimal lipophilicity and permeability. We applied these learnings to design a set of novel amide-to-ester-substituted, VHL-based BET degraders with the goal to increase permeability. Our ester PROTACs retained intracellular stability, were overall more potent degraders than their amide counterparts, and showed an earlier onset of the hook effect. These enhancements were driven by greater cell permeability rather than improvements in ternary complex formation. This largely unexplored amide-to-ester substitution provides a simple strategy to enhance PROTAC permeability and bioactivity and may prove beneficial to other beyond Ro5 molecules.
Highlights
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 the potential to expand the “druggable” proteome.1−7 These chimeric molecules typically contain a protein-of-interest (POI)-targeting ligand and a ligand that 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 subsequent targeted degradation of the POI.11
We used a short alkyl linker for compounds 1−7 to eliminate permeabilityaffecting intramolecular hydrogen bonds (IMHBs) that can be formed between poly(ethylene glycol) (PEG)-based linkers and amide −NHs in other parts of the molecule
We have previously shown that the PROTAC MZ1 forms highly cooperative, stable, and long-lived ternary complexes with bromodomain and extra terminal (BET) bromodomains/MZ1 and displays a preference for second bromodomains (BD2s) over first bromodomains (BD1s), for Brd4BD2, and these biophysical characteristics of the ternary complex underpin a high level of target ubiquitination and drive potent and fast degradation activity of MZ1 with Brd4.14,34,69 We wondered to what extent the improvements in cellular activity that we observed with our set of PROTACs might be contributed from the more favorable ternary complex formation
Summary
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 the potential to expand the “druggable” proteome.− These chimeric molecules typically contain a protein-of-interest (POI)-targeting ligand (or warhead) and a ligand that binds to an E3 ligase connected by a linker.− PROTAC-induced ternary complexes between the POI and E3 ligase are required for polyubiquitination and subsequent targeted degradation of the POI. 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 substoichiometric target occupancy. While PROTACs harbor several advantages as a new modality within drug discovery, their bifunctional nature and chemical composition mean that they are inherently larger than the warhead ligands on which they are based. This makes PROTAC compounds go beyond the “Rule of 5” (bRo5) and can impose hurdles to their pharmaceutical development.− efforts have been made recently to better understand the physicochemical properties and structure−property relationships of PROTACs to identify design parameters that may help guide development in this chemical space.− An important pharmacokinetic hurdle for high-molecular-weight compounds tends to be permeability.. This makes PROTAC compounds go beyond the “Rule of 5” (bRo5) and can impose hurdles to their pharmaceutical development.− efforts have been made recently to better understand the physicochemical properties and structure−property relationships of PROTACs to identify design parameters that may help guide development in this chemical space.− An important pharmacokinetic hurdle for high-molecular-weight compounds tends to be permeability. Uptake into cells occurs in competition with efflux, which is commonly a problem for large molecules. recently, we and others have established that PROTACs can show potent cellular activity despite exhibiting very low permeabilities compared to their individual ligand components and to more conventional inhibitors.− There is a great interest to develop strategies for improving cell permeability and other physicochemical properties of PROTACs
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