Systematic Investigation of the Permeability of Androgen Receptor PROTACs

Abstract

Bifunctional molecules known as PROTACs simultaneously bind an E3 ligase and a protein of interest to direct ubiquitination and clearance of that protein, and they have emerged in the past decade as an exciting new paradigm in drug discovery. In order to investigate the permeability and properties of these large molecules, we synthesized two panels of PROTAC molecules, constructed from a range of protein-target ligands, linkers, and E3 ligase ligands. The androgen receptor, which is a well-studied protein in the PROTAC field was used as a model system. The physicochemical properties and permeability of PROTACs are discussed.

Figure 1

(A) AR ligand 2 in complex with AR ligand binding domain (pdb id: 3v49). The linker attachment point is circled in red. (B) Structures of compounds 1–5, derived from AR ligands, for incorporation into PROTACs. The linker attachment point is indicated in red. (C) Proteolysis directing groups 6–9; the attachment point when incorporated into PROTACs is indicated in blue. (D) Lenalidomide bound to Cereblon E3 ubiquitin ligase (pdb id: 4ci2). The linker attachment point is circled in blue. (E) General AR PROTAC structure of PROTAC Sets 1 and 2.

Figure 2

(A) Helix 12 (H12, red) of AR protein (pdb id: 2pnu) occludes the entrance to the ligand-binding domain. The docked small molecule 3b (Figure S1) is indicated by green spheres. (B) A homology model of AR with helix-12 in an open conformation (red helix) was generated. The remodeled helix 12 allows access from the ligand-binding domain to the bulk solvent and accommodation of PROTAC linkers.

Figure 3

Structure, biophysical properties and biological activities of PEG-linked PROTAC Set 1. Coloring is added to aid visualization. (A) The general structure of a set of PROTACs (10–19, Set 1) is shown in the top left, comprised of AR ligands 1–4, proteolysis directing groups 6–8, and a PEG-linker. The PAMPA permeability for compounds 1–4 and 6–8 is shown in the outer edge of the table. The main body of the table shows the PROTAC compound number in bold (10–19), composed from AR ligand 1–4 and proteolysis directing group 6–8, followed by PAMPA permeability. The same table format is used in (B–C). BLQ = Below limit of quantification. (B) HSA % free determined by HPLC and as described in methods section. (C) chromLogD_7.4 determined by HPLC as described in the methods section.

Figure 4

(A) AR clearance activity of previously reported PROTAC 20a. (B) AR clearance activity of PROTACs 14 and 15. Plots show normalized AR protein levels as a function of PROTAC concentration. PROTAC 14 is inactive in this cell assay. A minor change of the Cereblon ligand leads to higher activity of PROTAC 15: a DC_MAX of 33% and a DC₅₀ of 10 nM. N = 5 (or more) independent experiments performed in triplicate (individual data points shown). Statistical significance by ANOVA (Dunnett’s) **** = p < 0.0001.