Fig. 4. Optimization of the structure of EPI-001.
a, Experimental set-up for the measurement of the partition coefficient of EPI-001 in condensed AR AD. LLPS, liquid–liquid phase separation. b,c, Chemical structures of EPI-002 and compounds, with a modified linker between the two aromatic rings of EPI-002. b, Schematic of the structures and the corresponding IC50 measured in androgen-induced PSA-luciferase assay. Purple and brown circles correspond to chemical groups depicted in c, in which hydrogens are at the R1 and R2 positions. d, Changes in 15N chemical shift (δN) in the NMR spectra of Tau-5* (60 μM) as a function of amino acid positions, caused by addition of 1 molar equivalent of EPI-001 (blue) or 1aa (red). Orange circles indicate aromatic amino acids positions in the sequence of Tau-5*. R1-3 (ref. 23) and polyP regions are highlighted in light and dark gray, respectively. Samples contained 200 mM NaCl and 2% DMSO-d6. e, Illustrated molecular dynamics (MD) snapshot of the AR AD interacting with 1aa. Helices are shown in dark and light blue; the loop between them is gray. 1aa is shown in green, and chlorine in purple. f, Per-residue contact probabilities observed in REST2 MD simulations between Tau-5 residues 391–446 and the compounds EPI-002 (blue) or 1aa (red). Contacts are defined as occurring in frames in which any non-hydrogen ligand atom is within 6.0 Å of a non-hydrogen protein atom. Orange circles represent the positions of aromatic residues. Values are presented as mean ± statistical errors from block averaging. g, Compounds developed from 1aa, and their corresponding potency in the androgen-induced PSA-luciferase assay. h, Correlation between the activity of the compounds in the PSA-luciferase assay and their hydrophobicity in terms of LogD determined by chromatography (ChromLogD). i, Dose-dependent inhibition of AR-V7 transcriptional activity by 1ae. j,k, Effect of 1 molar equivalent EPI-001 and 1ae on the Tc of AR AD (average ± s.d., n = 3 independent samples) (j) and on the distribution of droplet sizes (n > 4,000 droplets for DMSO, n > 2,500 for EPI-001 and n > 2,000 for 1ae), where boxes show the mean and the quartiles of all droplets (k).