Development of p300-targeting degraders with enhanced selectivity and onset of degradation

Abstract

p300 and CBP are paralogous epigenetic regulators that are considered promising therapeutic targets for cancer treatment. Small molecule p300/CBP inhibitors have so far been unable to differentiate between these closely related proteins, yet selectivity is desirable in order to probe their distinct cellular functions. Additionally, in multiple cancers, loss-of-function CREBBP mutations set up a paralog dependent synthetic lethality with p300, that could be exploited with a selective therapeutic agent. To address this, we developed p300-targeting heterobifunctional degraders that recruit p300 through its HAT domain using the potent spiro-hydantoin-based inhibitor, iP300w. Lead degrader, BT-O2C, demonstrates improved selectivity and a faster onset of action compared to a recently disclosed A 485-based degrader in HAP1 cells and is cytotoxic in CIC::DUX4 sarcoma (CDS) cell lines (IC₅₀ = 152-221 nM), significantly reducing expression of CDS target genes (ETV1, ETV4, ETV5). Taken together, our results demonstrate that BT-O2C represents a useful tool degrader for further exploration of p300 degradation as a therapeutic strategy.

Fig. 1. (A) p300 and CBP domain schematic (upper) with KAT domain sequence alignment for p300 and CBP (lower). Conserved regions are highlighted black and key residues mediating iP300w binding through H-bonds and Van der Waals interactions are underlined orange. Sequence alignment generated with using EMBL-EBI EMBOSS Needle pairwise sequence alignment tool (UniProt and NCBI accession numbers: Q09472 and NP_004371). (B) Chemical structures of iP300w and A 485, with the solvent facing exit vector region highlighted blue. (C) Co-crystal structure of p300 in complex with iP300w (PDBID 7LJE) with the acetamide exit vector indicated (black dashed circle).

Scheme 1. Synthesis of iP300w-based degraders. Reagents & conditions: (a) KCN, (NH₄)₂CO₃, EtOH, H₂O, 70 °C; (b) sodium 2-iodobenzenesulfonic acid, Bu₄N(HSO₄), Oxone, MeCN, 65 °C; (c) bromoacetyl bromide, DCM, RT; (d) K₂CO₃, DMF, 5 °C, followed by preparative chromatography; (e) tert-butyl 2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazol-1-yl]acetate Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, 1,4-dioxane, H₂O, 95 °C; (f) NaBH₄, THF, MeOH, 0 °C; (g) DAST, DCM, −70 °C; (h) HCl (4 M in 1,4-dioxane), RT; (i) [amino-linker-E3 ligand], HATU, DIPEA, DMF, RT.

Fig. 2. Summary of p300 and CBP degradation data using a HiBiT assay. Normalized protein abundance is represented as the area under the curve (AUC) over a 48 hour time course HiBiT degradation assay for CBP (left) and p300 (right). AUCs are normalized on a 0 to 1 scale, where 1 represents no degradation over the time course.

Fig. 3. Degradation profiles of p300 and CBP following compound treatment using a HiBiT assay in a HAP1 cell line. Degraders were dosed from 100 nM to 10 μM. Readout is normalized protein abundance (y axis) versus hours after degrader treatment (x axis). Briefly, p300 or CBP were endogenously N-terminal tagged with a HiBiT tag using CRISPR/Cas9 genome editing in a HAP1 cell line stably expressing LgBiT. A luminescence read-out was used to assess protein levels over a time period of 48 hours following treatment.

Fig. 4. Assessment of p300 and CBP degradation following compound treatment. Capillary electrophoresis (Wes™ platform, powered by Simple Western technology) measurements of p300 and CBP levels were made following treatment with (A) DMSO, dCPB-1, iP300w, JQAD1 or BT-O2C and (B) DMSO, BT-O2F, BT-O2H or BT-O2K. HAP1 cells were incubated with the compounds (or DMSO-only vehicle control) indicated at 3 concentrations (100 nM, 1 μM and 10 μM) for 24 hours prior to lysis and analysis. Vinculin is shown as the loading control for each.

Fig. 5. Evaluating mechanism of action for BT-O2C in HAP1 cells. Capillary electrophoresis (Wes platform, powered by Simple Western technology) measurements of p300 and CBP levels were made following treatment with vehicle (lane 1) or compound BT-O2C (100 nM) for 24 hours (lane 2). In lanes 3 and 4, MLN 4924 or carfilzomib were added after 22 hours incubation with BT-O2C (100 nM) to block neddylation or the 26S proteasome, respectively. Samples were lysed and analyzed after 24 hours. Vinculin is shown as the loading control for each.

Fig. 6. Effect of p300-targeting degraders in CIC-DUX4 sarcoma (CDS) cell lines. RT-qPCR for CIC::DUX4 target genes ETV1, ETV4, and ETV5, cell cycle regulation genes P21 and CCNE1, and c-MYC in NCC-CDS-X1 cells following 6 h treatment with compounds. Control is DMSO-only. Compound concentrations are: iP300w and A 485, 250 nM; JQAD1, BT-O2C and BT-O2C-N, 4 μM. Gene expression levels normalized to the expression of actin (ACTB). Data is presented as mean ± SEM; *p < 0.05 compared to the control, by one-way ANOVA (n = 4).