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. 2024 Jan 26;15(3):355–361. doi: 10.1021/acsmedchemlett.3c00490

Discovery and Characterization of Active CBP/EP300 Degraders Targeting the HAT Domain

Iván Cheng-Sánchez ∥, Katherine A Gosselé ∥,‡, Leonardo Palaferri ∥, Mariia S Kirillova ∥, Cristina Nevado ∥,*
PMCID: PMC10945562  PMID: 38505842

Abstract

graphic file with name ml3c00490_0005.jpg

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that simultaneously bind an E3 ligase and a protein of interest, inducing degradation of the latter via the ubiquitin-proteasome system. Here we present the development of degraders targeting CREB-binding protein (CBP) and E1A-associated protein (EP300)—two homologous multidomain enzymes crucial for enhancer-mediated transcription. Our PROTAC campaign focused on CPI-1612, a reported inhibitor of the histone acetyltransferase (HAT) domain of these two proteins. A novel asymmetric synthesis of this ligand was devised, while PROTAC-SAR was explored by measuring degradation, target engagement, and ternary complex formation in cellulo. Our study demonstrates that engagement of Cereblon (CRBN) and a sufficiently long linker between the E3 and CBP/EP300 binders (≥21 atoms) are required for PROTAC-mediated degradation using CPI-1612 resulting in a new active PROTAC dCE-1. Lessons learned from this campaign, particularly the importance of cell-based assays to understand the reasons underlying PROTAC performance, are likely applicable to other targets to assist the development of degraders.

Keywords: PROTAC, CBP, EP300, HAT, Degrader

Proteolysis Targeting Chimeras (PROTACs) are a novel class of small molecule drugs that trigger protein degradation by hijacking the cellular proteolysis machinery. These bifunctional probes typically consist of a binder for the protein of interest (POI) connected to an E3 ligase ligand via a suitable linker. Simultaneous binding of the POI and E3 ligase by a PROTAC, forming a so-called ternary complex, brings the two proteins into close proximity resulting in the polyubiquitination and subsequent proteasomal degradation of the POI.1−4 Compared to classical small molecule inhibitors, PROTACs offer several advantages: they can simultaneously abolish the activity of all domains for proteins with multiple functions; and they can enhance specificity for close homologues through additional protein–protein interactions (PPIs) between the POI and the E3 ligase.5−7

CREB-binding protein (CBP) and E1A-associated protein (EP300) are examples of such homologous multidomain proteins that work as important regulators of enhancer-mediated transcription8−10 through numerous PPIs11 and by acetylating histone and non-histone proteins.12,13 Given their central role and near ubiquitous expression, it is not surprising that CBP and EP300 have been implicated in a wide variety of human diseases such as cancer, inflammation, and developmental disorders.14−18 CBP and EP300 have been chemically targeted primarily through their bromo and histone acetyl transferase (HAT) domains. Although multiple nanomolar binders have been reported, selectivity between these two homologous proteins is yet to be attained due to the similarity of their binding pockets.19−26 Interestingly, the effects of either bromodomain or HAT inhibition were enhanced by co-inhibition of the other, demonstrating that PROTACs may bring additional efficacy compared to single domain binders.27 Three CBP/EP300 PROTACs, namely, dCBP-1,28 JQAD1,29 and JET-209,30 have been reported recently. dCBP-1 and JET-209 are based on the bromodomain inhibitors GNE-78119 and GNE-207,31 respectively. JQAD1 is the only PROTAC based on a HAT inhibitor, namely A-485.24 A recent work using close analogues of JQAD1 showed a different impact of linker length on degradation, but the basis for this improvement was not further investigated.32

Herein, we present the development of novel CBP/EP300 degraders derived from the HAT inhibitor CPI-1612, including a de novo asymmetric route to improve the chemical accessibility of this ligand. As part of this campaign, in cellulo ternary complex formation has been determined for the first time for CBP which sheds light on the reasons for PROTAC performance and aids in the design of PROTAC libraries. We show that PROTAC activity is highly dependent upon the E3 ligase and linker length, with only Cereblon (CRBN) binding compounds possessing a sufficiently long linker (≥21 atoms) acting as degraders. This work demonstrates the importance of cell-based assays beyond degradation to successfully inform compound design and thus improve the efficiency of PROTAC development campaigns. Further, our results highlight how degraders targeting different domains of a given protein or even the same domain but based on different parent inhibitors can display distinct phenotypical profiles.

To design CBP/EP300 targeting PROTACs, a recently described cell active HAT inhibitor, CPI-1612 (Figure 1A), was chosen as the starting point for our campaign.23 Analysis of its X-ray structure in complex with EP300 showed that the methyl group on the pyrazole moiety points outside the pocket, thus revealing an optimal vector for linker attachment (Figure 1B).33 We set out to replace the methyl group with an aliphatic chain bearing a terminal carboxylic acid to enable later coupling with an amine derivative of the corresponding E3 ligand (Figure 1C). The distance between the pyrazole and the edge of the binding pocket corresponds to ca. 3–5 carbon bonds (Figure S1 in the Supporting Information). As the resulting amide bond may significantly affect binding, we synthesized and determined the CBP and EP300 HAT inhibitory activities of three CPI-1612 derivatives (compounds 1, 2, and 3) featuring the amide—connected to a short PEG chain—at different positions with respect to the pyrazole (Figure 1D). In contrast to CPI-1612, these compounds were synthesized as mixtures of four stereoisomers. Hence, a 4-fold decrease in potency was expected due to the presence of three potentially nonbinding isomers. Since their affinities were not markedly lower than anticipated (ca. 4- to 10-fold), we assumed that both the exit vector and the chosen amide-linkage were well suited for subsequent derivatization. At this stage, we selected compound 2—in which the carbonyl group is four atoms away from the alkylated nitrogen of the pyrazole—as the target warhead for our PROTAC design campaign. In doing so, we aimed to keep the amide as close as possible to the pocket without compromising binding, to minimize interference with ternary complex formation, in terms of both rotational freedom and hydrogen bond formation.

Figure 1.

Figure 1

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(A) Molecular structure and cellular potency of CPI-1612. (B) CPI-1612 X-ray structure in complex with the HAT domain of EP300 (PDB: 6V8N). (C) PROTAC assembly strategy. (D) Screening of the amide position and its effect on HAT activity measured using a radiometric assay. CPI-1612 gave IC50 values of 116 and 11 nM for CBP and EP300, respectively, when measured in the same assay.

The reported synthesis of CPI-161223 requires purification by chiral HPLC in several steps, which significantly limits the overall synthetic accessibility of the compound (10 steps, <1.5% overall yield). Hence, a de novo convergent, asymmetric route was designed to access CPI-1612 and its corresponding PROTACs as single isomers (Figure 2A). The synthesis commenced with alcohol 4, which was prepared in enantiomerically pure form via Evan’s enolate according to a previously reported procedure.34 A sequence of straightforward functional group manipulations delivered methyl ester 5 in five steps (see Supporting Information for reaction details). After hydrolysis under basic conditions to the corresponding carboxylic acid, coupling with aniline derivative 6 using COMU furnished amide 7 in 67% yield as a 1:1 mixture of diastereomers. Separation of the two isomers by column chromatography was possible after the acid-mediated removal of the N-Boc protecting group to deliver derivative 8 in 46% yield over 2 steps. Notably, the undesired diastereomer (S,S)-8 could be quantitatively epimerized using 1,8-diazabicyclo(4.4.0)undec-7-ene (DBU) to regenerate and thus provide additional stock of (R,S)-8 (see Supporting Information for experimental details). Finally, basic hydrolysis of the methyl ester in (R,S)-8 delivered the carboxylic acid warhead 9, the key intermediate for subsequent PROTAC assembly.

Figure 2.

Figure 2

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(A) Synthesis of HAT warhead 9 and CBP/EP300 PROTACs. (B) PROTAC initial screen: structures and biological data. CBP (%) and EP300 (%) are the percentage of each protein remaining after 16 h treatment of LP1 cells with 5 μM compound relative to DMSO treated cells as determined by Western blotting (images in Figure S2). GI50 values were calculated as the concentration required to inhibit LP1 cell viability to 50% relative to DMSO treated cells after 3 days of treatment. All biological data shown are averages from 3 independent experiments. aFor experimental details, see the Supporting Information. bCompound 17 was conjugated to the C5 position of thalidomide.

Initially, PROTACs were prepared by combining 9 with various E3 ligands and linker types. The CRBN, Von Hippel Lindau (VHL), and Inhibitor of Apoptosis Proteins (IAP) E3 ligases were targeted using well established ligands, namely pomalidomide, VH032, and a recently reported IAP ligand, respectively (Figure 2B).35−38 Aliphatic and PEG linkers of various lengths were coupled to these E3 ligands and subsequently engaged in amide coupling reactions with 9 to deliver each of the PROTACs included in this initial screen (Figure 2A, bottom; for reaction details see Supporting Information). Dual probes 10–19 were evaluated by Western blotting to determine their ability to degrade CBP/EP300; however, no significant depletion of either protein was detected (Figure 2B and also Figure S2 in the Supporting Information).

At this point, we were concerned with the potential lack of cellular permeability of our compounds. Thus, considering that the multiple myeloma cell line LP1 is CBP/EP300 dependent and highly sensitive to CPI-1612 (GI50 = 4.8 nM, Figure 2B and also Figure S3 in the Supporting Information), we set out to determine the GI50 values of our compounds in this cell line. Compounds targeting CRBN were more active than those targeting VHL or IAP. Considering that pomalidomide itself has no significant effect on proliferation (GI50 > 10 μM)28 and that 18 and its negative analogue, which is unable to bind CRBN, have similar GI50 values (Figure S3 in the Supporting Information), the stronger antiproliferative effects of the CRBN-binding PROTACs are likely related to engagement of the CBP/EP300 HAT domains, indicating relatively higher cellular permeability. We thus chose to focus our further development campaign on CRBN-targeting PROTACs.

To assess CRBN cellular target engagement by compounds 16–19, a competition assay with another CRBN-targeting PROTAC, ARV-825,39 was carried out. ARV-825 degrades the bromodomain and extraterminal (BET) family member BRD4, which was quantified using a luminescence-based readout in HEK293T cells with an N-terminal HiBiT tag on endogenous BRD4.40 As expected, ARV-825-mediated BRD4 degradation could be blocked by cotreatment with dCBP-1 or pomalidomide, due to competition for CRBN binding, but not by CPI-1612 or the negative control PROTAC dCBP-1-bump, which bears an extra methyl group on the thalidomide ligand to block its binding to CRBN (Figure 3A). Compounds 16–19 were able to rescue BRD4 degradation to an extent similar to that of dCBP-1, indicating sufficient cellular engagement of CRBN and that the permeability of our PROTACs was not the limiting factor impeding degradation.

Figure 3.

Figure 3

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(A) Rescue of BRD4 degradation by 20 nM ARV-825 by competition with 10 μM ligand or PROTAC in HEK293T-BRD4-HiBiT cells. (B) Illustration of FluoPPI assay mechanism and fluorescence microscopy image of dCBP-1 treated cells. (C) Integrated density of fluorescent foci following a 6 h treatment with 2 μM compound, as a percentage of the response to dCBP-1.

We next assessed if our compounds were able to form a ternary complex between CRBN and the CBP catalytic core using fluorescent-based technology detecting protein–protein interactions (FluoPPI).41 Briefly, a tetrameric fluorescent protein (hAG) and an oligomeric assembly helper tag (Ash) are fused to CRBN and the catalytic core of CBP, respectively. Initially these are distributed evenly across the cell, but upon PROTAC induced ternary complex formation they associate with each other to form bright and condensed foci, an effect amplified through phase separation (Figure 3B). To the best of our knowledge, this is the first time ternary complex formation in cells has been explored for CBP/EP300 PROTACs. As expected, treatment with dCBP-1 induced foci (Figures 3A and C) while none were observed with the corresponding negative controls: dCBP-1-bump, pomalidomide, or CPI-1612. Unfortunately, treatment with 16–19 did not lead to foci formation, indicating that these compounds are unable to mediate ternary complex formation, which thus explains their lack of degradation activity.

We next questioned whether an insufficient linker length could be responsible for the results obtained for compounds 16–19, which were able to bind CBP/EP300 and CRBN individually but not form a ternary complex.42 Hence, a new series of CRBN PROTACs with increased linker lengths (4–7 PEG units ≡ z = 18–27 atoms, Figure 4A) was prepared. To our delight, PROTACs with linkers containing 5 to 7 PEG units (z = 21–27 atoms) were able to form foci in the FluoPPI assay (Figure 4B). Western blotting in LP1 cells demonstrated that these compounds are indeed able to degrade CBP/EP300, suggesting that the lack of ternary complex formation was the key issue underlying the inactivity of PROTACs with shorter linkers (Figures 4C and D). Interestingly, a bias toward CBP over EP300 degradation could be clearly observed despite a slightly better affinity of CPI-1612 for EP300 (IC50_EP300 = 11 vs IC50_CBP = 116 nM). Furthermore, compounds with longer linkers are required for efficient EP300 degradation compared to CBP.

Figure 4.

Figure 4

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(A) Molecular structure of compounds 20–23. (B) FluoPPI assay integrated density of fluorescent foci following 6 h treatment with 2 μM compound, as a percentage of the response to dCBP-1. (C) Western blotting in LP1 cells following 16 h treatment with 5 μM CRBN-PROTACs; quantification in D. Images used for quantification in Figure S4.

PROTAC 22 (dCE-1), featuring a 6 PEG unit linker (z = 24 atoms, Figure 5A), was selected for further characterization.43 To confirm that 22 acts through the expected mechanism, we synthesized a negative control, 22-bump, which is unable to bind CRBN. As expected, 22-bump did not show either ternary complex formation in the FluoPPI assay (Figure 5B) or degradation of CBP (Figure 5C). Furthermore, degradation by 22 (dCE-1) could be blocked by cotreatment with CPI-1612 or pomalidomide and by the proteasome inhibitor MG132, showing that degradation requires binding to both the HAT domain and CRBN and is dependent upon the proteasome system (Figure 5C). Compound 22 (dCE-1) induces foci formation in the FluoPPI assay with an EC50 of 1.2 μM (Figure 5B), which is in line with its DC50 value of 1.3 μM in LP1 cells (Figure S6 in the Supporting Information). Finally, 22 (dCE-1) successfully induces rapid degradation of CBP, with a maximal effect achieved within 8 h and maintained for at least 48 h (Figure 5D) and is also active in the multiple myeloma 1S (MM1S) cell line (Figure 5E). Interestingly, JQAD1,29 the only reported PROTAC binding the HAT domain of CBP/EP300, was unable to significantly degrade either protein in LP1 or MM1S cells, thus highlighting the cell-line-specific nature of degraders targeting the same domain of a given protein but stemming from different parent binders (Figures S9 and S10 in the Supporting Information).

Figure 5.

Figure 5

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(A) Structure of 22 (dCE-1) and 22-bump. (B) FluoPPI assay integrated density of fluorescent foci following 6 h compound treatment, as a percentage of the response to 2 μM dCBP-1; 22-bump tested at 2 μM. (C) Western blotting in LP1 cells. Left side 1 h pretreatment with indicated ligands (pom = 50 μM pomalidomide, CPI = 10 μM CPI-1612) followed by 16 h treatment with 5 μM 22 or 22-bump. Right side 30 min pretreatment with 10 μM MG132 followed by 6 h with 5 μM 22. (D) Time course CBP Western blot measurements following treatment with 5 μM 22 in LP1 cells; images used for quantification in Figure S7 in the Supporting Information. (E) CBP/EP300 degradation in MMS1 cells following 16 h treatment with 5 μM 22; images used for quantification in Figure S8 in the Supporting Information. (F) Percentile growth inhibition (relative to control) of various cancer cell lines treated with 22 in the NCI-60 screen.

As PROTAC 22 (dCE-1) has antiproliferative effects in the LP1 and MM1S cell lines (Figure S11 in the Supporting Information), we screened its effect on a wider panel of cell lines through the NCI-60 screen.44 Notably, this compound is able to inhibit growth of the vast majority of screened cell lines, with the strongest effect on leukemia cell lines (Figure 5F and Table S1 in the Supporting Information).

In conclusion, this work outlines the systematic and effective development of a new CBP/EP300 degrader, 22 (dCE-1), consisting of the HAT inhibitor CPI-1612 conjugated via a 24-atom linker to pomalidomide. First, we identified a viable vector for linker conjugation to CPI-1612 that retains affinity for the HAT domain. Next, we secured the accessibility of CPI-1612 and its derivatives as single isomers through an asymmetric, high yielding, and convergent route devoid of chiral HPLC. This, combined with the use of a late-stage amide coupling, provided a straightforward assembly strategy that is applicable not only to our PROTACs but also to other bifunctional probes based on this ligand. An early-stage screening of PROTACs based on ligands for CRBN, VHL, and IAP ligases did not lead to any active compounds, clearly demonstrating that blindly conjugating a potent cell permeable inhibitor to an E3 ligand is insufficient to develop active degraders. Additional cellular assays were performed to unravel the reasons underlying the inactivity of these compounds. These assays involved the first exploration of ternary complex formation in cells for CBP/EP300 PROTACs, as well as the use of FluoPPI technology in early PROTAC development. The results of these investigations guided us in the direction of CRBN-recruiting compounds and then toward increased linker length, which directly led to active CBP/EP300 degraders. Finally, our degraders can be used as tools to understand the biological differences of degrading CBP and EP300 through their catalytic or reader domains, also highlighting the importance of expanding the toolbox of degraders for underexplored, biologically relevant protein targets.

Acknowledgments

This work was funded by SNF-Sinergia (CRSII5_180345/1) and Krebsliga (KFS-4585-08-2018) grants. The Forschungskredit from the University of Zurich is acknowledged for financial support to I.C.-S. (FK-21-114). We would like to thank G. Morf for the preparation of some of the building blocks used in the synthesis of compound 9, E. Laul for the preparation of stock solutions for cellular assays, and A. Caflisch for enabling cloning experiments in his laboratory and for insightful discussions. We thank the Developmental Therapeutics Program at the National Cancer Institute (NCI) for performing the NCI-60-cell cytotoxicity assays.

Glossary

Abbreviations

PROTAC

Proteolysis Targeting Chimera

CBP

CREB-binding protein

EP300

E1A-associated protein

HAT

histone acetyltransferase

SAR

structure–activity relationship

FluoPPI

Fluorescent based technology detecting protein–protein interactions

POI

protein of interest

PPIs

protein–protein interactions

CRBN

Cereblon

VHL

Von Hippel Lindau

IAP

Inhibitor of Apoptosis Proteins

BET

bromodomain and extraterminal

COMU

(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino morpholino-carbenium hexafluorophosphate, 1-[(1-(cyan-2-ethoxy-2-oxoethylidenaminooxy)-dimethylamino-morpholino)]-uronium-hexafluorophosphate

DBU

1,8-diazabicyclo(4.4.0)undec-7-ene

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00490.

  • Distance from the pyrazole of CPI-1612 to the end of the binding pocket (PDB: 6V8N), Western blot images used for the quantification of CBP and EP300 degradation in Figures 2B, 4D, and 5E, dose-response antiproliferative effects of CPI-1612 and pomalidomide and of 18 and its negative control 18-bump in LP1 cells following 3 days of treatment, biological characterization of analogues of compound 22, dose-response of compound 22, Western blot images used for the time course of CBP degradation by 22 in Figure 5D, percentage of CBP/EP300 remaining after 16 h treatment of LP1 or MM1S cells with 5 μM 22 or 5 μM JQAD1 relative to DMSO treated cells, dose-response antiproliferative effects of 22 in LP1 and MM1S cells following 3 days of treatment, percent growth of cells treated with 10 μM 22 in the NCI-60 screen, methods, general procedures, experimental procedures, and compound characterization (1H NMR and 13C NMR and LCMS spectra) (PDF)

Author Contributions

† I.C.-S., K.A.G., and L.P. contributed equally. I.C.-S., K.A.G., L.P., and C.N. conceived the work, designed and interpreted the experiments, and wrote the manuscript. I.C.-S. and L.P. synthesized all compounds, and K.A.G. performed the cellular assays. M.S.K. contributed to the design of the asymmetric synthesis of compound 9. C.N. supervised the project.

The authors declare no competing financial interest.

Supplementary Material

ml3c00490_si_001.pdf (17.8MB, pdf)

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