CBP30, a Selective CBP/P300 Bromodomain Inhibitor, Suppresses Human Th17 Responses
Total Page：16
File Type：pdf, Size：1020Kb
CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses Ariane Hammitzscha, Cynthia Tallantb,c, Oleg Fedorovb,c, Alison O’Mahonyd, Paul E. Brennanb,c, Duncan A. Hayb,1, Fernando O. Martineza, M. Hussein Al-Mossawia, Jelle de Wita, Matteo Vecellioa, Christopher Wellsb,2, Paul Wordswortha, Susanne Müllerb,c, Stefan Knappb,c,e,3, and Paul Bownessa,3 aBotnar Research Institute, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, United Kingdom; bStructural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom; cTarget Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom; dBioSeek Division of DiscoveRx Corporation, South San Francisco, CA 94080; and eInstitute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, 60438 Frankfurt am Main, Germany Edited by Saadi Khochbin, Université Joseph Fourier, INSERM U823, Grenoble, France, and accepted by the Editorial Board July 13, 2015 (received for review January 29, 2015) Th17 responses are critical to a variety of human autoimmune BET bromodomain inhibitors exhibit antiinflammatory activity by diseases, and therapeutic targeting with monoclonal antibodies inhibiting expression of inflammatory genes (11). The pan-BET in- against IL-17 and IL-23 has shown considerable promise. Here, we hibitor JQ1 ameliorated collagen-induced arthritis and experimental report data to support selective bromodomain blockade of the autoimmune encephalomyelitis (17), both mouse models of hu- transcriptional coactivators CBP (CREB binding protein) and p300 as man inflammatory disease with major Th17 components. Th17 an alternative approach to inhibit human Th17 responses. We show cells, a subset of T helper cells producing IL-17A, IL-17F, IL-21, that CBP30 has marked molecular specificity for the bromodomains IL-22, and GM-CSF, have been identified as central effectors of of CBP and p300, compared with 43 other bromodomains. In un- several autoimmune diseases, including ankylosing spondylitis biased cellular testing on a diverse panel of cultured primary human (AS), psoriasis and psoriatic arthritis (PSA), rheumatoid arthritis, cells, CBP30 reduced immune cell production of IL-17A and other Crohn’s disease, and multiple sclerosis (18–20). AS is a common proinflammatory cytokines. CBP30 also inhibited IL-17A secretion ∼ – chronic inflammatory disease affecting 0.2 0.5% of adults in the INFLAMMATION by Th17 cells from healthy donors and patients with ankylosing United States. AS is characterized by inflammation of the sacro- IMMUNOLOGY AND spondylitis and psoriatic arthritis. Transcriptional profiling of human iliac and spinal joints leading to bony fusion (ankylosis), pain, and T cells after CBP30 treatment showed a much more restricted effect disability. Treatment options have until recently been limited on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by Significance CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials. Epigenetic inhibitors have shown considerable promise for the treatment of malignant and inflammatory diseases. We CBP/p300 | bromodomain | epigenetic inhibitors | Th17 | present here the detailed characterization of a potent and ankylosing spondylitis highly selective inhibitor of the bromodomains of CBP (CREB binding protein)/p300. Functional preclinical data studying cells derived from patients with ankylosing spondylitis and romodomain recognition of acetylated lysines in histones psoriatic arthritis (two human Th17-driven diseases) show plays a key role in the epigenetic control of gene expression B that selective inhibition of the CBP/p300 bromodomain with (1). Therapeutic targeting of bromodomains has recently been CBP30 strongly reduces secretion of IL-17A, without having recognized as an important potential therapeutic modality in the broader and potentially deleterious effects on cytokine human malignant and inflammatory diseases (2–4). production and gene transcription of the pan-BET (bromo and The transcriptional coactivators CBP [also known as cAMP extraterminal domain protein family) inhibitor JQ1. CBP/p300 responsive element binding protein (CREB) binding protein] play a significant role in IL-17A production, and CBP/p300 in- and the closely related p300 possess such a bromodomain (5). hibition is a promising therapeutic strategy in human type-17– Strategies targeting catalytic histone acetyl transferase (HAT) mediated diseases such as ankylosing spondylitis and psori- activity (6), the N-terminal domain (7), or the KIX (kinase- atic arthritis. inducible domain interacting) domain (8) have had variable efficacy. By contrast, most bromodomain acetyl-lysine binding Author contributions: A.H., F.O.M., P.W., S.K., and P.B. designed research; A.H., C.T., O.F., sites have good predicted druggability (9), confirmed experi- A.O’M., P.E.B., D.A.H., M.H.A.-M., J.d.W., M.V., C.W., and S.M. performed research; A.H., F.O.M., mentally by the recent development of potent and selective S.K., and P.B. analyzed data; and A.H., S.K., and P.B. wrote the paper. inhibitors targeting the bromo and extraterminal domain (BET) Conflict of interest statement: P.B. declares research support from Merck Research family (10–13). The promising effects of BET inhibitors ob- Laboratories. served in cancer cell lines have stimulated the rapid opti- This article is a PNAS Direct Submission. S.K. is a guest editor invited by the Editorial mization of these tool molecules to yield clinical candidate Board. Data deposition: The microarray data reported in this paper have been deposited in the drugs (2). A number of weak inhibitors, such as ischemin (KD = 25 μM), have been reported to target the CBP/p300 bromo- Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE71231). The models and structure factors have been deposited in the Protein Data domain (14). The synthesis and binding modes of more potent Bank, www.pdb.org {PDB ID codes 5BT3 [EP300], 5BT4 [BRD4(1)], and 5BT5 [BRD2(2)]}. CBP and BET/CBP inhibitors have been reported in the last 1Present address: Evotec (UK) Ltd., Milton Park, Abingdon OX14 4RZ, United Kingdom. year (15, 16). The most potent of these inhibitors, CBP30, has 2Present address: Nanotether Discovery Sciences Ltd., Cardiff University, Cardiff CF10 3AX, recently been shown to bind with low nanomolar affinity to the United Kingdom. bromodomains of CBP/p300 (15). However, this initial study 3To whom correspondence may be addressed. Email: [email protected] or did not examine the broader specificity of CBP30 for other [email protected]. bromodomain targets or look at the ability of CBP30 to inhibit This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. physiological cellular processes. 1073/pnas.1501956112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501956112 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 bound with highest affinity (KD of 885 nM), thus with 34-fold reduced selectivity compared with CBP (Table S2). Molecular Details of the Interaction of CBP30 with p300, BRD4, and BRD2. To understand the structural mechanisms for the selectivity of CBP30 for specific BET family members, we determined the cocrystal structure of CBP30 with the p300, BRD4(1), and BRD2(2) bromodomains. Crystals of the CBP30/p300 complex diffracted to 1.05 Å resolution (Table S3). As observed in the CBP bromodomain cocrystal structure (PDB ID code 4nr7), the CBP isoxazole oxygen acted as an acetyl-lysine mimetic moiety forming hydrogen bonds to the conserved asparagine (N1132) and a water-mediated interaction with the hydroxyl group of Y1089ofp300(Fig.2A). However, and in contrast to the CBP complex, we did not observe an induced fit binding mode of R1137, which has been reported to flip into the acetyl-lysine binding side forming a cation-π interaction. The reason for this difference in ligand coordination is due to the presence of a neighboring tyrosine Fig. 1. CBP30 is a selective and potent inhibitor of CBP/p300. (A)Chemical residue (Y1141) that engages into a hydrogen bond interaction structure of CBP30. (B) Temperature shift data covering 45 human bromodo- with R1137. In the CBP bromodomain, this position is occupied by mains. Screened targets are highlighted in black and targets that have not been a phenylalanine, allowing the arginine to stack with the aromatic screened in gray. Temperature shifts are represented by spheres. The gene name decoration of the inhibitor. This induced fit has also been observed CREBBP corresponds to CBP protein, and EP300 to the protein p300. (C)Iso- for other CBP bromodomain inhibitors (16). In the p300/CBP30 thermal titration calorimetry data measuring the binding of CBP30 to BRD4 complex, the benzimidazole ring system was shifted, resulting in ro- (blue), CBP (red), and p300 (black). (Upper) Raw injection heats for BRD4(1) and tation of the morpholine moiety and the 3-chloro-4-methoxyphenol p300. (Lower) Normalized binding heats [BRD4(1), CBP, p300]. Nonlinear least ring system (Fig. 2B). Additional comparison of CBP30 binding to squares fits to a single binding site model are shown as solid lines. p300 with BRD4(1) and BRD2(2)