Article pubs.acs.org/jmc
A Novel Cell-Permeable, Selective, and Noncompetitive Inhibitor of KAT3 Histone Acetyltransferases from a Combined Molecular Pruning/Classical Isosterism Approach Ciro Milite,† Alessandra Feoli,† Kazuki Sasaki,‡,§ Valeria La Pietra,∥ Amodio Luca Balzano,† Luciana Marinelli,∥ Antonello Mai,⊥ Ettore Novellino,∥ Sabrina Castellano,*,†,# Alessandra Tosco,*,† and Gianluca Sbardella*,† †
Epigenetic Med Chem Lab, Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano, Salerno, Italy ‡ Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan § Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ∥ Dipartimento di Farmacia, Università di Napoli “Federico II” Via D. Montesano 49, I-80131 Naples, Italy ⊥ Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le A. Moro 5, I-00185 Rome, Italy # Dipartimento di Medicina e Chirurgia, Università degli Studi di Salerno, Via Salvador Allende, I-84081 Baronissi, Salerno, Italy S Supporting Information *
ABSTRACT: Selective inhibitors of the two paralogue KAT3 acetyltransferases (CBP and p300) may serve not only as precious chemical tools to investigate the role of these enzymes in physiopathological mechanisms but also as lead structures for the development of further antitumor agents. After the application of a molecular pruning approach to the hardly optimizable and not very cell-permeable garcinol core structure, we prepared many analogues that were screened for their inhibitory effects using biochemical and biophysical (SPR) assays. Further optimization led to the discovery of the benzylidenebarbituric acid derivative 7h (EML425) as a potent and selective reversible inhibitor of CBP/p300, noncompetitive versus both acetyl-CoA and a histone H3 peptide, and endowed with good cell permeability. Furthermore, in human leukemia U937 cells, it induced a marked and time-dependent reduction in the acetylation of lysine H4K5 and H3K9, a marked arrest in the G0/G1 phase and a significant increase in the hypodiploid nuclei percentage.
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INTRODUCTION
predominantly cytoplasmic and acetylate free histones but not those already deposited into chromatin. Even if they are a more diverse family of enzymes than the type-Bs, nuclear (type-A) KATs can be classified into different groups by structural homology and biochemical mechanisms of actions: GCN5 (general control nonderepressible 5)-related N-acetyltransferases (GNATs) (including GCN5, PCAF, ELP3, HAT1, and HPA2), the MYST (MOZ, YBF2/SAS3, SAS2 and TIP60)
1
After 50 years from its discovery, lysine acetylation is far from being thoroughly understood and remains an intriguing topic for study. Among the other histone posttranslational modifications, this chemical modification neutralizes the positive charge of lysine side chains, weakening the interactions between histones and DNA and, consequently, relaxing the chromatin structure and making chromosomal DNA more accessible.2,3 The enzymes responsible for this transformation, lysine acetyltransferases (KATs),4 are generally classified into two major categories, type-A and type-B. The type-B KATs are © 2015 American Chemical Society
Received: December 19, 2014 Published: March 2, 2015 2779
DOI: 10.1021/jm5019687 J. Med. Chem. 2015, 58, 2779−2798
Article
Journal of Medicinal Chemistry
Figure 1. Indirect (top) and direct (bottom) inhibitors of p300. Selective inhibitors are indicated in blue.
distinctive facial features, and broad thumbs and first toes.12 Moreover, CBP and p300 were demonstrated to be involved in hematopoietic homeostasis, such that mutations in the CBP/ p300 interaction domain of different transcription factors were found in hematologic malignancies13,14 and chromosomal translocations involving CBP or p300 genes are associated with leukemia and lymphomas.15−17 CBP and p300 promote prostate cancer progression by activating androgen receptor-regulated transcription18 and colon cancer progression by microsatellite instability19 and are involved in the development of drug resistance.20 Apart from cancer, CBP/p300 interactions with transcription factors are involved in the development of a variety of diseases,21 comprising viral diseases,22,23 cognitive disorders, and Alzheimer’s disease,24−27 diabetes,28−30 and cardiovascular diseases.31−34 For all the above considerations, there is an urgent need for modulators of CBP/p300 activity not only as useful tools to dissect the role of these KATs in physiological and pathological mechanisms but also as potential leads for the development of drug candidates for specific diseases. The inhibitors of CBP/
family, p300 (adenoviral E1A-associated protein of 300 kDa)/ CBP (CREB, cyclic-AMP response element binding protein), general transcription factor KATs, and nuclear hormone receptor-related KATs.5,6 The two paralogues p300 and CBP (KAT3A and KAT3B, respectively; also called CBP/p300) were first described as binding partners of the adenovirus early region 1A (E1A) and the cAMP-regulated enhancer (CRE) binding protein, respectively,7,8 but it was later demonstrated that these two proteins contribute to transcriptional regulation through their histone acetyltransferase activity.9,10 The structure of their HAT domain suggests a “hit-and-run” (Theorell−Chance) catalytic mechanism in which, after binding of acetyl-CoA, the lysyl residue of the substrate peptide snakes through the p300 tunnel and reacts with the acetyl group.11 Interacting with a large number of transcription factors, CBP and p300 are involved in different cellular processes; this implies that the dysregulation of their activity leads to many human diseases, including cancer. Mutations in the CBP (rarely p300) gene causes Rubinstein−Taybi syndrome, characterized by a short stature, moderate to severe intellectual disability, 2780
DOI: 10.1021/jm5019687 J. Med. Chem. 2015, 58, 2779−2798
Article
Journal of Medicinal Chemistry
Figure 2. Flowchart of our molecular pruning approach.
Scheme 1a
Reagents and conditions: (a) malonic acid, Ac2O, MW (60 °C, 10 min); (b) Bz2O, MW (160 °C, 30 min), neat; (c) appropriate benzoic acid, DCC, DMAP, TEA, THF, room temperature, overnight; (d) aqueous 6 N HCl, methanol, 1 h; (e) appropriate benzaldehyde, ethanol, reflux, 1 h; (f) Zn, AcOH, room temp, 1 h. a
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DOI: 10.1021/jm5019687 J. Med. Chem. 2015, 58, 2779−2798
Article
Journal of Medicinal Chemistry Table 1. Effects of Compounds 1−9 on the Activity of p300 and PCAF
% inhibition (at 100 μM, mean ± SD)a,b,c d
compd
R1
R2, R3
p300/KAT3B
1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c 6a 6b 6c 7a 7b 7c 7d 7e 7f 7g 8a 8b 8c 8d 8e 8f 8g 9a 9b 9c 9d 9e 9f 9g curcumin AA C646
Bn Bn Bn i-Bu i-Bu i-Bu allyl allyl allyl Bn Bn Bn i-Bu i-Bu i-Bu allyl allyl allyl Bn Bn Bn Bn Bn Bn Bn i-Bu i-Bu i-Bu i-Bu i-Bu i-Bu i-Bu allyl allyl allyl allyl allyl allyl allyl
H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH H 4-OH 3-OH, 4-OH 3-OH, 4-OMe 3-OMe, 4-OH 3-OMe, 4-OMe 4-OMe H 4-OH 3-OH, 4-OH 3-OH, 4-OMe 3-OMe, 4-OH 3-OMe, 4-OMe 4-OMe H 4-OH 3-OH, 4-OH 3-OH, 4-OMe 3-OMe, 4-OH 3-OMe, 4-OMe 4-OMe
84.8 ± 0.2 54.4 ± 0.2 48.2 ± 2.2 34.1 ± 1.2 39.7 ± 0.9 8.4 ± 2.1 28.1 ± 3.8 19.1 ± 1.8 4.9 ± 1.9 83.9 ± 6.7 60.3 ± 1.1 59.9 ± 0.6 23.1 ± 4.0 64.6 ± 0.5 24.3 ± 2.4 28.9 ± 10.3 23.7 ± 0.6 1.1 ± 2.3 NTg 99.2 ± 0.6 96.15 ± 0.15 97.28 ± 0.22 97.06 ± 0.18 88.25 ± 0.85 93.59 ± 1.39 NTg 94.03 ± 0.17 91.85 ± 0.22 84.68 ± 0.35 96.43 ± 0.67 45.82 ± 2.91 53.78 ± 5.01 NTg 64.71 ± 0.71 61.65 ± 2.46 49.48 ± 2.10 67.33 ± 0.01 31.15 ± 0.48 59.85 ± 1.96 93.45 ± 0.81 86% @10 μMh
PCAF/KAT2B
d
IC50 (μM)e,f p300/KAT3Bd
−14.5 ± 7.8
19.3 ± 2. 4 3.4 ± 8.2 −13.2 ± 11.6 −2.7 ± 0.1 3.6 ± 1.8 5.0 ± 1.7
−10.5 ± 9.6 −7.5 ± 7.4 −10.5 ± 9.6 −0.6 ± 1.6 −2.3 ± 2.8 −162.4 ± 8.2 −55.6 ± 9.1 −25.8 ± 16.4 −8.2 ± 3.7 −22.2 ± 1.8
2.1 1.6 5.9 1.5 11.4 8.0
± ± ± ± ± ±
0.2 0.1 0.3 0.2 0.6 0.4
−34.1 ± 2.4 10.6 ± 2.0 9.3 ± 1.0 9.2 ± 0.4 3.6 ± 3.2 3.1 ± 1.4
8.5 5.4 31.4 4.2
± ± ± ±
0.3 0.2 0.9 0.3
3.3 ± 0.7 3.0 ± 1.2 5.1 ± 1.6 6.6 ± 0.5 11.7 ± 0.2 3.6 ± 3.2
26.4 ± 0.7
6.5 ± 0.2 102.9 ± 2.3 (IC50 33.9 ± 0.7)e,f
