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. 2022 Sep 9;23(18):10446.
doi: 10.3390/ijms231810446.

New Inhibitors of the Human p300/CBP Acetyltransferase Are Selectively Active against the Arabidopsis HAC Proteins

Chiara Longo  1 , Andrea Lepri  1 , Andrea Paciolla  1 , Antonella Messore  2 , Daniela De Vita  3 , Maria Carmela Bonaccorsi di Patti  4 , Matteo Amadei  4 , Valentina Noemi Madia  2 , Davide Ialongo  2 , Roberto Di Santo  2 , Roberta Costi  2 , Paola Vittorioso  1
Affiliations

New Inhibitors of the Human p300/CBP Acetyltransferase Are Selectively Active against the Arabidopsis HAC Proteins

Chiara Longo et al. Int J Mol Sci. .

Abstract

Histone acetyltransferases (HATs) are involved in the epigenetic positive control of gene expression in eukaryotes. CREB-binding proteins (CBP)/p300, a subfamily of highly conserved HATs, have been shown to function as acetylases on both histones and non-histone proteins. In the model plant Arabidopsis thaliana among the five CBP/p300 HATs, HAC1, HAC5 and HAC12 have been shown to be involved in the ethylene signaling pathway. In addition, HAC1 and HAC5 interact and cooperate with the Mediator complex, as in humans. Therefore, it is potentially difficult to discriminate the effect on plant development of the enzymatic activity with respect to their Mediator-related function. Taking advantage of the homology of the human HAC catalytic domain with that of the Arabidopsis, we set-up a phenotypic assay based on the hypocotyl length of Arabidopsis dark-grown seedlings to evaluate the effects of a compound previously described as human p300/CBP inhibitor, and to screen previously described cinnamoyl derivatives as well as newly synthesized analogues. We selected the most effective compounds, and we demonstrated their efficacy at phenotypic and molecular level. The in vitro inhibition of the enzymatic activity proved the specificity of the inhibitor on the catalytic domain of HAC1, thus substantiating this strategy as a useful tool in plant epigenetic studies.

Keywords: Arabidopsis thaliana; HAC proteins; p300/CBP inhibitors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of the cinnamoyl compounds 1a–f and of the saturated derivatives 2a–c.
Figure 2
Figure 2
Synthetic route to 2a–c derivatives. i: NaH 60% dispersion in mineral oil, N,N′-trimethyleneurea, dioxane dry, reflux, 24 h, 50% yield; ii: 1. 1-(1-cyclohexen-1-yl)pyrrolidine, DIPEA, CHCl3, reflux, 12 h, 2. HCl 1 N, reflux, 4 h, 16% yield; iii: BBr3 1 M in CH2Cl2, CH2Cl2 dry, −45 °C to rt, 2 h, 35–50% yield (see Section 4).
Figure 3
Figure 3
Identification of structurally and functionally conserved amino acid positions. Each residue of the catalytic subunit of Arabidopsis HAC1 is identified by a color representing the degree of evolutionary conservation, cyan to purple, corresponding to variable (grade 1) through conserved (grade 9) positions (first line, uppercase). Below each amino acid is reported the position in the 3D structure of the residue (second line, b and e), and the predicted functionality (third line, f and s). e corresponds to an exposed residue according to the neural network algorithm, b to a buried residue according to the neural network algorithm, f to a predicted functional residue (highly conserved and exposed), s to a predicted structural residue (highly conserved and buried) [35].
Figure 4
Figure 4
Selection of active compounds through a phenotypic screening. The hypocotyl length of wild type 6-day-old seedlings was treated with: (A) 1a (60, 120 μM), (B) 1b–f and 2a–c (60, 120 μM), (C) with the selected compounds 1b, 2b, and 2c compared to 1a (120 and 240 μM). All the samples were compared to the DMSO-treated control (MOCK). (D) Six-day-old seedlings treated with DMSO (MOCK) or with compounds 2b and 2c (120 and 240 μM). Scale bar, 1 cm. The values of the hypocotyl length are the mean of two biological replicates (50 seedling each) presented with SD values. Significant differences from the mock were analyzed by t-test (* p ≤ 0.01).
Figure 5
Figure 5
HAC inhibitors result in stronger phenotypes of hac mutants, in the presence of ACC. (A) The hypocotyl length of wild type 6-day-old seedlings untreated, or treated with the selected compounds 1b, 2b, 2c and 1a (120 and 240 μM), in the presence of ACC (10 μM), compared to the DMSO-treated control (MOCK). (B) The hypocotyl length of untreated (no ACC) or treated (+ACC) wild type, hac1 and hac5 single mutants compared to the 2b- and 2c-treated wild type (+ACC). (C) Six-day-old dark-grown untreated (no ACC) or treated (+ ACC) wild type, hac1 and hac5 single mutants compared to the 2b- and 2c-treated wild type (+ ACC). Scale bar, 1 cm. The values of the hypocotyl length are the mean of two biological replicates (50 seedling each) presented with SD values. Significant differences from the mock were analyzed by t-test (* p ≤ 0.01; ** p ≤ 0.005).
Figure 6
Figure 6
Treatment with 2b and 2c affects expression of target genes. Relative expression level of: (A) HLS1, ORG1, LTP5 and LSH6 in 2b- and 2c-treated 6-day-old dark-grown wild type seedlings, (B) ERF4 (left) and ERF6 (right) in 2b- and 2c-treated 6-day-old dark-grown hac1 seedlings. Treated samples were compared to DMSO-treated wild type seedlings (MOCK), which was set to 1. RT-qPCR assays were performed with 1 μL of the diluted cDNA, along with the specific primers, listed in Table S1. Relative expression levels were normalized with the GAPA1 (AT3G26650) and UBQ10 (AT4G05320) reference genes. The values of relative expression level are means of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (* p ≤ 0.01; ** p ≤ 0.005).
Figure 7
Figure 7
HAC1 and HAC5 mutually regulate their expression. Relative expression level of HAC1 (A) and HAC5 (B) in 6-day-old dark-grown wild type (control, ctrl), hac5 and hac1 mutant seedlings, respectively. RT-qPCR assays were performed with 1 μL of the diluted cDNA, along with the specific primers, listed in Table S1. Relative expression levels were normalized with the GAPA1 (AT3G26650) and UBQ10 (AT4G05320) reference genes. The values of relative expression level are means of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (** p ≤ 0.005).
Figure 8
Figure 8
Compound 2c inhibits acetylase activity of HAC1. HAT activity of partially purified HAC1 catalytic site. Purified protein extracts treated with 6 µM compound 2c compared to untreated samples. Similar results were obtained from two independent biological replicates, and one representative experiment is presented. The value of HAT activity is the mean of two technical replicates presented with SD values. Significant differences from the untreated samples were analyzed by t-test (** p ≤ 0.005).
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