Developing selective histone deacetylases (HDACs) inhibitors through ebselen and analogs

Figures & data

Table 1 Compound structures and IC₅₀ values in the biochemical HDAC assays

Compound ID	Compound structure	HDAC1	HDAC2	HDAC3	HDAC4	HDAC5	HDAC6	HDAC7	HDAC8	HDAC9	HDAC10	HDAC11
		IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM	IC50 µM
Ebselen		>10	>10	>10	>10	3.49	6.99	>10	1.21	3.75	>10	>10
Ebselen oxide		2.26	>10	4.18	4.70	1.49	2.98	1.88	0.20	2.37	>10	>10
Thr101		3.62	>10	4.24	6.07	1.57	4.14	3.43	0.66	3.77	>10	>10
RBC-2004 (Ebsulfur)		1.65	>10	2.72	4.12	1.49	2.89	1.51	0.19	1.70	>10	>10
Benzisothiazol		>10	>10	>10	>10	>10	3.20	>10	>10	>10	>10	>10
RBC-2005		>10	>10	>10	>10	>10	>10	>10	>10	>10	>10	>10
RBC-2006		>10	>10	>10	>10	>10	>10	>10	>10	>10	>10	>10
RBC-2008		0.25	>10	3.70	5.40	0.94	0.003	4.10	0.064	3.80	>10	>10
RBC-2009		>10	>10	>10	>10	>10	>10	>10	>10	>10	>10	>10
RBC-2010		6.58	>10	5.01	>10	4.79	11.32	6.08	0.63	6.19	>10	>10
RBC-2011		4.00	>10	5.30	9.90	3.30	>10	4.10	0.56	4.60	>10	>10
RBC-2012		8.80	>10	8.20	>10	5.50	>10	8.79	1.03	>10	>10	>10
SAHA		0.293	0.683	0.435	27.400	12.200	0.018	>10	1.130	>10	0.604	0.597

Abbreviations: HDAC, histone deacetylase; IC₅₀, half maximal inhibitory concentration.

Figure 1 Dose-dependent inhibition of HDACs by ebselen (A), ebselen oxide (B), Thr101 (C), and RBC-2008 (D) in a biochemical assay. The activity against all the 11 HDACs was assessed by using the acetylated AMC-labeled peptide substrates. HDAC enzyme was incubated with test compound for 10 min, and then, the substrate was added to start the reaction. After the reaction was completed, a developer was added to digest the deacetylated substrate, and the fluorescence generated was detected with excitation (Ex) at 360 nM and emission (Em) at 460 nM by using the EnVision Multilabel Plate Reader (PerkinElmer, Santa Clara, CA, USA). The IC₅₀ curves were plotted, and IC₅₀ values were calculated by using the GraphPad Prism 4 program based on a sigmoidal dose–response equation. Data shown here represent one of three independent experiments.

Abbreviations: AMC, 7-Amino-4-methylcoumarin; HDAC, histone deacetylase; IC₅₀, half maximal inhibitory concentration.

Table 2 Effects of ebselen and ebsulfur analogs on SIRT functional activities

Compound ID	SIRT1	SIRT2	SIRT3	SIRT5
	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)
Ebselen	1.12E–06	4.96E–06	>1.00E–05	6.06E–06
Ebselen oxide	3.78E–07	3.74E–06	>1.00E–05	4.39E–06
Thr101	1.23E–07	2.75E–06	>1.00E–05	4.07E–06
RBC-2004	3.43E–07	2.99E–06	>1.00E–05	4.88E–06
RBC-2008	1.19E–06	7.35E–06	>1.00E–05	7.55E–06
RBC-2010	2.83E–07	3.70E–06	>1.00E–05	3.22E–06

Note: Data shown here represent one of three independent experiments.

Abbreviations: IC₅₀, half maximal inhibitory concentration; SIRT, silent mating-type information regulation 2′.

Figure 2 Mechanisms of HDAC inhibition by RBC-2008.

Notes: (A) Kinetic analysis. 2.6 ng of purified HDAC6 was incubated with a wide range of concentrations of substrate from 0 to 100 µM in the presence of the vehicle DMSO or RBC-2008 at 10 nM and 50 nM for 1 h of reaction. The data were then fitted based on the kinetics model of Michaelis–Menten plot. The Km of HDAC6 was determined to be 29 µM with DMSO, 65 µM with 10 nM RBC-2008, and 266 µM in the presence of 50 nM of the compound. The V_max was not dramatically changed by the RBC-2008. The V_max was ~12 (nM/min/mg) with DMSO, in comparison with 6.8 and 5.9 nM/min/mg in the presence of 10 nM or 50 nM of compound, respectively. (B) Effect of dilution on the inhibition of HDAC6 by RBC-2008. 2.6 ng of HDAC6 was incubated with DMSO vehicle or 300 nM RBC-2008 for 1 h. Then, a portion of the mixtures was diluted 100-fold, and a portion of the mixtures was not diluted to serve as a positive inhibition control for RBC-2008. Then, 50 µM substrate was added to the mixtures to start the reaction. The reactions were stopped at indicated time points up to 3 h by adding 10 µM trichostatin A, and a developer was added to digest the deacetylated substrate, and the fluorescence generated was detected with excitation (Ex) at 360 nM and emission (Em) at 460 nM by using the EnVision Multilabel Plate Reader (PerkinElmer). Data shown here represent one of two independent experiments.
Abbreviations: DMSO, dimethyl sulfoxide; HDAC, histone deacetylase.

Figure 3 Modulation of α-tubulin acetylation by RBC-2008 in PC-3 cells.

Notes: PC-3 cells were treated with the indicated concentrations of RBC-2008, HDAC inhibitor tubastatin A, or ACY-1215 for 18 h. The whole cell lysates were subject to Western blot analyses with anti-acetylated-tubulin antibody and anti-α-tubulin antibody. Data shown here represent one of three independent experiments.
Abbreviations: DMSO, dimethyl sulfoxide; HDAC, histone deacetylase.

Table 3 Activities of compounds on human tumor cell viability

Compound ID	A549	HTC116	MiaPaCa-2	SKOV-3	SW480	PC-3	SK-BR-3	K562	MDA-MB-231	A498	HT-29	Jurkat
	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)	IC50 (M)
Ebselen	>1.00E–04	2.59E–05	4.03E–05	7.81E–05	9.24E–05	>1.00E–04	9.27E–05	1.30E–05	>1.00E–04	>1.00E–04	6.35E–05	1.38E–05
Ebselen oxide	>1.00E–04	>1.00E–04	3.41E–05	>1.00E–04	1.10E–04	>1.00E–04	>1.00E–04	1.51E–05	8.36E–05	4.71E–05	9.35E–05	7.95E–05
Thr101	1.82E–05	9.03E–06	1.07E–05	1.98E–05	6.29E–06	2.52E–05	1.58E–05	4.50E–06	2.17E–05	3.00E–05	2.06E–05	1.24E–05
RBC-2004	3.89E–05	9.64E–06	3.00E–05	2.44E–05	7.53E–06	>1.00E–04	3.37E–05	5.94E–06	2.67E–05	4.01E–05	2.61E–05	1.36E–05
RBC-2008	>1.00E–04	2.96E–05	1.81E–05	>1.00E–04	>1.00E–04	>1.00E–04	2.81E–05	1.71E–05	3.16E–05	>1.00E–04	7.93E–05	6.40E–06
RBC-2010	>1.00E–04	2.04E–05	1.82E–05	2.20E–05	8.35E–06	2.08E–05	4.10E–05	4.90E–06	1.86E–05	4.51E–05	3.12E–05	7.57E–06
ACY-1215	>1.00E–04	6.56E–06	7.38E–06	1.42E–05	9.55E–06	>1.00E–04	1.09E–05	1.12E–05	1.58E–05	6.21E–05	1.86E–05	4.40E–06
Tubastatin A	>1.00E–04	2.31E–05	1.96E–05	3.86E–05	3.31E–04	>1.00E–04	6.28E–06	3.42E–05	1.68E–05	>1.00E–04	>1.00E–04	.1.00E–04
SAHA	2.00E–05	6.55E–07	3.23E–06	1.28E–05	3.54E–06	2.15E–05	1.23E–06	2.36E–06	3.85E–06	1.38E–05	3.74E–06	7.92E–07

Abbreviations: IC₅₀, half maximal inhibitory concentration; SAHA, suberoylanilide hydroxamic acid.

Figure 4 Effects of RBC-2008 on the human tumor cell viability in CellTiter-Glo^® assay. Approximately 1,000 of U266 cells, REH cells, K562 cells, KMS-11 cells, RPMI-8226 cells, and MM.1S cells were incubated with the indicated concentrations of RBC-2008 in 384-well assay plates at 37°C, 5% CO₂ for 72 h. Then, 25 µL of CellTiter-Glo^® reagent was added to the wells of the assay plates and incubated at a room temperature for 10 min with gentle shaking. The luminescent signal within plates were measured by using EnVision Multilabel Reader. The IC₅₀ curves were plotted, and the IC₅₀ values were calculated by using the GraphPad Prism 4 program based on sigmoidal dose–response equation. The IC₅₀ values of RBC-2008 were calculated to be 14.5 µM, 1.8 µM, 13.1 µM, 2.5 µM, 3.8 µM, and 2.1 µM on U266 cells, REH cells, K562 cells, KMS-11 cells, RPMI-8226 cells, and MM.1S cells, respectively. Data shown here represent one of three independent experiments.

Abbreviation: IC₅₀, half maximal inhibitory concentration.

Figure S1 ¹H NMR spectrum for RBC-2008.

Abbreviation: NMR, nuclear magnetic resonance.

Figure S2 ¹³C PENDANT NMR spectrum for RBC-2008.

Abbreviation: NMR, nuclear magnetic resonance.

Figure S3 Matrix-assisted ionization-high-resolution mass spectrum for RBC-2008.

Note: Predicted spectrum for C₁₅H₁₂N₂O₃S+H is shown at the bottom.