methods (supplement) molecular...

Methods (Supplement)

Molecular Modeling

HDAC6 structure prediction: As the crystal structure is not available for HDAC6, we

constructed a homology model using the program Model (1). The input alignment for the

Modeller was obtained with ClustalW (2) based on the sequence of the human catalytic

domain of human histone deacetylase HDAC7 (PDB: 2NVR). The model was refined

further by energy minimization for 10,000 cycles with a consistent valence force field

(cff91) using the DISCOVER module of INSIGHT II (Accelrys Inc., San Diego, CA, USA).

The quality of the refined model was checked with PROCHECK (3). We again compared

the model with the HDAC7 structure to ensure that catalytic binding site residue side

chains are in the proper orientations after remodeling. This final predicted model was

used for docking simulations.

Docking simulations were carried out with the SurFlexDock and AutoDock 4.0.

However manual docking were done in the case HDAC6 model as the predicted

structure is not precise enough to initiate automatic docking simulations. Molecular

models were then compared with the SAHA·HDAC8 (PDB: 1T69) and SAHA·HDAC7

(PDB: 3C0Z) structure to ensure that the compounds as well as catalytic binding site

residue side chains are in the proper orientations after remodeling. Docking simulations

generated the conformations slightly different conformation. After optimally oriented

conformations of the compounds through manual intervention, minimization was carried

for 10,000 cycles.

These minimized models were subjected to molecular dynamic (MD) simulations to

relax the structure as described below. The zinc atom within the catalytic binding site

was included in the simulations. Minimization was carried out using DISCOVER, Insight

II (Accelrys, Inc. San Diego) with a cff91 force field, cutoff for non-bonded interaction

energies set to ∞ and dielectric constant set at 4. All atoms within 7.0 Å of the inhibitor

were allowed to relax during the minimization. Brief MD simulations were also performed

in the NVE ensemble consisted of an initial equilibration of 25 ps and followed by a

production run of 100 ps dynamics at 300 K. The final complex structure at the end of

the MD simulations was subjected to minimization. A distance-dependent dielectric

constant and non-bonded distance cutoff of 12 Å were used. MD simulations were

performed using the AMBER8.0 package (4) with the General Amber Force Field (GAFF)

and RESP charge models.

As shown in Figure 1 (B-D), compound 2 forms similar interaction patterns with

HDAC6, 7 and 8. However, the low conformational flexibility and entropic penalty of

compound 2 against HDAC6 confers high potency. This is due to the arrangement of

residues lining the zinc catalytic site, and channel shape of HDAC6 is slightly narrowed

but sufficient enough to fit the ligand tightly and confer less flexibility. In the case of

HDAC6, the amide group of the sulphonamide forms H-bonds with water on one side,

and SO2 forms water mediated contact with D626 on the other side. In the case of

HDAC8, the sulphonamide group interacts differently than do the HDAC6 and 7 isomers.

The reason may be due to the broader binding pocket of HDAC8, implying that

additional flexibility arising for compound 2 allow it to rotate freely. In this case, SO2

makes contact with water, and the amide group contacts with D101 through H-bonds. In

the case of HDAC6, compound 2 has more beneficial contacts to the cap region. The

additional hydrophobic contact of the aromatic ring of the dansyl group contained in

compound 2 and HDAC7 did not provide greater potency as compared to a lack of this

interaction in HDAC6. Moreover, marked differences in the H-bond capability of the

sulphonamide moiety of compound 2 and a glysine residue may provide an explanation

of the decreased HDAC8 potency. Additionally, the enhanced potencies of HDAC6 and

7 are explained by the dansyl planar rings of compound 2 preferentially occupying the

relatively planar and hydrophobic portions of the cap region of the enzyme as shown in

Figure 1B-D.

Chemical Synthesis and Analysis

NMR spectra were recorded using a Varian-400 spectrometer for 1H (400 MHz) and

13C (100 MHz). Chemical shifts (δ) are given in ppm downfield from tetramethylsilane,

as internal standard, and coupling constants (J-values) are in hertz (Hz). Purifications by

flash chromatography were performed. Analytical high pressure liquid chromatography

(HPLC) and liquid chromatography / mass spectrometry (LC/MS) analyses were

conducted using Shimadzu LC-20AD pumps and a SPD-20A UV-vis detector. MS

detection was performed with a Micromass platform LC spectrometer. Reverse phase

HPLC was performed on Water µBondapak C18 (300 × 3.9 µm) using two Shimazu LC-

20AD pumps and a SPD-20A-vis detector set at 330 nm: Method A, 50% acetonitrile

plus 0.1% acetic acid in H2O(v/v), with a flow rate at 1 mL/min over 10 mins; method B,

50% 2-propanol + 0.1% acetic acid in H2O (v/v), with a flow rate at 1 mL/min over 10

mins. High-resolution mass spectra (HMRS) were recorded on a QSTAR Elite mass

spectrometer.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-pentanoic acid 5: To a stirred

solution of 6-Aminopentanoic acid (1.0 g, 8.54 mmol) in 1 M NaHCO3 (45 mL) was

added dansyl chloride (1.84 g, 6.83 mmol) in acetone (10 mL) and triethylamine (2 mL).

The solution was stirred for 1 h, acidified to pH 3 with 2 N HCl, extracted with ethyl

acetate (3 × 15 mL). The organic layer was washed with water (15 mL) and brine (15

mL), dried over Na2SO4. The solvent was evaporated and purified by flash

chromatography using CH2Cl2-MeOH to afford 5 as green sticky oil (2.08 g, 87%). 1H

NMR (CDCl3, 400 MHz) δ 9.60 (br, 1H), 8.48 (d, 1H, J = 8.8 Hz), 8.30 (d, 1H, J = 8.8

Hz), 8.19 (dd, 1H, J = 7.2, 1.2 Hz), 7.47 (m, 2H), 7.12 (d, 1H, J = 7.6 Hz), 5.60 (t, 1H, J =

6.0 Hz), 2.86 (m, 2H), 2.82 (s, 6H), 2.13 (t, 2H, J = 7.2 Hz), 1.43 (m, 4H); 13C NMR (100

MHz) δ 178.46, 151.74, 134.91, 130.31, 129.80, 129.62, 129.40, 128.36, 123.24,

118.99, 115.28, 45.39, 42.73, 33.17, 28.85, 21.47.

The same procedure was used as above for the synthesis of compounds 6, 7 and 8.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-hexanoic acid 6: green sticky oil

(2.06 g, 98%). 1H NMR (CDCl3, 400 MHz) δ 10.66 (br, 1H), 8.46 (d, 1H, J = 8.4 Hz),

8.33 (d, 1H, J = 8.4 Hz), 8.19 (d, 1H, J = 7.2 Hz), 7.44 (m, 2H), 7.09 (d, 1H, J = 7.6 Hz),

5.66 (br, 1H), 2.85 (m, 2H), 2.79 (s, 6H), 2.08 (t, 2H, J = 8.2 Hz), 1.32 (m, 4H), 1.12 (m,

2H); 13C NMR (100 MHz) δ 179.29, 151.67, 135.03, 130.18, 129.72, 129.58, 129.25,

128.26, 123.16, 119.01, 115.20, 45.30, 42.88, 33.98, 29.07, 25.74, 24.01.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-heptanoic acid 7: green sticky oil

(0.76 g, 92%). 1H NMR (CDCl3, 400 MHz) δ 8.52 (d, 1H, J = 8.4 Hz), 8.31 (d, 1H, J = 8.4

Hz), 8.23 (dd, 1H, J = 7.6, 1.4Hz), 7.52 (m, 2H), 7.17 (d, 1H, J = 7.6 Hz), 5.66 (t, 1H, J =

6.2 Hz), 2.88 (m, 8H), 2.20 (t, 2H, J = 7.4 Hz), 1.38 (m, 4H), 1.12 (m, 4H); 13C NMR (100

MHz) δ 179.46, 152.02, 134.95, 130.49, 129.97, 129.77, 129.68, 128.48, 123.34,

118.98, 115.35, 45.54, 43.22, 33.90, 29.37, 28.44, 26.09, 24.47.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-heptanoic acid 8: green sticky oil

(1.76 g, 89%). 1H NMR (CDCl3, 400 MHz) δ 8.52 (d, 1H, J = 8.4 Hz), 8.31 (d, 1H, J = 8.8

Hz), 8.23 (dd, 1H, J = 7.4, 1.4Hz), 7.52 (m, 2H), 7.17 (d, 1H, J = 7.6 Hz), 5.10 (t, 1H, J =

6.0 Hz), 2.87 (m, 8H), 2.25 (t, 2H, J = 7.6 Hz), 1.48 (m, 2H), 1.35 (m, 2H), 1.11 (m, 6H);

13C NMR (100 MHz) δ 179.71, 152.07, 135.06, 130.53, 130.04, 129.85, 129.75, 128.53,

123.40, 119.06, 115.39, 45.61, 43.36, 34.12, 29.56, 28.89, 28.70, 26.32, 24.65.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-pentanoic acid benzyloxy amide 9:

To an ice bath cooled solution of 5 (1.9 g, 5.02 mmol) in THF (50 mL) was added 1-

hydroxybenzotriazole (HOBt, 0.81 g, 6.02 mmol), 4-dimethylamino pyridine (DMAP, 0.74

g, 6.02 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,

1.21 g, 6.31 mmol), followed by O-Benzylhydroxylamine (0.72 g, 5.82 mmol). The

mixture was stirred at room temperature for 48 h. Water (15 mL) was added to the

mixture and stirred at room temperature for 10 minutes. The mixture was extracted with

ethyl acetate (3 ×15 mL). The organic layer was washed with sat. NaHCO3 (15 mL),

water (15 mL) and brine (15 mL), dried over Na2SO4. The solvent was evaporated and

the residue was purified by flash chromatography using CH2Cl2-MeOH to afford 9 as

pale yellow sticky oil (1.5 g, 68%). 1H NMR (CDCl3, 400 MHz) δ 8.53 (d, 1H, J = 8.4 Hz),

8.22 (d, 1H, J = 8.8, Hz), 8.25 (d, 1H, J = 7.2 Hz), 7.52 (m, 2H), 7.36 (m, 5H), 7.17 (d,

1H, J = 7.6 Hz), 5.01 (t, 1H, J = 6.2 Hz), 4.85 (s 2H), 2.88 (s, 6H), 2.84 ((t, 2H, J = 6.4

Hz), 1.92 (m, 2H), 1.54 (m, 2H), 1.40 (m, 2H).

Same procedure was used as above for the synthesis of compounds 10, 11 and 12.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-hexanoic acid benzyloxy amide

10: green sticky oil (0.64 g, 94%). 1H NMR (CDCl3, 400 MHz) δ 9.04 (br, 1H), 8.47 (d,

1H, J = 8.4 Hz), 8.29 (d, 1H, J = 8.4 Hz), 8.17 (dd, 1H, J = 7.4, 1.0 Hz), 7.47 (m, 2H),

7.27(m, 5H), 7.12 (d, 1H, J = 7.6 Hz), 5.40 (br, 1H), 4.81 (s 2H), 2.82 (s, 6H), 2.79 (m,

2H), 1.83 (m, 2H), 1.33 (m, 4H), 1.09 (m, 2H); 13C NMR (100 MHz) δ 170.90, 157.35,

151.96, 135.02, 130.39, 129.93, 129.72, 129.50, 129.22, 128.64, 128.56, 128.45,

123.31, 119.00, 115.31, 78.13, 45.49, 43.02, 33.92, 29.13, 25.69, 25.04.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-heptanoic acid benzyloxy amide

11: orange sticky oil (1.53 g, 90%). 1H NMR (CDCl3, 400 MHz) δ 9.31 (br, 1H), 8.46 (d,

1H, J = 8.4 Hz), 8.31 (d, 1H, J = 8.4 Hz), 8.16 (d, 1H, J = 6.8 Hz), 7.44 (m, 2H), 7.26 (m,

5H), 7.10 (d, 1H, J = 7.6 Hz), 5.57 (br, 1H), 4.81 (s 2H), 2.78 (m, 8H), 1.85 (m, 2H), 1.30

(m, 4H), 1.01 (m, 4H); 13C NMR (100 MHz) δ 171.02, 151.81, 135.46, 135.00, 130.23,

129.77, 129.60, 129.27, 129.07, 128.47, 128.40, 128.31, 123.18, 118.91, 115.18, 77.96,

53.56, 45.36, 43.07, 33.67, 29.22, 28.18, 25.76, 24.97.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-octanoic acid benzyloxy amide 12:

pale yellow oil (2.11 g, 95%). 1H NMR (CDCl3, 400 MHz) δ 9.47 (br, 1H), 8.44 (d, 1H, J =

8.0 Hz), 8.32 (d, 1H, J = 8.4 Hz), 8.16 (d, 1H, J = 6.8 Hz), 7.44 (m, 2H), 7.25 (m, 5H),

7.08 (d, 1H, J = 7.2 Hz), 5.62 (br, 1H), 4.81 (s 2H), 2.78 (m, 8H), 1.93 (m, 2H), 1.30 (m,

4H), 1.01 (m, 6H); 13C NMR (100 MHz) δ 171.16, 151.99, 134.95, 130.43, 129.93,

129.76, 129.65, 129.33, 128.75, 128.67, 128.49, 123.35, 118.97, 115.30, 78.20, 45.55,

43.18, 33.13, 29.83, 29.36, 28.76, 28.35, 26.07, 25.17.

6-(5-Dimethylaminonaphthalene-1-sulfonylamino)-hexanoic acid hydroxyamide 1:

To a solution of 9 (1.17 g, 2.57 mmol) in methanol (40 mL) was added 10% palladium

on carbon (0.27 g, 0.257 mmol), the apparatus was degassed and then filled with

hydrogen under atmosphere pressure. The reaction was allowed to stir at room

temperature until starting material disappeared completely by TLC (CH2Cl2 / MeOH:

10:1, Rf = 0.5). The suspension was then filtered through a pad of celite and

concentrated under reduced pressure, the residue was purified by flash chromatography

using CH2Cl2-MeOH to afford 1 as yellow soft solid (0.91 g, 97%). 1H NMR (CDCl3, 400

MHz) δ 9.84 (br, 1H), 8.46 (d, 1H, J = 8.4 Hz), 8.23 (m, 2H), 7.43 (m, 2H), 7.08(m, 1H, J

= 6.8 Hz), 6.13 (br, 1H), 2.83 (m, 8H), 2.11 (m, 2H), 1.52 (m, 4H); 13C NMR (100 MHz) δ

172.08, 151.67, 135.01, 130.16, 129.77, 129.62, 129.17, 128.41, 123.32, 119.18,

115.31, 45.45, 42.78, 32.24, 28.91, 22.47; HPLC: Method A, retention time = 3.31 min;

Method B, retention time = 4.80 min; LC-MS: m/z 366 (MH+) ; HRMS (MH+) calculated:

366.1488; Found, 366.1471.

Same procedure was used as above for synthesis of compounds 2, 3 and 4.


yellow soft solid (0.38 g, 94%). 1H NMR (CDCl3, 400 MHz) δ 9.65 (br, 1H), 8.47 (d, 1H, J

= 8.4 Hz), 8.30 (d, 1H, J = 4.4 Hz), 8.17 (d, 1H, J = 6.0 Hz), 7.44 (m, 2H), 7.10(d, 1H, J

= 6.8 Hz), 5.85 (br, 1H), 2.83 (m, 8H), 2.04 (m, 2H), 1.38 (m, 4H), 1.18 (m, 2H); 13C NMR

(100 MHz) δ 172.05, 151.92, 135.14, 130.39, 129.97, 129.79, 129.44, 128.54, 123.40,

119.22, 115. 42, 45.57, 43.04, 32.60, 29.10, 25.70, 24.76; HPLC: Method A, retention

time = 3.32 min; Method B, retention time = 4.50 min; LC-MS: m/z 380 (MH+) ; HRMS

(MH+) calculated: 380.1644; Found, 380.1648.


yellow soft solid (1.53 g, 90%). 1H NMR (CDCl3, 400 MHz) δ 9.72 (br, 1H), 8.47 (d, 1H, J

= 7.2 Hz), 8.25 (m, 2H), 7.45 (m, 2H), 7.11(m, 1H), 5.78 (br, 1H), 2.82 (m, 8H), 2.02 (m,

2H), 1.27 (m, 8H); 13C NMR (100 MHz) δ 171.94, 151.68, 135.06, 130.15, 129.71,

129.58, 129.17, 128.31, 123.23, 119.05, 115. 20, 45.37, 43.06, 32.52, 29.19, 28.13,

25.73, 25.08; HPLC: Method A, retention time = 3.08 min; Method B, retention time =

4.69 min; LC-MS: m/z 394 (MH+) ; HRMS (MH+) calculated: 394.1801; Found, 394.1746

(MH+).


yellow soft solid (1.24 g, 96%). 1H NMR (CDCl3, 400 MHz) δ 8.47 (d, 1H, J = 7.2 Hz),

8.25 (m, 2H), 7.46 (m, 2H), 7.11(m, 1H), 5.76 (br, 1H), 2.82 (m, 8H), 2.10 (m, 2H), 1.34

(m, 4H), 1.04 (m, 6H); 13C NMR (100 MHz) δ 171.16, 151.92, 135.17, 130.42, 129.95,

129.81, 129.50, 128.57, 123.45, 119.23, 115. 42, 45.63, 43.33, 32.97, 29.50, 28.71,

28.50, 26.15, 25.36; HPLC: Method A, retention time = 3.26 min; Method B, retention

time = 4.53 min; LC-MS: m/z 408 (MH+) ; HRMS (MH+) calculated: 408.1957; Found,

408.1954 (MH+).

Immunohistochemistry

Rabbit Antibody (ab19845-100, abcam) was applied to detect HDAC 1 in PC3 cells at

1:1000 dilution, and visualized with secondary antibody against rabbit, conjugated with

Alex Fluor (R) 488 ( 4412S Cell signaling), after treating the cell for 24 hours at 1μM.

Western Blot

Cells were treated with a dose range of 100 nM to 50 μM of compound 2 for 24 hours

and lysed with Ripa buffer. After resolution on Page Gel, different HDAC members were

detect by appropriate antibodies (9928S, Cell Signaling), at a dilution of 1:2000.

Supplement References

1. Fiser A, Do RK, Sali A. Modeling of loops in protein structures. Protein Sci 2000;

9: 1753-73.

2. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of

progressive multiple sequence alignment through sequence weighting, position-

specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:

4673-80.

3. Laskowski RA, Macarthur MW, Moss DS, Thornton JM. Procheck - a Program to

Check the Stereochemical Quality of Protein Structures. J Appl Cryst 1993; 26:

283-91.

4. Case DA, Darden TA, Cheatham III TE, et al. AMBER 8. San Fransisco:

University of California; 2004.

Supplemental Figure Legends

Supplemental Figure 1. Synthesis of fluorescent HDAC inhibitors.

Supplemental Figure 2. Fluorescent HDAC inhibitor 2 in PC-3 and DU-145 cells.

Images were taken at 695 nm with a multiphoton laser. Cells treated with 20 μM of

compound 2 for 60 minutes, A-C (in PC-3). A. Compound 2 fluoresces in the cytoplasm.

B. Differential interference contrast (DIC) image. C. Merged A and B. D-F (in Du145) D.

Compound 2 fluoresces in cytoplasm. E. DIC image. F. Merged D and E.

Supplemental Figure 3. Effects on HDAC isomers 1, 2, 3, 4 and 5 over dose

ranging concentrations of compound 2 for 24 hrs. Antibodies purchased from Cell

Signaling (9928S). Primary antibody concentration (1:2000).

Supplemental Figure 4. A. Detection of HDAC1 in PC3 cells without compound 2

treatment. B. Nucleus stain with DAPI in PC3 cells without compound 2 treatment. C.

Detection of HDAC1 in PC3 cells with compound 2 treatment. D. Nucleus stain with

DAPI in PC3 cells with compound 2 treatment. Rabbit Antibody (ab19845-100, abcam)

was applied to detect HDAC 1 in PC3 cells at 1:1000 dilution, and visualized with

secondary antibody against rabbit, conjugated with Alex Fluor (R) 488 ( 4412S Cell

signaling), after treating the cell for 24 hours at 1μM.

Supplemental Figure 1.

H2NCOOH( )n

N

SO

OCl

N

SO

ONH

COOH

( ) n

N

SO

ONH

( ) n

ONH

O

N

SO

ONH

( ) n

ONH

OH

+a

b c

Condition: a) 1M NaHCO3, acetone/Et3N. 5 (n = 1), 87%; 6 (n = 2), 98%; 7 (n = 3), 92%; 8 (n = 4), 89%. b) benzyloxy amide,HOBT/EDCl, D MAP/TH F. 9 (n = 1), 68%; 10 (n = 2), 94%; 11 (n = 3), 90%; 12 (n = 4), 95%. c) H2, 10%P d-C, MeOH. 1 (n = 1),97%; 2 (n = 2), 94%; 3 (n = 3), 90%; 4 (n = 4), 96%

n = 1-4 5-8 (n = 1-4)

9-12 (n = 1-4) 1-4 (n = 1-4)


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