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Volumn 17, Issue 19, 2007, Pages 5300-5309

The discovery of 6-amino nicotinamides as potent and selective histone deacetylase inhibitors

Author keywords

Benzamides; Epigenetics; Histone acetylation; Histone deacetylases; Nicotinamides

Indexed keywords

4 N ACETYLDINALINE; 6 AMINONICOTINAMIDE; BENZAMIDE; HISTONE DEACETYLASE 1; HISTONE DEACETYLASE 8; HISTONE DEACETYLASE INHIBITOR; HYDROXAMIC ACID; MGCD 0103; N (2 AMINOPHENYL) 4 (3 PYRIDINYLMETHOXYCARBONYLAMINOMETHYL)BENZAMIDE; N (2 AMINOPHENYL)NICOTINAMIDE; NICOTINAMIDE; UNCLASSIFIED DRUG; VORINOSTAT;

EID: 34548563726     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2007.08.023     Document Type: Article
Times cited : (49)

References (50)
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    • note
    • 2, and 0.1 mg/mL BSA. HDAC enzymatic activities were determined by the following procedure: 3× serial dilutions of a 10 mM solution of inhibitor were performed in DMSO followed by a 20× dilution into Assay buffer. 20 μl HDAC was preincubated with 5 μL diluted compound at RT for 10 min. The reaction was initiated by the addition of 25 μL of the appropriate substrate (HDACs 1, 2, 3, and 6: fluor-de-Lys substrate KI-104; HDAC8: fluor-de-Lys HDAC8 substrate KI-178), incubated 15 (HDAC8) or 60 (HDACs 1, 2, 3, and 6) minutes at 37 °C, before adding 50 μL of the appropriate development solution. The development solution for HDACs 1, 2, 3, and 6 was 167×-diluted 20× Developer Concentrate (BIOMOL: KI-105) plus 10 μM SAHA. For HDAC8, the development solution was 100×-diluted 5× Developer Concentrate (BIOMOL: KI-176) plus 10 μM SAHA. The assay was read in a VictorV plate reader (Perkin-Elmer, Wellesley, MA) at Ex 360 nm/Em 460 nm. The final substrate concentration was 30 μM and final HDAC concentrations in the reaction were 1 nM for HDACs 1, 3, and 6), 2 nM for HDAC2, and 15 nM for HDAC8. Carboxy-terminal FLAG-tagged human HDACs 1, 2, 3 (co-expressed with the domain of SMRT), and 6 were overexpressed in mammalian cells and affinity purified using an anti-Flag antibody matrix, eluted from the matrix with 100 μg/mL of a competing FLAG peptide in 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10% glycerol, and protease inhibitor cocktail (Roche cat. # 1836153).
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    • note
    • 2, viable cells were quantitated using Vialight Plus (Cambrex) according to manufacturer's instructions.
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    • note
    • + 446.2, exp. 446.2.
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    • Comparative protein structure modeling
    • An alignment of the two crystal structures and the amino acid sequence for hHDAC1 (retrieved from SwissProt, accession ID: Q13547) was accomplished using the sequence/structure alignment tools in MOE (Chemical Computing Group, Montreal, Canada). This alignment was then used in Quanta/Modeler5 (Accelrys, San Diego, CA, USA) to generate a set of homology models. That with the least number of violations was further energy minimized using CHARMm 200 steps steepest descents without the catalytic Zn or any waters present:. Webster D.M. (Ed), Humana Press, New Jersey
    • An alignment of the two crystal structures and the amino acid sequence for hHDAC1 (retrieved from SwissProt, accession ID: Q13547) was accomplished using the sequence/structure alignment tools in MOE (Chemical Computing Group, Montreal, Canada). This alignment was then used in Quanta/Modeler5 (Accelrys, San Diego, CA, USA) to generate a set of homology models. That with the least number of violations was further energy minimized using CHARMm 200 steps steepest descents without the catalytic Zn or any waters present:. Sánchez R., and Sali A. Comparative protein structure modeling. In: Webster D.M. (Ed). Protein Structure Prediction: Methods and Protocols Vol. 143 (2000), Humana Press, New Jersey 97-129
    • (2000) Protein Structure Prediction: Methods and Protocols , vol.143 , pp. 97-129
    • Sánchez, R.1    Sali, A.2
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    • Distance Geometry and Molecular Conformation
    • The 150 conformers were generated and energy minimized using MMFFs with a distance-dependent dielectric of 2r:. Bawden D. (Ed), Research Studies Press, Wiley, New York
    • The 150 conformers were generated and energy minimized using MMFFs with a distance-dependent dielectric of 2r:. Crippen C.M., and Havel T.F. Distance Geometry and Molecular Conformation. In: Bawden D. (Ed). Chemometrics Research Studies Series (1988), Research Studies Press, Wiley, New York
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    • note
    • During the course of energy minimization within the context of the enzyme model, only slight differences in the presentation of the co-minimizing residues (those within 5 Å of the inhibitor) were noted between the starting and final enzyme structure. The catalytic Zn and chelating residues were virtually unchanged during the minimization process.
  • 47
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    • note
    • 2 for 24 hours with either 0.2% DMSO or increasing concentrations of 7i dissolved in DMSO (final DMSO concentration = 0.2%). Whole cell lysates were made by disrupting cell membranes with 2% deoxycholate followed by sonication. Using these HCT116 lysates as antigen, H2BK5 acetylation was quantified using an indirect ELISA and normalized to total H2B levels obtained using a second indirect ELISA. Primary antibodies used were rabbit anti-acetylated H2BK5 (Cell Signaling) and sheep anti-histone H2B (Abcam). Goat anti-rabbit IgG HRP (Bio-Rad) and rabbit anti-sheep-HRP (Jackson ImmunoResearch) were used as secondary antibodies, respectively. TMB substrate (Pierce) was used for detection with absorbance read at 450 nm.


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