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Identifying functional mimetics of histone deacetylase using isothermal titration calorimetry (LB242)
Author(s) -
Robinson Sophia,
Jin Lihua,
Grice Kyle,
Karver Caitlin
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb242
Subject(s) - isothermal titration calorimetry , chemistry , titration , methanol , tris , chelation , binding constant , stoichiometry , amine gas treating , zinc , solubility , nuclear chemistry , inorganic chemistry , organic chemistry , binding site , biochemistry
Inhibition of histone deacetylase (HDAC) is known to induce tumor cell death, making it a promising approach for the treatment of cancer. To determine the energetic contribution of the active‐site Zn 2+ to HDAC interaction with inhibitors as superoylanilide hydroxamic acid (SAHA), we have explored Zn 2+ chelators as functional mimetics of HDAC as an alternative to HDAC enzyme. Our goal was to first identify Zn 2+ chelators that are capable of forming tight 1:1 (Zn 2+ :chelator) binary complex and then to determine the energetics of inhibitor binding to the binary complex. One chelator, N ‐(2‐(1‐methylimidazolyl)methyL)iminodiacetic acid (DA2Im), was synthesized and three others, Nitrilotriacetic acid (NTA), Tris(2‐pyridylmethyl)amine (TPA) and Tris(2‐aminoethyl)amine (TREN), were purchased from commercial sources. Their Zn 2+ binding stoichiometry and thermodynamic parameters were determined using isothermal titration calorimetry (ITC) in either a NEM buffer (pH 6.8) or a methanol:buffer mixture (pH 6.8) with the methanol added to increase the solubility of the less polar chelators. DA2Im and NTA were found to bind with a stoichiometry equivalent to three chelators for every two Zn 2+ and an equilibrium constant of (1.2±0.3)×10 8 and (2.1±0.6)×10 7 M ‐1 , respectively, in the buffer without methanol. Conversely, TREN and TPA were found to bind Zn 2+ with the desired 1:1 stoichiometry and an equilibrium constant of (1.5±0.1)×10 6 and >10 7 M ‐1 , respectively, in the methanol:buffer mixture. We conclude that TPA and TREN are promising candidates as HDAC functional mimetics for studying Zn 2+ inhibitor interactions. ITC experiments are currently underway to use the Zn 2+ :chelator binary complexes to obtain the energetic contribution of active site Zn 2+ to HDAC interaction with inhibitors.