1. HDAC6
Synthesis and Characterization of a
Thiol-Based HDAC Inhibitor
Sarah Lopez, Shrasta Tamrakar, Youya Gao, Lihua Jin* and Caitlin E. Karver*
Department of Chemistry, DePaul University, Chicago IL, 60614
DePaul University Department of Chemistry
Acknowledgments
Method
Background
Summary and Conclusions
Abstract
References
Histone deacetylase (HDAC) is an enzyme involved in histone modification resulting in changes in gene
expression. All NAD+-independent HDACs deacetylate lysine residues on histones and contain a catalytic
zinc ion in their active sites. HDAC inhibitors are appealing anticancer agents because they hinder the
formation of tumors, prevent cell proliferation, and induce terminal differentiation of tumor cells. However,
they have been problematic for their off-target effects as well as poor bioavailability. Thiol-based HDAC
inhibitors are potent metabolically stable small molecules which may be able to combat some of these
issues. The thiol moiety of these inhibitors functions by coordinating zinc ions preventing it from initiating
catalysis. In this work 7-mercapto-N-(4-phenyl-2-thiazolyl)hexanamide was synthesized, purified and
characterized. Its binding energetics with zinc were analyzed by isothermal titration calorimetry (ITC).
Preliminary ITC data indicate complications due to sulfhydryl group oxidation in solution which is
mitigated by zinc chelation. Namely, we see much more oxidation occurring in control runs where the
HDAC inhibitor is titrated into a buffer without zinc. Efforts are underway to select solution conditions that
minimize oxidation, thus, increase accuracy of resulting binding parameters. In summary a novel HDAC
inhibitor has been synthesized. Through investigating binding energetics with zinc, information is obtained
regarding the significance of metal chelation on HDAC inhibition, furthering the development of antitumor
agents.
•Histone acetyl transferase (HAT) relaxes
chromatin; histone deacetylases (HDACs), in
contrast, changes chromatin into a closed
phase.
•Cancerous cells have histone
hypoacetylation. Furthermore, HDACs
promote cancerous cells by preventing the
expression of certain genes.
•This work aims to develop the need for a
variety of compounds to target HDAC as
well as measure binding thermodynamics of
a synthesized HDAC inhibitor.
Figure 1. HDACs role in cancer (K. Garber,
Nature Biotechnology, 2004. 22: p. 364.)
Cancer
•11 out of the 18 HDACS in humans are metal
dependent (typically Zn2+).
•Here a focus is on HDAC6.Consequently,
treatment is directed toward a specific disease,
minimizing the side effects and increasing
potency.
Figure 2. HDAC tetrahedral transition state
Synthesis
Results
ITC is commonly used to study the interaction between two molecules or ions. ITC is capable of
providing a complete thermodynamic profile including stoichiometry (n), binding constant (Ka) (thus,
Gibbs free energy change, ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) values. During an
ITC experiment, one ligand is titrated in small aliquots into a sample cell containing the other ligand
in a controlled fashion with the use of a motor-operated syringe. ITC studies were performed on a
microcalorimeter VP-ITC instrument (MicroCal Inc.). All solutions were prepared gravimetrically in
buffer or a DMSO:buffer mixture. The buffer consisted of 50 mM Tris and 0.10 M NaCl at pH 7.4
and the DMSO:buffer mixture was a 80:20 by volume mixture with buffer being 50 mM MES, 0.10
M NaCl, pH 6.0.
Figure 3. ITC
Results
•To conserve the thiol product, L-cysteine
was used first to determine suitable
solution conditions.
•In addition, control runs for titrant into
buffer were completed.
•Table 1 values reflect an average of three
independent trials.
1. Suzuki, T., A. Kouketsu, A. Matsuura, A. Kohara, S. Ninomiya, K. Kohdaa, and N.
Miyataa, Thiol-based SAHA analogues as potent histone deacetylase inhibitors Bioorg.
Med. Chem. Lett., 2004. 14: p. 3313-3317.
2. Chekmeneva, E., R. Prohens, J. M.Díaz-Cruz, C. Ariño, and M. Esteban,
Thermodynamics of Cd2+ and Zn2+ binding by the phytochelatin(γ-Glu-Cys)4-Gly and its
precursor glutathione Analytical Biochemistry, 2008. 375: p. 82–89.
3. Glozak, M.A., and E. Seto, Histone deacetylases and cancer Oncogene, 2007. 26: p.
5420–5432.
Figure 5. Raw data and binding
isotherm for 10 mM thiol product
titration in 50 mM MES, 0.10 M
NaCl, pH 6.0 (20% by v), DMSO
(80%) into buffer
n(Zn2+/Cys) Ka (M-1) ΔG° (kcal mol-1) Kd (µM) ΔH° (kcal mol-1) TΔS°(kcal mol-1)
0.204 ± 0.008 1.7 ± 0.3 × 105 -7.1 ± 0.1 6 ± 1 -78 ± 1 -48 ± 2
Table 1. Thermodynamic parameters for Zn2+ binding to L-cysteine
Figure 4. Raw data and binding isotherm for 0.6 mM Zn2+ solution
titration into 0.2 mM cysteine in 50 mM Tris, 0.10 M NaCl buffer, pH 7.4
1.We determined the best solution conditions for the thiol product as 80:20 (% by
volume) DMSO:buffer with the buffer being 50 mM MES, 0.10 M NaCl, pH 6.0.
2. A large control heat implies that the majority of heat is associated with dilution in this
solvent mixture. Furthermore, experiments with Zn2+ titration into the thiol product
indicates weak binding affinity.
3. A related–SH containing compound, cys, was more soluble allowing progress for
optimization in Tris buffer.
•Binding curves in the DMSO:buffer mixture for the interaction of Zn2+ and the thiol
product was far from complete and has a small magnitude of heat. Hence, the binding
affinity was too weak to be determined using ITC. This may be explained by the main
solvent in the mixture, i.e., DMSO, being a much weaker base than water, greatly
lowering the acidity of the –SH group thus its ability to coordinate metal ions.
•Success in using 50 mM Tris, 0.10 M NaCl, pH 7.4 to study L-cysteine thermodynamic
parameters.