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.