Thermodynamics of Binding of Structurally Similar Ligands to Histone Deacetylase 8 Sheds Light on Challenges in the Rational Design of Potent and Isozyme-Selective Inhibitors of the Enzyme

Abstract

Among the different histone deacetylase (HDAC) isozymes, HDAC8 is the most highly malleable enzyme, and it exhibits the potential to accommodate structurally diverse ligands (albeit with moderate binding affinities) in its active site pocket. To probe the molecular basis of this feature, we performed detailed thermodynamic studies of the binding of structurally similar ligands, which differed with respect to the “cap”, “linker”, and “metal-binding” regions of the suberoylanilide hydroxamic acid (SAHA) pharmacophore, to HDAC8. The experimental data revealed that although the enthalpic (ΔH°) and entropic (ΔS°) changes for the binding of individual SAHA analogues to HDAC8 were substantially different, their binding free energies (ΔG°) were markedly similar, conforming to a strong enthalpy–entropy compensation effect. This effect was further observed in the temperature-dependent thermodynamics of binding of all SAHA analogues to the enzyme. Notably, in contrast to other metalloenzymes, our isothermal titration calorimetry experiments (performed in different buffers of varying ionization enthalpies) suggest that depending on the ligand, its zinc-binding group may or may not be deprotonated upon the binding to HDAC8. Furthermore, the heat capacity changes (ΔCp°) associated with the ligand binding to HDAC8 markedly differed from one SAHA analogue to the other, and such features could primarily be rationalized in light of the dynamic flexibility in the enzyme structure in conjunction with the reorganization of the active site resident water molecules. Arguments are presented that although the binding thermodynamic features described above would facilitate identification of weak to moderately tight-binding HDAC8 inhibitors (by a high-throughput and/or virtual screening of libraries of small molecules), they would pose major challenges for the structure-based rational design of highly potent and isozyme-selective inhibitors of human HDAC8.

A distal regulatory region of a class I human histone deacetylase

“…In contrast to SAHA and DCPI, binding of the inhibitor Trichostatin A (TSA) with an unsaturated chain and a more polar cap-group, led to substantial and distinct chemical shift changes; Fig. 1c, e. Previous investigations have shown that TSA binds to HDAC8 in a 1:1 stoichiometry in solution 31 . Upon sub-stoichiometric addition of TSA the cross-peaks split into two fractions, revealing that the binding reaction is in the intermediate-to-slow exchange regime 32 in agreement with previous reports 33,34 .…”

“…1c, e . Previous investigations have shown that TSA binds to HDAC8 in a 1:1 stoichiometry in solution 31 . Upon sub-stoichiometric addition of TSA the cross-peaks split into two fractions, revealing that the binding reaction is in the intermediate-to-slow exchange regime 32 in agreement with previous reports 33 , 34 .…”

Mechanism of N-Acylthiourea-mediated Activation of Human Histone Deacetylase 8 (HDAC8) at Molecular and Cellular Levels

“…All of the structural, kinetic, and thermodynamic data suggest that SAHA binds to the active site pocket of HDAC8 (28,29,32,39). Because TM-2-51 does not obliterate the binding of SAHA to the enzyme (Fig.…”

“…6), it implies that both the activator and the inhibitor (used to serve as a substrate analog) can simultaneously bind to the enzyme. This is not surprising in view of the marked malleability/flexibility of the active site pocket of HDAC8, as evident from its ability to interact with structurally diverse ligands with modest binding affinities (6,10,32,39). The kinetic (Fig.…”

Inhibition mechanism of SAHA in HDAC: a revisit

“…Very recently, Wiest 18 further put forward a viewpoint that the deprotonation of SAHA in HDAC8 is "Model-dependent" (namely "QM-size" dependent) in their static QM/MM (ONIOM) calculation, in which negative SAHA is more stable if the two Asp residues (see conserved D-H dyads as shown in Figure 1) was considered in the QM region while neutral SAHA is more stable if using a smaller QM region as we used in previous study. 12 Meanwhile, on the basis of the Isothermal Titration Calorimetry (ITC) experiments in 2014, Srivastava et al 19 suggested that SAHA may release a proton to the exterior buffer upon binding to HDAC8, but as the determined enthalpy and entropy difference is tiny and thus still difficult for definitely identifying the feasibility of the proton transfer reaction between SAHA and His142 (as shown in Figure 1). Nevertheless, it is the first time to clarify the SAHA's protonation state in the enzyme-inhibitor complex experimentally.…”