We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the Km value of the enzyme’s substrate and increase in the catalytic turnover rate of the enzyme
Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ = 0.4 ns) and bound NADH (τ = 6.0 ns). We used this technique to investigate the effects of Mg2+ on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD for NADH with ALDH2 ranged from 468 µM to 12 µM for Mg2+ ion concentrations of 20 µM to 6000 µM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4 s−1 (6000 µM Mg2+) to 8.3 s−1 (0 µM Mg2+) as determined by addition of excess NAD+ to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 µM−1s−1 over the entire Mg2+ ion concentration range and demonstrate that Mg 2+ ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ = 11.2 ns).
Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, Suberoylanilide hydroxamic acid (SAHA) has been approved by the FDA for treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, Trichostatin A (TSA), which has a higher in vitro inhibitory-potency against HDAC8, reportedly shows a poor efficacy in clinical settings. In order to gain the molecular insight into the above discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA to the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both the inhibitors to the enzyme showed the biphasic profiles, which represented an initial encounter of enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with the above inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step; which is solely contributed by entropic changes. The standard binding-enthalpy (ΔH0) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2–3 kcal/mol higher as compared to TSA. The experimental data presented herein suggests that SAHA serves as a preferential (target-specific/selective) HDAC8 inhibitor as compared to TSA. Arguments are presented that the detailed kinetic and thermodynamic studies may guide in the rational design of HDAC inhibitors as therapeutic agents.
Histone deacetylase 4 (HDAC4) is a high priority drug target for the treatment of various human diseases including Huntington’s disease and various cancers. Pan‐HDAC inhibitors, such as Suberoylanilide Hydroxamic Acid (SAHA) have already been screened and approved by the FDA as a treatment for some forms of cancer. BNG 3‐36 is a novel, thiol based compound that binds to HDAC4 utilizing a unique slow‐binding feature, inhibiting the catalytic mechanism of the enzyme. When added to HDAC8, however, this particular inhibitor shows normal competitive features. When the inhibitor is added to HDAC8, a member of another class of HDACs, the slow‐binding feature is not seen and instead is a normal competitive. We show that the inhibitor has a slow‐binding competitive feature when used with HDAC4 and has a normal competitive feature when used with HDAC8. Grant Funding Source: Supported by the NIH
Silent information regulator2 (sir2) proteins, or sirtuins, are nicotinamide adenine dinucleotide dependent lysine deacetylase. There are 7 human sirtuins, which have linked to a broad range of biological functions including aging, metabolism, cell cycle and apoptosis, and are considered as therapeutic targets. SIRT5 is the only enzyme that also possesses NAD+ dependent desuccinylase activity, which is important for modulating the urea cycle. Crystallization data shows that the binding of a sirtuin inhibitor, suramin, with SIRT5 leads to protein dimerization. Here we describe the detailed binding and inhibition mechanism of suramin to SIRT5. We also did comparative study with half suramin, which binds to SIRT5 with lower affinity. The data suggest that suramin bridging leads to dimerization of SIRT5 and improves the binding affinity. SIRT5 oligomerization state varies accordingly to the change of suramin/SIRT5 ratio. Our kinetic data also shows that suramin competes with both the succinylated lysine and NAD+, which explains the discrepancy between the binding affinity (2.5µM) and inhibition constant (40µM). The overall study may provide insight for the rational design of more potent inhibitors against sirtuins.
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