Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

Abstract

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-N2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this ''gain of function phenotype'' could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.catalytic domain ͉ enzymatic activity ͉ trifluoroacetyl-lysine ͉ gain of function

Inhibitors of Class 1 Histone Deacetylases Reverse Contextual Memory Deficits in a Mouse Model of Alzheimer's Disease

“…Previous studies have reported that VPA, butyrate, and SAHA have a pan-isoform profile with half-inhibitory concentration (IC 50 ) values ranging from low micromolar to millimolar ranges. However, it has become clear recently that immunoprecipitated and even recombinantly expressed and purified class IIa HDACs (eg, HDAC4) associate with class I HDACs and that these co-purifying enzymatic activities can occlude the true selectivity and potency of inhibitor treatments (Lahm et al, 2007;Jones et al, 2008).…”

Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors

“…This pocket is formed as a consequence of a tyrosine-histidine substitution. 7 We now report the discovery of tetrasubstituted cyclopropane hydroxamic acid class IIa HDAC inhibitors, with additional substitution at C1 (Figure 1). These compounds exhibited improved pharmacokinetic profiles, and so may provide a further means for evaluating efficacy in preclinical in vivo HD disease models.…”

Vorinostat-Like Molecules as Structural, Stereochemical, and Pharmacological Tools

“…1,7,[12][13][14] In addition, complete data regarding the factual HDAC profile for most of the early discovered agents are not available, since the development of effective isozyme-based assays is relatively recent. [5][6][7][15][16][17][18] As a consequence of the complex, pleiotropic nature of cancer, promiscuous HDAC inhibitors such as Vorinostat (1) (SAHA, suberoylanilide hydroxamic acid) 19 ( Figure 1) seem superior and as safe in the clinic as compared to the few class-specific agents available. [5][6][7]15 Ligand-based approaches are a first-choice solution to delineate the structural requirements for HDAC selectivity, especially with the emergence of ω-aryl alkanoyl hydroxamates such as Vorinostat as clinical candidates.…”

“…16 In view of this possibility, we deemed it necessary to extend our assay to include this method of HDAC profiling with (R/S)-2. The in vitro test was performed in the presence of the class IIa-specific fluorogenic substrate acetyl-Lys-(trifluoroacetyl)-AMC, using Vorinostat and TSA as the reference compounds (Table 3; see also the Supporting Information).…”