Abstract

Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC(50) values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K(i) of approximately 14 nm, 15 times lower than the K(i) for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.

Highlights

  • The NADϩ-dependent enzymes [9, 10]

  • Hydroxamate-based inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroamic acid (SAHA; Fig. 1A) are believed to be pan-Histone deacetylase (HDAC) inhibitors (14 –16); recent studies have shown that TSA and SAHA are class I-specific inhibitors, and previous results reporting inhibition of class II HDACs were due to class I HDAC contamination [17,18,19]

  • HDAC Inhibition Assays—We assayed each of the recombinant class I (HDACs 1, 2, 3, and 8) and representative class II (HDACs 4, 5, and 7) enzymes with the pimelic diphenylamide HDAC inhibitor 106 (Fig. 1A)

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Summary

Introduction

The NADϩ-dependent enzymes (class III or sirtuins) [9, 10]. HDACs 1, 2, 3, and 8 belong to class I, showing homology to the yeast enzyme RPD3. To determine the mechanism and associated kinetic values, a series of inhibition progression curves for HDACs 1, 2, and 3, at different concentrations of inhibitor 106, were generated by adding 100 ng of each enzyme into separate reaction mixtures containing 50 ␮M acetyl-Lys(Ac)-AMC substrate (5 times the Km) and 2 milliunits of Lys-C peptidase developer.

Results
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