Design, Synthesis, and Evaluation of Polyamine Deacetylase Inhibitors, and High-Resolution Crystal Structures of Their Complexes with Acetylpolyamine Amidohydrolase

Polyamines are essential aliphatic polycations that bind to nucleic acids and accordingly are involved in a variety of cellular processes. Polyamine function can be regulated by acetylation and deacetylation, just as histone function can be regulated by lysine acetylation and deacetylation. Acetylpolyamine amidohydrolase (APAH) from <i>Mycoplana ramosa</i> is a zinc-dependent polyamine deacetylase that shares approximately 20% amino acid sequence identity with human histone deacetylases. We now report the X-ray crystal structures of APAH–inhibitor complexes in a new and superior crystal form that diffracts to very high resolution (1.1–1.4 Å). Inhibitors include previously synthesized analogues of <i>N</i>⁸-acetylspermidine bearing trifluoromethylketone, thiol, and hydroxamate zinc-binding groups [Decroos, C., Bowman, C. M., and Christianson, D. W. (2013) <i>Bioorg. Med. Chem. 21</i>, 4530], and newly synthesized hydroxamate analogues of shorter, monoacetylated diamines, the most potent of which is the hydroxamate analogue of <i>N</i>-acetylcadaverine (IC₅₀ = 68 nM). The high-resolution crystal structures of APAH–inhibitor complexes provide key inferences about the inhibition and catalytic mechanism of zinc-dependent deacetylases. For example, the trifluoromethylketone analogue of <i>N</i>⁸-acetylspermidine binds as a tetrahedral gem-diol that mimics the tetrahedral intermediate and its flanking transition states in catalysis. Surprisingly, this compound is also a potent inhibitor of human histone deacetylase 8 with an IC₅₀ of 260 nM. Crystal structures of APAH–inhibitor complexes are determined at the highest resolution of any currently existing zinc deacetylase structure and thus represent the most accurate reference points for understanding structure–mechanism and structure–inhibition relationships in this critically important enzyme family

Compromised Structure and Function of HDAC8 Mutants Identified in Cornelia de Lange Syndrome Spectrum Disorders

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly disorder resulting from mutations in genes that encode the core components of the cohesin complex, SMC1A, SMC3, and RAD21, or two of its regulatory proteins, NIPBL and HDAC8. HDAC8 is the human SMC3 lysine deacetylase required for cohesin recycling in the cell cycle. To date, 16 different missense mutations in HDAC8 have recently been identified in children diagnosed with CdLS. To understand the molecular effects of these mutations in causing CdLS and overlapping phenotypes, we have fully characterized the structure and function of five HDAC8 mutants: C153F, A188T, I243N, T311M, and H334R. X-ray crystal structures reveal that each mutation causes local structural changes that compromise catalysis and/or thermostability. For example, the C153F mutation triggers conformational changes that block acetate product release channels, resulting in only 2% residual catalytic activity. In contrast, the H334R mutation causes structural changes in a polypeptide loop distant from the active site and results in 91% residual activity, but the thermostability of this mutant is significantly compromised. Strikingly, the catalytic activity of these mutants can be partially or fully rescued <i>in vitro</i> by the HDAC8 activator <i>N</i>-(phenylcarbamothioyl)­benzamide. These results suggest that HDAC8 activators might be useful leads in the search for new therapeutic strategies in managing CdLS

Biochemical and Structural Characterization of HDAC8 Mutants Associated with Cornelia de Lange Syndrome Spectrum Disorders

Cornelia de Lange Syndrome (CdLS) spectrum disorders are characterized by multiple organ system congenital anomalies that result from mutations in genes encoding core cohesin proteins SMC1A, SMC3, and RAD21, or proteins that regulate cohesin function such as NIPBL and HDAC8. HDAC8 is the Zn²⁺-dependent SMC3 deacetylase required for cohesin recycling during the cell cycle, and 17 different HDAC8 mutants have been identified to date in children diagnosed with CdLS. As part of our continuing studies focusing on aberrant HDAC8 function in CdLS, we now report the preparation and biophysical evaluation of five human HDAC8 mutants: P91L, G117E, H180R, D233G, and G304R. Additionally, the double mutants D233G–Y306F and P91L–Y306F were prepared to enable cocrystallization of intact enzyme–substrate complexes. X-ray crystal structures of G117E, P91L–Y306F, and D233G–Y306F HDAC8 mutants reveal that each CdLS mutation causes structural changes that compromise catalysis and/or thermostability. For example, the D233G mutation disrupts the D233–K202–S276 hydrogen bond network, which stabilizes key tertiary structure interactions, thereby significantly compromising thermostability. Molecular dynamics simulations of H180R and G304R HDAC8 mutants suggest that the bulky arginine side chain of each mutant protrudes into the substrate binding site and also causes active site residue Y306 to fluctuate away from the position required for substrate activation and catalysis. Significantly, the catalytic activities of most mutants can be partially or fully rescued by the activator <i>N</i>-(phenylcarbamothioyl)-benzamide, suggesting that HDAC8 activators may serve as possible leads in the therapeutic management of CdLS