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

Abstract

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 Mycoplana ramosa 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 N(8)-acetylspermidine bearing trifluoromethylketone, thiol, and hydroxamate zinc-binding groups [Decroos, C., Bowman, C. M., and Christianson, D. W. (2013) Bioorg. Med. Chem. 21, 4530], and newly synthesized hydroxamate analogues of shorter, monoacetylated diamines, the most potent of which is the hydroxamate analogue of N-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 N(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.

Figure 1

Substrates and inhibitors of APAH from Mycoplana ramosa. Substrates include: acetylputrescine, acetylcadaverine, N¹-acetylspermidine, N⁸-acetylspermidine, and N¹-acetylspermine. Previously described³³ inhibitors of APAH are analogues of the substrate N⁸-acetylspermidine bearing different metal-binding groups: trifluoromethylketone (1), thiol (2), or hydroxamic acid (3). New inhibitors reported in this study (4–7) are shorter hydroxamic acids with varying chain lengths.

Figure 2

(a) Simulated annealing omit map showing trifluoromethylketone 1 bound as a gem-diol in the active site of APAH (monomer B, contoured at 3.5σ). Atomic color codes are as follows: C = wheat (protein, monomer B), brown (monomer A), or green (inhibitor), N = blue, O = red, F = light cyan, Zn²⁺ = magenta sphere. Water molecules are represented as red spheres. Metal coordination and selected hydrogen bond interactions are shown as solid black or dashed black lines, respectively. (b) Stereoview of the N⁸-acetylspermidine substrate bound in the active site of the inactive mutant APAH H159A (PDB accession code 3Q9C, monomer A). Atomic color codes are identical to those in (a); C atoms in brown correspond to adjacent monomer I of the APAH dimer. We note that in monomer J only of this structure, the secondary amine of N⁸-acetylspermidine donates a hydrogen bond to E117. Additionally, a water molecule makes a bridging interaction in some monomers between Y19 and the terminal amino group of the substrate. (c) Simulated annealing omit map of thiol 2 bound in the active site of APAH (monomer A, contoured at 3.0σ). Atomic color codes are identical as in (a), with S = gold.

Figure 3

(a) Simulated annealing omit map of hydroxamate 4 bound in the active site of APAH (monomer A, contoured at 3.5σ). Atomic color codes are as follows: C = wheat (protein, monomer A), brown (monomer B), or green (inhibitor), N = blue, O = red, Zn²⁺ = magenta. Water molecules are represented as red spheres. Metal coordination and selected hydrogen bond interactions are shown as solid black or dashed black lines, respectively. (b) Simulated annealing omit map of hydroxamate 5 bound in two conformations in the active site of APAH (monomer B, contoured at 3.0σ). Atomic color codes are identical to those in (a). (c) Simulated annealing omit map of hydroxamate 6 bound in the active site of APAH (monomer A, contoured at 4.0σ). Atomic color codes are identical to those in (a). (d) Simulated annealing omit map of hydroxamate 3 bound in the active site of APAH (monomer B, contoured at 3.0σ). Atomic color codes are identical to those in (a).

Figure 4

Superposition of APAH complexes with hydroxamate inhibitors 3–6 (monomer A for all). Atomic color codes are as follows: C = light grey (protein), teal (hydroxamate 3), orange (hydroxamate 4), yellow (hydroxamate 5), or green (hydroxamate 6), N = blue, O = red, F = light cyan, Zn²⁺ = magenta.

Figure 5

Proposed mechanism of APAH. The substrate binding mode is based on the complex of N⁸-acetylspermidine with the inactive mutant APAH H159A (PDB accession code 3Q9C), and the binding of the tetrahedral intermediate is mimicked by the binding of trifluoromethylketone 1 (Figure 2a).

Scheme 1. Synthesis of new APAH inhibitors 4–7

Reagents and conditions: (a) Carbonyldiimidazole (1.5 equivalents) in tetrahydrofuran, (rt, 1 h), then NH₂OH·HCl (2 equivalents; room temperature, overnight); (b) Anhydrous HCl (1 M) in ethyl acetate, (room temperature, overnight).