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. 2020 Aug 21;15(8):2154-2163.
doi: 10.1021/acschembio.0c00362. Epub 2020 Jul 23.

Structural Basis for the Selective Inhibition of HDAC10, the Cytosolic Polyamine Deacetylase

Corey J Herbst-Gervasoni  1 , Raphael R Steimbach  2   3 , Michael Morgen  3 , Aubry K Miller  3   4 , David W Christianson  1
Affiliations

Structural Basis for the Selective Inhibition of HDAC10, the Cytosolic Polyamine Deacetylase

Corey J Herbst-Gervasoni et al. ACS Chem Biol. .

Abstract

The cytosolic class IIb histone deacetylase HDAC10 is an emerging target for drug design. As an inducer of autophagy, its selective inhibition suppresses the autophagic response that otherwise attenuates the efficacy of cytotoxic cancer chemotherapy drugs. HDAC10 is a zinc-dependent polyamine deacetylase exhibiting maximal catalytic activity against N8-acetylspermidine. As revealed in the structure of Danio rerio (zebrafish) HDAC10, two conserved structural motifs direct this narrow substrate specificity: a 310 helix containing the P(E,A)CE motif that sterically constricts the active site and an electrostatic "gatekeeper," E274, that confers selectivity for cationic polyamine substrates. To accelerate drug design efforts targeting human HDAC10, we now report the preparation of "humanized" zebrafish HDAC10 in which two amino acid substitutions, A24E and D94A, yield an active site contour more similar to that of human HDAC10. X-ray crystal structures of this HDAC10 variant complexed with Tubastatin A and indole analogues bearing pendant tertiary amines reveal that inhibitors capable of hydrogen bonding with gatekeeper E274 exhibit high affinity and selectivity for HDAC10 over HDAC6 (the other class IIb isozyme). Moreover, these structures reveal that the P(E,A)CE motif helix can shift by up to 2 Å to accommodate the binding of bulky inhibitors. Thus, slender polyamine-like inhibitor structures are not exclusively required for selective, high affinity binding to HDAC10. Indeed, the flexibility of the P(E,A)CE motif helix could conceivably enable the binding of certain protein substrates.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
(a) Ribbonplot of HDAC10 showing the catalytically active deacetylase domain (cyan) with a bound analogue of substrate N8-acetylspermidine (CPK model, C = yellow, N = blue, O = red) coordinating to the active site Zn2+ ion (gray sphere). The “PEACE” motif (P(E,A)CE, magenta) is a 310 helix unique to HDAC10 that sterically constricts the approach to the active site so as to favor the binding of long, slender polyamine substrates. Opposite the PEACE motif is E274 (red stick figure), which provides electrostatic complementarity to cationic polyamine substrates. The catalytically inactive pseudo-deacetylase domain is shown in tan. (b) HDAC10 inhibitors Tubastatin A and recently reported analogues. Dissociation constants (Kd) were measured here by isothermal titration calorimetry using “humanized” A24E-D94A zebrafish HDAC10 (n.d., not determined). Human HDAC10 IC50 values and HDAC10/HDAC6 selectivities were previously reported. The tertiary amino groups of Tubastatin A, inhibitor 1, and inhibitor 2 are drawn in their protonated forms as they would exist at physiological pH.
Figure 2.
Figure 2.
Measurements using recombinant TwinStrep-HDAC10 fusion protein alone (black), with DMSO (red), Tubastatin A (green), and oxa-Tubastatin A (blue). Protein was tested at 3 μM and inhibitors at 6 μM, giving a protein:inhibitor ratio of 1:2. For the two control experiments (black and red), n = 1. For the inhibitor experiments (blue and green), n = 2; the data shown represent the mean of the two experiments.
Figure 3.
Figure 3.
(a) Stereoview of a Polder omit map of Tubastatin A bound in the active site of HDAC10 (contoured at 5σ). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (b) Active site surface of HDAC10 color-coded by electrostatic potential (−5kT (red) to +5kT (blue)). The side chain of E24 in the humanized PEACE motif plays an important role in accommodating the tricyclic tetrahydro-γ-carboline capping group. (c) Superposition of the HDAC10–Tubastatin A complex (gray) and the HDAC10–AAT complex (blue) reveals the significant shift (up to 1.9 Å) of the PEACE motif helix containing E24/A24 that accommodates the bulky capping group of Tubastatin A.
Figure 4.
Figure 4.
(a) Stereoview of Tubastatin A complexed with catalytic domain 2 of HDAC6 (PDB 6THV). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (b) Stereoview of Tubastatin A complexed with HDAC10 oriented in identical fashion to HDAC6 in plate (a). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.
Figure 5.
Figure 5.
(a) Stereoview of polder omit maps of inhibitor 1 bound in the active site of HDAC10 (contoured at 5.5σ). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (b) Stereoview of the superposition of the HDAC10–1 complex (colored as in plate a) and the HDAC10–Tubastatin A complex (protein = blue, inhibitor = mauve). Note the conformational changes of active site gatekeeper E274 and PEACE motif residue E24 that accompany the binding of each inhibitor.
Figure 6.
Figure 6.
(a) Stereoview of a Polder omit map of inhibitor 2 bound in the active site of HDAC10 (contoured at 5σ). (b) Stereoview of a Polder omit map of inhibitor 3 bound in the active site of HDAC10 (contoured at 4σ). In each plate, metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.
Figure 7.
Figure 7.
(a) Stereoview of Polder omit maps of inhibitor 4 and E24 in the active site of HDAC10 (contoured at 5.5σ). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. Electron density for the E24 side chain is weak or absent, indicative of conformational disorder. (b) Stereoview of the superposition of the HDAC10–4 complex (colored as in plate a; a modeled conformation of the disordered E24 side chain is shown for reference as a semi-transparent image) and the HDAC10–Tubastatin A complex (protein = blue, inhibitor = mauve). Note the conformational change of active site gatekeeper E274 that accompanies the binding of each inhibitor. (c) Stereoview of the superposition of the HDAC10–4 complex (colored as in plates a and b) and the recently reported structure of the HDAC10 complex with a bis-hydroxamate derivative.
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