Variable Active Site Loop Conformations Accommodate the Binding of Macrocyclic Largazole Analogues to HDAC8

Abstract

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn2+ ion, as observed in the X-ray crystal structure of the HDAC8–Largazole complex [Cole, K. E.; Dowling, D. P.; Boone, M. A.; Phillips, A. J.; Christianson, D. W. J. Am. Chem. Soc. 2011, 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a 6-membered pyridine ring is also substituted (with two different orientations) for the 5-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn2+ ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in the L1 and L2 loops flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

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

“…29−31,34,44,49 Accordingly, P91 is approximately 27–29 Å away from the active site Zn 2+ ion, depending on the conformation of the L2 loop. With a residual catalytic activity of 86% compared with the wild-type enzyme (Table 1), P91L HDAC8 is the least functionally compromised of the mutants described herein.…”

General Base–General Acid Catalysis in Human Histone Deacetylase 8

“…23–30 Indeed, HDAC8 is an excellent model system for understanding catalysis by all metal-dependent HDACs. Based on the crystal structure of an HDAC-like protein from Aquifex aeolicus reported five years prior to the structure determination of HDAC8, a catalytic mechanism was proposed for the metal-dependent HDACs in which tandem histidine residues H142 and H143 serve as a general base-general acid catalyst pair.…”

Real-time monitoring of conformational transitions of single-molecule histone deacetylase 8 with nanocircuits

“…11,21 For SAHA-inhibitors, the hydroxamate acid moiety chelates the metal ions (Zn 2+ ) in the active site pocket, while the aliphatic chain and the capping moiety interact with the hydrophobic pocket and the protein surface. 22,23 Crystallographic and computational studies have identified that two loops (L1 and L2) located in the vicinity of the active site pocket entrance are highly malleable to effectively adopt and catalyze structurally different substrates.…”