Dose-Dependent Inhibition of Histone Deacetylases Reprograms Gene Expression through Global Remodeling of the Enhancer Landscape

Largazole is a macrocyclic depsipeptide originally isolated from the marine cyanobacterium Symploca sp. , which is indigenous to the warm, blue-green waters of Key Largo, Florida (whence largazole derives its name). Largazole contains an unusual thiazoline! thiazole ring system that rigidifies its macrocyclic skeleton, and it also contains a lipophilic thioester side chain. Hydrolysis of the thioester in vivo yields largazole thiol, which exhibits remarkable antiproliferative effects and is believed to be the most potent inhibitor of the metal-dependent histone deacetylases (HDACs). Here, the 2.14 Å-resolution crystal structure of the HDAC8! largazole thiol complex is the first of an HDAC complexed with a macrocyclic inhibitor and reveals that ideal thiolate! zinc coordination geometry is the key chemical feature responsible for its exceptional affinity and biological activity. Notably, the core structure of largazole is conserved in romidepsin, a depsipeptide natural product formulated as the drug Istodax recently approved for cancer chemotherapy. Accordingly, the structure of the HDAC8! largazole thiol complex is the first to illustrate the mode of action of a new class of therapeutically import nt HDAC inhibitors. H deacetylases (HDACs) catalyze the hydrolysis of acetylated lysine side chains in histone and nonhistone proteins, and these enzymes are implicated in a number of biological processes such as cell differentiation, proliferation, senescence, and apoptosis.1! 3 The metal-dependent enzymes are classified by amino acid sequence relationships as class I HDACs (1, 2, 3, and 8), class IIa HDACs (4, 5, 7, and 9), class IIb HDACs (6 and 10), and the class IV enzyme, HDAC11. These HDACs adopt the R/β fold first observed in arginase, a metalloenzyme that utilizes a Mn2 cluster to catalyze L-arginine hydrolysis. 5 However, the metal-dependent HDACs utilize only a single metal ion, either Zn or Fe in vivo, for catalytic function. Aberrant HDAC activity is found in various diseases, most notably cancer, making these enzymes critical targets for therapeutic intervention.7! 9 HDAC inhibitors block the proli eratio of tumor cells by inducing cell differentiation, cell cycle arrest, and/or apoptosis, and these compounds comprise some of the leading therapies approved or in clinical trials for cancer chemotherapy.7! 11 The primary affinity determinant of anHDAC inhibitor is a functional group that coordinates to the active site Zn ion, such as a hydroxamic acid. A hydroxamic acid will ionize to form an exceptionally stable 5-membered ring chelate with the active site Zn ion, as first demonstrated in a thermolysin! hydroxamate complex. Perhaps the best known hydroxamic acid inhibitor of theHDACs is suberoylanilide hydroxamic acid (Zolinza), which was the first HDAC inhibitor approved for cancer chemotherapy. The Zn-binding moiety of an HDAC inhibitor is tethered to a “capping group” that interacts with the mouth of the active site cleft. The most structurally complex capping groups are found in macrocyclic peptide and depsipeptide inhibitors (a depsipeptide contains both amide and ester linkages). For example, romidepsin (Istodax, Figure 1) is a macrocyclic depsipeptid that was rec ntly approved for the treatment of cutaneous T-cell lymphoma. Romidepsin itself is actually a prodrug; upon disulfide bond reduction in vivo, one of the romidepsin thiol side chains is proposed to coordinate to the active site Zn ion. However, no crystal structure is available to confirm this proposal. The 16-membered macrocyclic ring of romidepsin is comparable to that of the recently identified marine natural product largazole (Figure 1), a cyclic depsipeptide originally isolated from the cyanobacterium Symploca sp. indigenous to Key Largo, Florida. In contrast with romidepsin, largazole contains Figure 1. The disulfide bond of romidepsin is reduced, while the thioester linkage of largazole is hydrolyzed to form potent depsipeptide thiol inhibitors of HDACs. Structurally identical portions of each inhibitor are highlighted in red. Received: June 27, 2011 Published: July 26, 2011 r 2011 American Chemical Society 12474 dx.doi. org/10.1021/ja205972n | J. Am. Chem. Soc. 2011, 133, 12474–12477 COMMUNICATION