Exploring Structural Determinants of Inhibitor Affinity and Selectivity in Complexes with Histone Deacetylase 6

Abstract

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.

Strategies To Design Selective Histone Deacetylase Inhibitors

“…Recently, one of the most active and selective oxazole derivatives published by Senger et al (IC 50 = 0.06 μM on HDAC6, IC 50 = 14 μM on HDAC1 and HDAC8) JS28 (41, Figure 3), which is similar to compound 38 but has a para-bromophenyl substitution on the oxazole hydroxamic acid, has been co-crystallized with zebrafish HDAC6 (PDB ID: 6Q0Z). [75,76] The solved crystal structure of JS28 corresponded to the predicted binding mode obtained by molecular docking into the homology model of human HDAC6 (RMSD 0.8 Å). Namely, the hydroxamic acid chelates the zinc ion in a bidentate manner and undergoes hydrogen bonds with the side chains of the nearby conserved amino acid residues H573, H574 and Y745.…”

“… [74c,85] A number of these highly selective as well as several less selective inhibitors and compounds with undetermined selectivity of the same chemotype were co‐crystallized with zebrafish HDAC6: nexturastat A (PDB IDs: 5G0I, 5G0J), HPOB (PDB ID: 5EF7), HPB (PDB ID: 5WGK), peptoid‐based inhibitors ( 6 and others, PDB IDs: 6DVL, 6DVM, 6DVN), ACY‐1083 (PDB ID: 5WGM), bavarostat (PDB ID: 6DVO), imidazopyridine derivative MAIP‐032 (PDB ID: 6CGP), phenothiazine derivative KV70 (PDB ID: 5W5K), dual HDAC‐proteasome inhibitor RTS‐V5 ( 68 , PDB ID: 6CW8), broad‐spectrum HDAC inhibitor AR‐42 ( 69 , PDB IDs: 6UO3, 6UO5, 6UO7), givinostat ( 70 , PDB ID: 6UOC), bishydroxamic acid ( 71 , PDB ID: 6VNR) and diverse inhibitors (compounds 72 – 74 , PDB IDs: 6PZO, 6PZS, 6PZU; Figure 4). [18g–i,76,85h,j,86] The experimentally obtained binding modes shed light on the protein‐ligand interactions and structure–activity relationships of these compounds. Strikingly, and in contrast to typically observed bidentate binding of hydroxamic acids with alkyl and vinyl linkers, all listed benzhydroxamic acids are bound in a monodentate fashion except bavarostat ( 63 ), AR‐42 ( 69 , PDB ID: 6UO3) and givinostat ( 70 ).…”

“…Independent on the HDAC isoform, it is stacked between the aromatic amino acid residues of the substrate binding tunnel (F583 and F643 in zebrafish HDAC6). The cap group is mostly directed toward the groove defined by the L1 loop, but examples exist where it interacts with the L2 pocket, between these pockets or in both of them (inhibitors with bifurcated cap groups) [76] . The cap group also often interacts with the unique S531 amino acid residue (zebrafish HDAC6 numbering), which plays a significant role in HDAC6‐substrate recognition and might influence the gain of selectivity toward HDAC6.…”

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Inhibitors of Neuroblastoma Cell Colony Growth That Increase Lysosome Accumulation

“…Noteworthy, crystal structures of drHDAC6 in complex with hydroxamic acid derivatives have shown that the inhibitors can chelate the catalytic zinc ion in either mono-or bidentate fashion. 54,[80][81][82][83] Hence, two different settings were used for docking of the hits into HDAC6 structure to investigate plausible binding modes.…”

“…Further preparations of the protein structures were done using the Protein Preparation Wizard of Schrödinger software. 82,96 Bond orders were assigned and hydrogen atoms added, and the H-bond network was subsequently optimized. The protonation states at pH 7.0 were predicted using the Epik-tool in Schrödinger.…”