In the previous section, very few structural information were reported for histone deacetylase 6 (HDAC6). Indeed, no crystallographic data was available on the Protein Data Bank (PDB) in 2015, with respect to the zinc finger domain. Two years later, the structural knowledge on HDAC6 was expanded with twenty-eight crystallographic structures available on the PDB (accessed on November, 6th 2017), from both zebrafish and human species, characterizing bound and apo forms of several isolated HDAC6 domains (section 1.6, Table S2). HDAC6 differentiates itself from the other HDAC isoforms at both the structural and the biological levels. Although HDAC6 phylogenetically belongs to the histone deacetylases family, it is mainly involved in cytosolic processes, which opens up the debate on whether the effects promoted by HDAC6 are of epigenetic nature. Waiting for a consensus on what characterizes the overall landscape of epigenetic regulation 2, here the definition of “epigenetic enzyme” presented earlier will be kept all along the manuscript. Therefore, HDAC6 is here defined as an epigenetic enzyme (capable of deacetylating histones for chromatin remodeling in some cases), without excluding its important functions of deacetylating cytosolic proteins, as well as its protein-protein interactions 115, 116. Containing 1215 residues that define eight domains and signaling sequences, HDAC6 is the largest isozyme of the histone deacetylase family (Fig. 6). Its predominant cytosolic localization is due to its two nuclear export signals (NES), the Ser-Glu rich domain (SE14), and the acetylation of its nuclear localization signal (NLS) 115, 117. The first discovered HDAC6 substrate was the α-tubulin subunit of the microtubule, thus regulating the microtubules dynamics 118, 119. The acetylation status of the α-tubulin is now commonly used as a cellular marker for HDAC6-selective modulation.
Figure 6. HDAC6 functional domains. NLS: nuclear localization signal; NES 1 and 2: nuclear export signals; DMB: dynein motor binding domain; SE14: Ser-Glu rich domain. Catalytic domains 1 and 2 (CD1 and CD2) comprise one zinc ion each for catalytic activity, whereas the ubiquitin-binding domain (ZnF-UBP) is a zinc finger region that contains three structural zinc atoms. Figure adapted from Batchu et al.120 and built using Servier Medical Art under the Common Creative License 3.0.
More specifically, HDAC6 is the only one, among its protein family, to bear tandem catalytic domains CD1 and CD2, and an ubiquitin-binding domain (ZnF-UBP) 34, 110, 121. The first catalytic domain CD1 specifically hydrolyzes the C-terminus of acetyllysine substrates (i.e. with free α-carboxylate group), and may act as a microtubule-binding region 121, 122. Conversely, the second catalytic domain CD2 displays a broader substrates spectrum, and is responsible for the α-tubulin deacetylase activity 34,
121, 123, 124. Through, at least, both its CD1 and ZnF-UBP, HDAC6 is a key player in misfolded proteins degradation: it recognizes and binds ubiquitinated misfolded proteins, and transports them along the microtubules through dynein binding, to reach perinuclear aggresome formation areas. Indeed, the ubiquitin tag is a molecular signal for proteolysis, and dynein is a motor protein that hydrolyzes ATP to progress along the microtubules 110, 113, 125, 126. Nevertheless, there are still controversial results on which function is proper to which functional domain, and their possible combined mode of action remains to be fully explored. Besides, intact tandem catalytic domains have been shown critical for HDAC6 full
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catalytic activity 121. A summary of the substrates for which the interacting domain was clearly identified is provided in the section 1.6 (table S3).
Having ubiquitin as a chosen protein partner makes HDAC6 a pivotal protein in autophagic clearance and aggresome formation, the latter being exploited by the influenza virus for host cell entry 127. Among others, the chaperone heat shock protein HSP90, cortactin, and the microtubule-associated protein Tau are also substrates of HDAC6 113, 128, 129. The function of regulatory T-cells (Tregs) are influenced by HDAC6, as its knock-out enhanced the suppressive activity of Tregs in vitro and in vivo (murine model)
130. Altogether these non-epigenetic mechanisms fuel the interest for HDAC6-selective modulation in drug discovery targeting cancer, neurodegenerative disorders, autoimmunity and inflammation.
HDAC6-selective catalytic modulation
The two first HDAC6-selective catalytic inhibitors were tubacin and tubastatin A (Fig. 7). These two compounds follow the pharmacophoric scheme for histone deacetylase inhibition (zinc-binding group (ZBG), linker and cap parts. Tubacin was discovered in 2001 by combinatorial chemistry methods, but is only used to probe HDAC6 activity in vitro because of its non-drug-like structure and its multistep chemical synthesis 91, 123, 131. Tubastatin A was discovered nine years later through a structure-based drug design approach (HDAC6 homology modeling) combined to in vitro enzymatic screening 132. Tubastatin A is also widely used as a research tool. Numerous other HDAC6 catalytic inhibitors, with various HDAC selectivity profile, have been reported in the literature to get insights into HDAC6-related pathways and in an attempt to develop potential drug candidates. Chemical modulations were done at each level of the HDAC pharmacophoric scheme. Having a hydroxamate as the ZBG is not mandatory to reach HDAC6 selectivity, others like pyrimidinethione or benzoic acid are possible isosters, although inhibitors that bear hydroxamates are usually very potent (Fig. 7). The linker can also be modified and may contribute to selectivity as the constraints it provides to the molecule orient the cap group for more or less favored interactions with the active site. Nevertheless, there was no consensus about the spacer optimal length and composition, and contradictory studies were reported 36, 133, 134. Finally, the cap group, which was hypothesized to be critical for isoform selectivity, has also underwent extended variations in terms of scaffold and rigidity (Fig. 7) 135-140. Briefly, HDAC6 selectivity has been attributed to bulky ligands.
Indeed, until 2016, no crystallographic structure of the catalytic domains of HDAC6 was available, and virtual screening or molecular docking used to study and report inhibitors were performed using a homology model of HDAC6 CD2. The CD2 active site is characterized, like in other isoforms, by a tunnel-like pocket having the catalytic zinc ion located at its bottom, and being chelated by three residues (Asp649, His651 and Asp742). Unlike HDAC8, the presence of a structural water molecule in the zinc first chelation shell was unknown 63, 97, 98, 141. The CD2 catalytic pocket had been predicted wider than in other HDACs, especially at the channel rim level, which was considered as an area of diversity 132, 136. This observation supported the biological data that showed a trend for bulky ligands favored by HDAC6.
The recently released crystallographic structures of CD2 has confirmed that the compounds designed by the scientific community did target this catalytic domain. Surprisingly, these crystallographic structures displayed a much more constricted channel than expected by homology modeling calculations, rising questions about a potential plastic behavior, as observed for HDAC8, a fact that could be exploited for selectivity (Fig. 8) 121, 124, 132.
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Figure 7. A selection of HDAC6-selective catalytic inhibitors. The pharmacophoric scheme for HDAC inhibition is reminded: zinc-binding group (ZBG), linker and cap parts are highlighted.
Chemical structures and enzymatic inhibition data are coming from Butler et al., Huang et al.,Santo et al., Kozikowski et al., Suzuki et al., Inks et al., Sodji et al., and Bergman et al. for Tubastatin A and Tubacin, ACY-241, ACY-1215, PMID_18642892_3, Nexturastat A, NQN-1, the thiolate derivative, the 3-hydroxypyrimidine-2-thione derivative, and the 4-hydroxybenzoic acid derivative, respectively 132, 139, 142-147.
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Figure 8. Comparison between the HDAC6 homology model and the crystallographic structure (CD2). (A) Active pocket view of a homology model of the HDAC6 CD2 (provided by Butler et al.
132). The protein is shown as yellow ribbons. The pocket was predicted shallower and wider than other HDACs catalytic site which typically display a 11 Å deep pocket. (B) Active pocket view of the crystallographic structure reported by the 5EDU PDB code 121. The protein is shown as light blue ribbons. Conversely to the homology model, the pocket is deeper and more constricted, showing a more rugged channel rim. Molecular surface is drawn in grey 12 Å around the zinc ion to provide a general view of the active sites landscape. Catalytic pockets are highlighted with violet dotted lines, and zinc ions are labelled and positioned. Pictures were generated with MOE 2015.10 148.
The main applications for HDAC6-selective catalytic inhibitors are, so far, in oncology. In 2017, six clinical trials were underway with three compounds investigated either alone or in combination, with immunomodulatory drugs or proteasome inhibitors, in either solid tumors or hematological malignancies (Fig. 7: compounds ACY-1215, ACY-241 and KA2507 for which the structure has not been released yet; clinical trials NCT03008018, NCT02935790, NCT02635061, NCT01323751, NCT02091063, and NCT02632071). Growing evidence have showed that HDAC inhibition re-sensitizes cancer cells to chemo- and radiotherapy 13. Although transcriptional regulation is one of the mechanisms underlying the efficacy of HDAC inhibitors, part of the mode of action of pan-HDAC inhibitors (i.e. non-selective) has been associated with the catalytic inhibition of HDAC6 for multiple myeloma treatment 52, 149. Moreover, extended exposure to the FDA-approved pan-HDAC inhibitors lead to severe side effects (cardiac and hematological toxicities, profound fatigue, dose-limiting nausea and vomiting) 13, 49-52, so focusing on isoform selectivity let one hope in reducing these adverse events . From a biological point of view, the rational for HDAC6-selective inhibition in combination to the two immunomodulatory drugs nivolumab (programmed cell death 1 PD1 inhibitor) and ipilimumab (cytotoxic T lymphocyte antigen 4 CTLA4 inhibitor) in solid cancers is to slow tumor growth by cell cycle arrest in G1, and to increase tumor antigens expression in order to potentiate the immunotherapy treatment 150-153. Clinical trials for multiple myeloma and lymphoma treatment use ACY-1215 combined to a proteasome inhibitor. This strategy exploits another specificity of HDAC6-related molecular mechanism, being its central role in the degradation of aggregated and misfolded proteins through the aggresome pathway. The combination of ACY-1215 with bortezomib aims at synergism between the two drugs. Indeed, among other mechanisms, when the proteolytic barrel 26S proteasome is impaired (here pharmacologically inhibited by the bortezomib), the aggresome pathway is stimulated as an escape
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mean for cancer cells to survive. Selectively interfering with HDAC6 is thus expected to overcome cancer cells resistance and to induce their apoptosis 52, 110, 113, 154-157.
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