One of the first use of the word “epigenetic” was the illustration of the genes influence on cell fate decision through an “epigenetic landscape”. Indeed, Waddington, working on the fruit fly developmental biology, linked epigenesis, a theory of embryology that describes specialized tissue developing from non-specialized states, and genetics to coin the concept of an epigenetic landscape 1-5. Nanney, who worked with a bacterial model in which he assumed that epigenetic mechanisms were related to cellular memory, reconsidered Waddington’s theory in 1958 6. This idea was further developed by Riggs and Holliday 7, who described DNA methylation as a potential transcriptional regulatory mechanism that could be maintained by cell division, insisting on the cellular memory mechanism previously assumed by Nanney. 2,8. It is noteworthy that, by defining DNA methylation as an epigenetic modification, the term has evolved from a cellular development mechanism (involved in what is investigated in the current field of developmental biology) into a molecular mechanism of gene regulation 9. Although some debate exists about its rigorous definition 2, epigenetics can be translated from its direct Greek etymology meaning the inheritance of variation (-genetics) above, and beyond (epi-) changes in the DNA sequencing 10. Its expanded definition currently includes not only DNA methylation, but also the molecular mechanisms involved in chromatin remodeling, such as histone modifications 11,12.
The mediation of the DNA accessibility through its folding/unfolding around the nucleosomes (the fundamental subunits of chromatin), and how nucleosomes are positioned along the DNA material is at the heart of the epigenetic regulation of gene expression (Fig. 1) 13. The molecular mechanisms involved in this process are ruled by protein families that, either catalyze reversible covalent modifications (methylation, acetylation, phosphorylation, ubiquitination, or SUMOylation, among others), also called the “writers” and the “erasers”, or that “read” these marks on DNA (e.g. cytosine methylation) and on histones (e.g. histone acetylation) (Fig.1). As a matter of clarification in the context of the present thesis, “epigenetic enzymes” are considered as those proteins catalyzing the reactions involved in the selective addition or removal of chemical marks on DNA and chromatin, thereby altering chromatin and DNA accessibility towards the transcriptional machinery. Accordingly, “epigenetic drugs” refer to drugs used to modulate the activity of epigenetic enzymes.
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Figure 1. DNA packaging and selected examples of epigenetic writers, readers and erasers. The DNA material is not free within the nucleus, but highly packaged through the chromatin fiber by wrapping around nucleosome cores. One nucleosome unit is composed of 147 base pairs of DNA which are wrapped around an octamer of histone proteins that contains 2 copies of H2A, H2B, H3 and H4 histone proteins. Histones are globular proteins comprising an N-terminal domain that is the place for post-translational modifications (lysine methylation or acetylation, arginine methylation being the most abundant marks). Epigenetic proteins which add these marks are called “writers”
(HATs: histone acetyl-transferases; HMTs: histone methyltransferases; PRMTs: protein arginine methyltransferases), those which remove these marks are the “erasers” (HDACs: histone deacetylases; KDMs: lysine demethylases) whereas the proteins that recognize and bind such modifications are the “readers”. This picture has been built according to Arrowsmith et al.11, Falkenberg et al.14, Azad et al.13, Schmauss 2017 15, and Luo et al.16, and using Servier Medical Art, under the Common Creative license 3.0. Note: only a selection of epigenetic enzymes, mainly histones modifiers, are given for scheme clarity and because this thesis focuses on HDAC6. A more complete list of epigenetic enzymes is provided in section 1.6, table S1.
Epigenetic state in disease and epigenetic drugs
The epigenetic state of a cell, and its corresponding phenotype, evolves along with the differentiation processes and the development stage of the organ and the organism it belongs to. Because epigenetic changes are believed to allow cell plasticity in response to the environment (toxins, stress, diet, etc.), they have also been considered as a target for dysregulation associated to diverse diseases such as cancer. The first link between epigenetic processes and human disease was established in cancer cells showing less 5-methylcytosine than the corresponding normal cells 17. Increased DNA methylation was observed in the promoters of tumor suppressor genes that appeared to be silenced 18. Epigenetic aberrations, originating from loss or gain of function of epigenetic regulatory proteins, significantly take part in the onset and progression of human cancers.
Since then, there has been a broad enthusiasm around the manipulation of epigenetic enzymes in an attempt to reverse altered processes for cellular reprogramming, as demonstrated by the huge body of data found when entering the key words “epigenetics” and “drug discovery” in search engines with a gradually increase these last 4 years (551 articles on PubMed with 76, 105, 111 and 104 items found from 2014 to 2017, respectively, https://www.ncbi.nlm.nih.gov/pubmed, accessed on October, 25th 2017; 119,000 research articles found on Google Scholar, https://scholar.google.fr/, accessed on
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October, 25th 2017). More than fifty small molecules targeting one of the epigenetic protein families (Fig. 1, Table S1) were under clinical trials in 2017 19. Among them, two families are heavily targeted for hematological malignancies treatment: the histone deacetylases having sixteen compounds under clinical trials and four drugs approved (vorinostat - Zolinza®, Merck, 2006 20; romidepsin - Istodax®, Celgene, 2009 21; belinostat - Beleodaq®, Spectrum Pharmaceuticals, 2014 22; panobinostat - Farydaq®, Novartis, 2015 23) by the Food and Drug Administration (FDA), and the DNA methylases with three candidates under clinical trials and two FDA-approved drugs (azacitidine - Vidaza®, Celgene, 2004 24; decitabine - Dacogen®, MGI Pharma, 2006 25).
The histone deacetylase (HDAC) proteins family
One of the most abundant post-translational modifications in epigenetics, and one of the most studied (after DNA methylation), is histone acetylation 11. This histone mark is balanced by two protein families: the histone acetyl-transferases (HAT, Fig.1) that add acetyl groups to lysine residues on histone amino-terminal regions, and the histone deacetylases that remove these groups. These 2 counterparts were first viewed as direct modulators of the transcription through the chromatin compacting/de-compacting effects driven by the neutralization of the lysine basic charge that the acetyl group produces.
However, this was a simplistic picture, and it’s now widely recognized that a more sophisticated interplay between several epigenetic mechanisms and marks is involved to regulate gene transcription
14, 26, 27. While DNA methylation state can be in some cases inherited and maintained through mitosis and meiosis (contributing to cell memory mechanisms described in the definition of epigenetic regulation), the relevance of histone modifications to cellular memory is uncertain 2.
HDACs’ name originally stems from their discovery in the mid-1990s, being the identified enzymes responsible for histone deacetylation and a contributor to gene regulation 28-30. Actually, it is nowadays more justified to call them lysine deacetylases as this family of enzymes has many more substrates, other than histones, such as transcriptional factors (p53, Rb among more than sixty others), cytoskeleton proteins (α-tubulin, actin, cortactin) and a number of proteins implicated in cell signaling, apoptosis, DNA repair and replication, chaperones, and viral infection 31. The HDAC family comprises eleven + seven members classified in subgroups according to phylogenetics, homology to yeast and co-factors (Fig. 2) 11,32. Class I HDACs (1-3, 8, homologous to yeast Rpd3-like proteins 33, 34), class II HDACs (IIa: 4, 5, 7, and 9, IIb: 6 and 10, homologous to Hda1-like proteins 34) and class IV HDAC (11 is the sole member) contain zinc-dependent catalytic domain(s), whereas the class III HDACs, also called sirtuins (Sirt1-7) are a structurally and mechanistically unrelated group of NAD+-dependent hydrolases. Class I, II and IV hydrolyze the amide bond to release acetate from their substrates through a histidine tandem, one being a single general acid – base catalyst, and the other one remaining protonated along the catalytic cycle to be employed as an electrostatic catalyst. The process is assisted by a water molecule that is bound to the metal to be deprotonated by the neighboring histidine residue , hence activating it for a nucleophilic attack on the substrate carbonyl (Fig. 2) 35.
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Figure 2. HDACs phylogenetic classes, cellular localizations, and catalytic mechanism. (A) Phylogenetic tree of the HDAC family, reproduced from Bradner et al.32 and from Arrowsmith et al.11. (B) Simplified view of HDACs cellular localization 31. HDACs 1-3 are nuclear, whereas HDAC8, class IIa and class IV HDACs are able to shuttle between the cytoplasm and the nucleus.
Conversely, class IIb HDACs are mainly found in the cytoplasm. The figure was built according to Roche and Bertrand 2016 31 and using Servier Medical Art, under the Creative Common License 3.0.
(C) Catalytic scheme of HDACs. The residues numbering corresponds to HDAC8 isoform, as this new mechanism was proposed by Gantt et al. 35. His143 has a dual role: it serves both as a general base to catch a proton from the water molecule that, in turn, attacks the substrate carbonyl. The tetrahedral oxyanion is stabilized by the neighboring Tyr306 and the zinc ion. Finally the substrate catches a proton from His143 (which, this time, plays the role of a general acid) to release the acetate byproduct. Scheme reproduced and adapted from Gantt et al. 35.
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HDAC enzymes share a high degree of homology (sequence identity > 50% within class I isoforms, 22% of sequence similarity between HDAC1 and HDAC6, 33% of sequence similarity between HDAC6 and HDAC11), particularly when considering their catalytic site where only punctual differences characterize them 36-38. Because their cellular localizations and substrates are diverse (Fig.
2), HDAC enzymes have been shown to be involved in diverse diseases. Indeed, this family is implicated in multiple biomolecular pathways and cell signaling at multiple levels. HDACs have roles in brain function regulation and neurological development 15. As an example of their high degree of intricacy, HDAC1 and 2 are important for synaptic transmission in immature neurons, whereas only the altered expression of HDAC2 takes part in pathogenesis in mature neurons 39-41. Additionally, HDACs play a role in both inflammation and viral infection processes, in which class I HDACs (1-3) are in part responsible for the regulation of the innate immunity (cytokines production regulation), whereas class IIa isoforms are hypothesized to be central for adaptive immunity (influences on T-cell functions).
Overlaps between the two immune responses were also reported 42-45. What originally triggered the development of HDACs inhibitors was their large role in cancer onset and progression 14. HDACs function and/or expression is aberrant in a broad variety of cancers, and is often correlated with poor prognosis 46. However, similarly to the other physio-pathological fields in which HDACs are involved, there are paradoxes at the molecular level. The function of HDACs 1-3 remains unclear: although their inhibition demonstrated efficacy in the clinic, they might also have a tumour-suppressor role 14.
Figure 3. The HDAC catalytic inhibitor pharmacophoric scheme. The four FDA-approved compounds are also reported.
Nevertheless, sufficient efficacy profiles in cutaneous T-cell lymphoma, relapsed or refractory peripheral T-cell lymphoma, and multiple myeloma were reached, leading to FDA approval of four HDAC catalytic inhibitors (Fig. 3) 47, 48. Many others are under clinical trials, still for oncology purposes
19. These catalytic inhibitors all follow the same pharmacophoric scheme which is composed of three regions: a zinc-binding group (ZBG, usually a hydroxamic acid) to chelate the catalytic zinc ion located at the bottom of the active site, a surface recognition domain (also called the cap group) that occupies the pocket entrance (or channel rim), and a hydrophobic linker to connect the two and that fits into the tunnel-like catalytic pocket (Fig. 3).
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The FDA-approved compounds are either non-selective (Vorinostat, Belinostat, and Panobinostat) or class I selective (Romidepsin).While effective, they are subject to strong side effects, mainly attributed to class I HDACs catalytic inhibition (cardiac and hematopoietic toxicities, dose -limiting diarrhea and vomiting) 13, 49-52. Moreover, the hydroxamic acid ZBG is subject to rapid hydrolysis, hence weakening the pharmacokinetic profile of these drugs 53, 54. Although HDAC inhibitors represent a hope for patients suffering from leukemia, they are still poorly successful in solid tumours. Research efforts are thus ongoing to build the new generation of HDAC inhibitors. One of the possibilities to do so is reaching isoform selectivity. HDAC enzymes do not operate alone but are very often embedded in multiple subunits complexes. And promising compounds that are found active on one isolated purified isoform are not necessarily active when confronted to the corresponding HDAC-containing complex 55. Thus, unravelling the HDACs levels of complexity starts with in-depth structural knowledge on each isoform, especially regarding their respective flexible behavior. This requires selective molecular probes to study the first molecular level of HDACs, being their catalytic domain.
Indeed, selectively targeting one isoform begins with structurally knowing its adversaries. For this purpose, in the next section, information from class I and IIa HDACs, co-crystallized with various compounds, were gathered and analyzed altogether to highlight the catalytic domains plasticity regarding the isoform and the ligand properties.
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1.2 Structural aspects of HDAC isoforms relevant to ligand binding and selective catalytic