Contexto Histórico

Acetylation is a major post translational modification found in over 1700 proteins within the cell, and at least 3600 acetylation sites in total that are differentially acetylated upon treatment with HDACi². The ubiquity of the 11 Zn²⁺ depedent HDACs across a wide range of cellular pathways and processes demonstrates their importance in life. They perform many more functions than their name suggests.

In addition to deacetylating histone tails, they deacetylate non-histone proteins, both cytosolic and nuclear. Further to their role as catalytic hydrolases HDACs also mediate interactions with other proteins within multiprotein complexes. Often these complexes enable regulation of activity and substrate specificity of a particular HDAC isoform.

A great number of disease states have been linked to changes in regulation of expres-sion (or activity) of particular HDAC isoforms or HDAC binding partner. For this reason HDACs have been major targets for the development of therapeutic treat-ments for decades with four HDACi currently approved for the treatment of certain cancers. The search for effective HDACi therapies has been hampered by the severe side effects caused by HDACi. These side effects are so bad that HDACi are only used in late stage cancers which have resisted previous chemotherapy. These side effects are partly a consequence of the non-isoform selectivity of HDACi meaning that all 11 Zn²⁺ dependent HDACs are targeted in a non-selective manner. The side effects are also partly due to the promiscuous metal binding hydroxamic acid ZBG found in the majority of HDACs which are known to bind other metalloproteins.

To reduce side effects associated with HDACi therapies selective HDACi must be found that can specifically target individual HDAC isoforms, additionally the selec-tive HDACi should also utilise a ZBG that does not bind proteins other than HDACs as the hydroxamic acids that are currently used do.

The search for isoform selective HDACi has predominantly been through modifica-tion of the surface binding cap-group. This is because the internal core of the protein is thought to be fairly invariant between isoforms whereas the surface residues show a great deal less conservation. To some degreee this direction of research has been successful, many class selective inhibitors have been reported and some of these class selective HDAC inhibitors are showing promise in clinical trials.

The significance of HDAC8 within the cell is becoming more apparent as more of its functions are discovered. With roles in development of the skull, p53 expression and chromatid separation, HDAC8 functions both as a deacetylase and as a scaffold, facilitating the co-localisation of other proteins. HDAC8 has been shown to play a significant role in certain types of cancer, specifically in neuroblastoma which accounts for 15% of all pediatric cancer deaths. It has been shown that abolishing HDAC8 activity in these cancers leads to cell cycle arrest, terminal differentiation and apoptosis.

Despite its class I classification HDAC8 is evolutionarily divergent from HDAC1, HDAC2, and HDAC3 and is poorly inhibited by class I specific inhibitors as a

result. It follows that those same features of HDAC8 that make it poorly inhibited by class I inhibitors should be able to be targeted to create highly selective HDAC8 inhibitors.

There is a great deal of structural information about HDAC8 to be gained from the 32 structures deposited in the PDB, ligands are bound in all these structures suggesting that HDAC8 is a flexible and dynamic protein in its ligand-free form.

Certain key residues within the substrate binding tunnel and ligand binding L1 and L2 loops assume multiple conformations depending on the bound ligand, further demonstrating the dynamic nature of the protein which is thought to be significantly greater than other class I HDACs. The residues lining the substrate tunnel are thought to be more rigid in other HDACs and the L2 ligand binding loop is two residues shorter which may alter its dynamic characteristics.

HDACi have been reported which show selectivity, however these selective inhibitors are either relatively low affinity (SB-379278A, 22) or retain the undesirable hydrox-amic acid ZBG (PCI-34051, 1 and cpd6 20). There is a definite need for a potent non-hydroxamic acid HDAC8 selective inhibitor.

An HDAC8 selective inhibitor 4 has been reported which utilises a novel α-amino amide ZBG, showing 18-fold selectivity for HDAC8 over other class I HDACs. A second novel property of this inhibitor compared to other HDAC8 inhibitors is the presence of a group which occupies the acetate release pocket. This inhibitor lacks a cap-group and so its HDAC8 selectivity must arise from the properties of the substrate tunnel and acetate release pocket which provide interactions to this unique inhibitor. Little information exists about α-amino amide HDACi and so it is an ideal lead compound for rational design to improve its potency.

A commonly used flourogenic assay substrate is a suitable for assessment of inhibitor potency against HDAC8. Both the commercially available Fluor-de-Lyssubstrate and the “in-house” synthesised MAL substrate are equally suitable as they both report the relative activity of HDAC8 and show Michaelis-Menten kinteics, this makes for easy calculation of the inhibitor dissociation constants, and therefore good comparisons between inhibitors can be obtained.