Biografía

1.5.1 Cellular assays

The study of HDACs and their inhibition requires tools and techniques to monitor their activity. In a cellular context this usually comes in the form of detection of acetylation epitopes such as histone tails by Western blot. This is useful for investigations on the functions of HDACs when one isoform is knocked down or inhibited. Since over 1700 proteins are known to be deacetylated² detection of acetylation levels is a very rudimentary technique that may not detect a vast number of acetylation sites due to non-uniform affinity of anti-acetyl antibodies to different acetylation sites. Also cellular techniques cannot isolate individual HDAC functions since complex compensatory mechanisms exist and can cause knock-on effects. In an oncology setting the effect of HDACi on cancer cell lines is assessed much the same as other potential anticancer drugs, for example: flow cytometry, colony forming and migration assays.

1.5.2 in vitro Assays

No high affinity substrates (KM >100µM) are currently available for use in in vitro assays, this is primariy because information about the in vivo sequence specificity of HDAC8 is scarce.

Radiolabelling assays are an old technique used to monitor HDAC activity. Histone tails are labelled by chemically peracetylating histone tail peptides with ³H-labelled acetate¹⁸⁷. After purification by high performance liquid chromatography (HPLC) these can be used as an HDAC substrate. Following incubation with HDACs the hydrolysed acetate can be separated from the reaction mixture by extraction into organic solvent and the amount of radioactive acetate quantified through scintilation counting. This technique only quantifies HDAC activity against histone tails and so has very limited scope given the numerous non-histone HDAC substrates, especially when considering evaluation of a specific inhibitor.

Fluorescence based HDAC assays utilising the amino methyl coumarin (AMC) flu-orophore are however the most common technique used to assess HDAC activity and screen for inhibition due to their ease, speed and need for small amounts of enzyme. At the most basic level, the assay substrate MAL (27) consists of an aminomethylcoumarin (AMC) fluorophore conjugated to an acetylated N -Boc-lysine residue. Figure 1.19 shows the steps occurring during the assay, deacetylation of the substrate renders the lysine-AMC peptide bond suceptible to hydrolysis by trypsin which is added as part of a developer solution, cleavage of the AMC fluorophore is detected as an increase in fluoresence at 460 nm¹⁸⁸. Because this is a small, single residue substrate, its KM is very high (at ∼20 mM for HDAC8, see Section 1.6).

In a mass spectrometry based assay, HDAC8 was shown to have activity on histone H4 tail peptides, although only a longer 11 residue sequence from the H4 tail (Lys8-Arg19) produced detectable deacetylation¹⁸⁹. This suggests that distal regions of the substrate contribute to binding of and deacetylation by HDAC8. Similar exper-iments have shown a fragment of the p53 tail (residues 372-389) can be a substrate for HDAC8, albeit a very poor one¹⁹⁰.

O O

NH O HN

HN O O

O

O O

N H O H N

NH₃ O

O O

O H₂N

O H N

NH₃ O

O

OH

HDAC Trypsin

-O O

27

Figure 1.19: Steps occuring the an MAL-HDAC assay, HDAC activity removes an acetyl group from MAL (27) which makes the deacetylated substrate suitable for tryptic hydrolysis releasing the AMC fluorophore in the developer step.

AMC

AMC Tyr100

Asp101

Tyr100 Asp101

Lys-Ac

Lys-Ac

Figure 1.20: HDAC8-Fluor-de-Lys structure with aromatic stacking interactions being shared between two HDAC8 monomers and two Fluor-de-Lys substrate molecules

A further sequence specificity study, showed the preference of HDAC8 substrates to have Arg at the -1 position and Phe at the +1 position¹⁹¹. This preference of HDAC8 substrates to have an aromatic residue at the +1 position is supported by the sequence of an HDAC8 optimised assay substrate (Lys, 2). The Fluor-de-Lys substrate consists of an ArgHisFluor-de-Lys(Ac)Fluor-de-Lys(Ac) tetrapeptide sequence identified from the p53 tail with a C-terminal aromatic AMC reporter (at the +1 position), the inclusion of the extra ArgHisLys(Ac) sequence lowers the KM to 1.5 mM¹⁹² from ∼20 mM for the MAL substrate (see Section 1.6 in results). In the absence of the aromatic AMC fluorophore however, HDAC8 is unable to deacetylate the Fluor-de-Lys substrate¹⁹¹. A structure has been solved of inactive HDAC8 mutants with the Fluor-de-Lys substrate Figure 1.20. In these structures the aromatic AMC fluorophore can be seen to clearly form aromatic ring stacking interactions with Tyr100⁸⁵Figure 1.20. These fluorescent substrates are a useful tool in the laboratory for HDAC activity and inhibition assays but do not provide insight into bonafide in vivo substrates of HDAC8.

The catalytic efficiency (KM/kcat) of HDAC8 is very low (7500 M⁻¹s⁻¹ with the Fluor-de-Lys substrate¹⁰⁹) in comparison to other homologous enzymes such as HDAC1 (which are on the order of 10⁵ M⁻¹s⁻¹ with a Ac-GlyAlaLys-AMC HDAC1 optimised substrate)¹⁹³, and is far from the diffusion limited rate seen in other enzymes of up to 1.5 x 10¹⁰ M⁻¹s⁻¹¹⁹⁴. Binding partners such as scaffolds or co-activators may play a role in increasing binding affinity and positioning of substrates (and therefore reducing their KM and increasing the kcat in vivo).

Development of specific HDAC inhibitors requires a tool to detect activity of single isoforms in isolation. To this end purified individual HDAC isoforms are obtained, either recombinantly expressed or immunoprecipitated from cell cultures and studied in vitro.

Modifications have been made to the fluorescence based HDAC assays to improve affinity¹⁹⁵, to allow detection of inhibition by highly fluorescent inhibitors by derivati-sation of deacetylated product¹⁹⁶ and both improved affinity and derivatisation of products⁹⁵. A continuous protease coupled assay has also been developed that pro-vides a more efficient method for measurement of reaction rates¹⁹⁷. A thioaectylated

lysine peptide has been described as a novel HDAC8 specific assay substrate which quantifies released thioacetate using Elmann’s reagent (5,5’-dithiobis(2-nitrobenzoate))⁹⁵. Some direct detection HDACi-binding assays have used techniques such as fluores-cence resonance energy transfer (FRET) to, or fluoresfluores-cence polarisation/anisotropy of, internal tryptophan residues. These techniques suffer from low signal to noise due to the fact that many inhibitors (eg. Vorinostat, 5 and TSA, 3) do not have fluores-cent groups. Fluoresfluores-cent analogues are therefore required which sometimes quench the internal tryptophan fluorescence¹⁹⁸. Given the known positive effect of coumarin on the binding of the in vitro HDAC substrate Fluor-de-Lys⁸⁴, a fluorescent probe was designed adding an AMC fluorophore to Vorinostat (5). The fluorescence of the probe is quenched when bound to HDAC but is detectable when it is free in solution.

Displacement of the probe by a competitive HDACi can therefore be detected as an increase in fluorescence. This technique also allows the detection of kon and kof f

rates of HDACi.

1.6 Preliminary Assessment of Substrate K