6. Conclusions and Future Work
6.2 Future Work
The most interesting findings of this work establish the ability of piano-stool complexes to be tuned towards selectivity between HDAC enzyme isoforms. While the selectivity factors are by no means exceptional, their novelty provides sufficient reason for continued development and warrants further exploration. Other examples of piano-stool complexes utilised as drug candidates are subjected to extensive scrutiny in the literature - far beyond that tested in this work - to elucidate a more informed account of the complex’s behaviour. It would therefore be logical to undertake additional biological testing accordingly.
Due to the different toxicity profiles exhibited by complex Ru1L2 towards the H460 and MCF7 cell lines, it would be applicable to test the cytotoxicity of the complexes against a broader range of cancer cell lines, including those resistant to cisplatin, in an attempt to observe any specificity. Additionally, healthy cells such as HBL-100 should be tested to determine whether these metal-based HDAC inhibitors show any cancer-cell selectivity. Uptake and localisation studies could be expanded upon to enlighten the different results between the L2 and L3 containing
complexes. Transmission electron microscopy has been previously exploited by Dyson et al. to visualise the localisation of RAPTA complexes at a subcellular level.61 This technique, coupled with nanoscale secondary ion mass spectrometry (NanoSIMS), could discern if off-target localisation of L3 complexes is a source of their poor cytotoxicity, relative to their good Ru uptake. As described by Sadler et al., contrasting cellular uptake pathways could also be investigated. These assays include monitoring the extent of efflux and inhibition of efflux by co-administration of verapamil (an L-type calcium channel blocker enabling efflux evasion by the P-gp pathway – a pathway known to favour cationic and highly lipophilic drug molecules).68 Pursuing such analysis would supply more information with regard to the complex’s behaviour at a cellular level and highlight any influence of lipophilicity associated pathways.
As the molecular target, the HDAC enzyme activity could be further explored by following a variety of avenues. The extent of HDAC6 inhibition could be quantified using Western blotting to measure the acetylation levels of α-tubulin – the primary substrate for HDAC6 – in comparison to a control.142 The protein α-tubulin is linked to the regulation of the cytoskeleton, cell migration and plasticity; all features associated with aberrant HDAC activity and metastasis. Besides localisation, this could provide evidence of HDAC6 inhibition-generated cytotoxicity. Moreover, migration and invasion cellular assays could be undertaken to observe any antimetastasis effects brought about from HDAC6 inhibition. Specific HDAC enzyme interactions could be compared through
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subsequent modelling of both HDAC1 and HDAC6 with complexes Ru1L2 and Ru1L3, to enable a
more succinct comparison of the potential binding modes. This could display particular interactions that give rise to the observed selectivity between the isoforms. As modelling is a binding prediction based on a set of parameters, enzyme-drug crystallisations could also be reattempted to afford a more accurate representation of interactions within the biological system.
Figure 6.2 Alternative R1 substituents.
Future development of the L2 and L3 containing complexes, beyond the investigation into their behaviour in biological systems, would be through structural modifications. Firstly, having now modelled a catalytic domain homologous to HDAC6, discrete surface amino acid residues can be visualised and arene design can be refined to improve interactions with the enzyme surface. The favoured pose of complex Ru1L3, docked in the HDAC6 homologue, displayed a selection of residues in close proximity to the arene motif for such interactions such as Asp460, not reached by the p-cymene of Ru1L3. To tune the arene, specific amino acid side chain groups such as those found in tyrosine, tryptophan or aspartate (Figure 6.2) could be incorporated using their commercially available carboxylic acid derivatives.
Without further biological testing and aside from assumptions based on lipophilicity, it remains unclear as to why the L3 complexes are able to accumulate efficiently in the cell and yet display low levels of cytotoxicity. Both the differences in the size of the arene as well as the amide linker have been suggested as contributing to the complexes inability to reach the target or responsible for off-target binding. Given the amide linker shows no apparent residue interactions with the enzyme surface, this feature of the molecule could be changed. An alkene linker would reduce the number of hydrogen bond donors and acceptors within the arene motif and depending on its substituent, potentially enhance the lipophilicity in a desirable fashion. While the alkene would require more synthetic steps than the amide functionality, exchanging this moiety could prove advantageous. Finally, the HDAC assays discussed in Chapter 4 displayed the ability of the L3 containing
complexes to partially inhibit HDAC4 activity. To improve selectivity towards the class IIa HDAC enzyme, the zinc-binding group could be modified to a trifluoromethylketone, a functional group already noted for possessing a penchant for the HDAC4 active site compared to the hydroxamic
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acid.207 This small structural change could provide the first example of a class IIa selective metal- based HDAC inhibitor and would therefore be worth pursuing.