Cell Viability Tests

Cell viability tests or viability assays are studies which determine the ability of cells to maintain or recover their viability.^[291] The cultured cells can be used to determine the cytotoxicity of chemical compounds and for drug screening. The synthesised ligands and also the final multifunctional tools should be at least suitable for in vivo studies or in best case used as drugs.

Thus, the toxicity of the compounds had to be determined. The utilised test was a colorimetric MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test for cell viability based on the activity of the mitochondrion of living cells. Cell viability was determined by a mitochondria enzyme dependent reaction of MTT as described elsewhere.^[292] MTT (a yellow tetrazole) is reduced by the mitochondrial reductase to a purple farmazan (Scheme 22). The mitochondria play an important role in the respiration cycle of living cells and thus their activity can be used as a measure of enzymatic activity and cell viability, respectively. The colour change, induced by the metabolised MTT, can be measured by UV/Vis-spectroscopy. The purple farmazan has an absorption maximum at 570 nm, which can be shifted by solvatochromism

6.5 Cell Viability Tests

75 (500-600 nm). MTT was added to the neuronal cells (human tumor cells SH-SY5Y) at final concentration of 5 mg/ml at 37 °C for 4 h, 24 h after testing the compound. Undifferentiated human neuroblastoma SH-SY5Y cells were grown under standard culture conditions in an incubator containing a humidified atmosphere with 5 % CO2 at 37 °C. The medium used was DMEM (Dubelcoo’s Modified Eagle’s Medium) supplemented with 15 % foetal bovine serum (FBS). The reaction was terminated by removal of the supernatant and addition of 1.5 ml DMSO to dissolve the formazan product and afterwards, the absorbance was measured. The experiment was performed three times with three batches per measurement. The error margin for cell studies like the presented MTT assay can be up to 20 % and in this study only a low number of batches were conducted. Thus, the cell studies should be reconsidered as a first indication of the reactivity of the compounds with respect to cellular conditions. The Cell studies were performed by Isabelle Sasaki in the laboratories of the Institut de Pharmacologie et de Biologie Structurale (IPBS) in Toulouse, France.

Scheme 22 Reduction of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mitochondria.

The toxicity of the ligands L^3Me, L^5Me and L^8H (L^8H was used due to the better solubility than L^8Me) were determined by the MTT viability assay (Figure 36). L^3Me and L^5Me show no toxic behaviour.

Mainly all cells survived in the case of L^3Me and L^5Me. Unfortunately, the ligand L^8H, which has the highest affinity towards Cu⁺ and also shows the highest activity in the inhibition of ascorbate consumption, seems to by cytotoxic. The reason for the toxic behaviour of L^8H is not known and can only be speculated. The comparison with L^3Me and L^5Me leads to the hypothesis that the specific toxic behaviour could be induced by the alcohol function of L^8H. After coupling with the benzothiazole, the alcohol function would be protected and probably the generated multifunctional compound would show no cytotoxicity. Since no measurements were available to follow the pathway of the ligand, the cytotoxic mode of action remains unknown. One hypothesis is that since the ligand L^8H is relative nonpolar and highly lipophilic, it should be able to penetrate the cells and react with proteins or enzymes in the cell. Such reactivity could disturb the natural respiration of the cell and could than lead to cell death. Another possibility for the low survival rate is an interaction of L^8H with the FBS and a disturbance of the sensitive cell environment.

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By modification of the experiment it could be used to measure the protective function against metal induced ROS. The ROS can be artificially generated by adding Cu²⁺ and ascorbate to the cell medium (Figure 37). In this experiment only the ligands were examined, which have shown no toxic behaviour. Thus, the experiment was only performed with L^3Me and L^5Me. The first, black bar is the average survival rate of cells treated with ascorbate and copper, and serves as reference. The results show for both chelators a slight protective function. This result directly corresponds with the determined stability constants and also with the performed ascorbate consumption experiment. Thus, L^3Me and L^5Me seem to be able, even though only weakly, to stabilise Cu⁺ in aqueous solution. Thus, with this study a first impression is given for the metal-induced ROS protective function of the tridentate ligand systems.

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Figure 37 Modified viability assays to with L^3Me and L^5Me. Percentage of living cells after the treatment with 5 uM

Cu⁺, 100 uM ascorbate and 10 uM of the compounds.

6.6 Conclusion

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6.6 Conclusion

For L^3H, L^5H, L^8H and their methyl protected derivatives were brutto stability constants determined. The values indicated that all ligands should be able to compete with Aβ1-16. However, in the performed ¹H-NMR metal exchange experiment only L^8H was able to extract the copper from the Cu⁺(Aβ1-16) complex. The tripodal ligand system can interact with the metallo protein complex, but their affinity is too low to displace the Aβ1-16. The ligands and their derivatives were further investigated with respect to their redox-inhibition capacity in an metal mediated ascorbate consumption experiment. Out of the series only L^8H is able to slow down the ascorbate consumption more than the Aβ1-16. For L^3H and L^5H only a minor deceleration of the ascorbate consumption could be observed. Since the final aim is an in vivo application, the ligand systems were tested of their cytotoxicity. The performed cell viability test revealed that L^3Me and L^5Me are not toxic, whereas L^8H shows a toxic behaviour. The specific toxic mechanisms are not known, but it is possible that the free alcohol function is responsible for this reactivity and therefore it could be possible that the final molecule shows a different behaviour. However, the obtained results in the ascorbate consumption experiment and the determined stability constants are very promising. All three ligands were therefore introduced in a multifunctional tool.

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Chapter 7

Synthesis and Characterisation of Multifunctional Tools

for Applications in AD

7.1 Introduction

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7.1 Introduction

Since the discovery of Aβ plaques as one of the main features of AD, several dyes have been developed for Aβ plaques to ensure an early AD-therapy. Small fluorescent molecules like Congo red, Thioflavin or Stilbene and their derivatives become of great interest in this context, since they can intercalate in the β-sheet structure of the Aβ plaques and allow the use of tomographic techniques (PET or SPECT).[56,64,293]

One of the best known representatives of these Aß dyes is Thioflavin T, which stands out due to its spectral alteration upon binding to Aβ fibrils. Free ThT absorbs at 342 nm and has a characteristic fluorescence excitation at 430 nm. Binding to the fibrils induces a red shift of the absorbance to 442 nm and also a shift of the fluorescence excitation to 482 nm.^[62,294] One further developed ThT derivative is the Pittsburgh Compound B (PIB), where the benzothiazole moiety is slightly modified (Figure 38). However, this small variation remarkably increases the binding affinity to Aβ plaques and provides other advantages, such as a short clearance time from healthy brain tissues.^[57,58] Responsible for the different affinity and improved intercalation is most likely the combination of aromatic π-π-interactions and hydrogen bridges between PIB and the fibrils.

Figure 38 Hydrophobic and hydrophilic sides of PIB

AD is a very complex disease with various hallmarks like miss-regulation and dyshomeostasis of transition metals, formation of Aβ plaques, hyperphosphorylation of the tau protein. Classical therapeutic strategies which target only one of the hallmarks have shown low efficiency.^[217]

Certain symptoms can be treated with these drugs, but not the cause of AD. Thus, the trend goes to drugs, which do not target the symptoms but the cause itself.^[222,227] In order to provide these therapeutic strategies, a more profound understanding of the disease is necessary. To enlighten the specific mechanisms involved in the AD, molecules become favoured that comprising more than one active functional group.^[295] Thus, the compounds can modulate multiple targets that characterise the neurodegenerative disease. In the last decade multifunctional systems of different types were synthesised and evaluated for their efficiency in the treatment of AD (Figure 39). For the first type a proven active molecule gets an additional functionalisation to increase its activity or introduce a new beneficial property (e.g. solubility increase or crossing the BBB). The second type consists of single molecules which combine multiple functions, to allow interaction with more than one target. The third and last type are compounds with two molecules or functional groups with intrinsic activity, which are bridged by a linker, resulting in hybrid molecules. Depending on the nature of the spacer both active sites can interact or react completely independently.

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Figure 39 General representation of different multifunction types developed in the last decade. a) functionalisation, b) incorporation and c) coupling over an spacer.

The second type of multifunctional systems has high potential for applications due to its ability to “kill two birds with one stone”.^[295] Therefore, they were announced as most promising compounds in comparison with the other types.^[295] Their advantage to interact with multiple targets, is a major disadvantage in medical applications, since the observed activity cannot be assigned to a certain functional group or target.

Aim of this study is the synthesis of a multifunctional tool which can enlighten the interaction of Cu⁺ with Aβ. For this task, a multifunctional type three system was developed, containing a Cu⁺ chelator and an Aβ plaque sensitive unit. The synthesis and characterisation of suitable chelators for Cu⁺ recognition was reported in the previous chapters. Due to the promising results for L^3H, L^5H and L^8H, these systems will be used in the multifunctional tools. In this chapter, the synthesis of the Aβ recognition subunit ─ a benzothiazole analogue of PIB ─ and coupling attempts with the ligands will be presented. Coupling of these two molecules via a linker, should form a hybrid with the following attributes: i) selective coordination of Cu⁺, ii) selectivity towards Aβ plaques and iii) luminescence properties.