DOCUMENTO DE REFERENCIA.

Efforts to advance from in vitro antimicrobial screening towards in vivo screening in mammalian models can be expedited and de-risked by first attempting to understanding the in vitro toxicity of the compounds to mammalian cell lines, and lower metazoan animals such as insects.

3.5.1 Insect model

To assess the general toxicity of class Ia flexicates towards a metazoan animal, a straightforward toxicity assay was employed by Dr Nicholas Waterfield and co- workers (Microbiology and Infection Division, University of Warwick) using

Galleria mellonella larvae: commonly called “wax worms”, which have been shown to be a useful model organism for in vivo toxicology and pathogenicity testing, making them an alternative to small mammals in early experiments.50, 51

Figure 3.8: G. mellonella larvae injected with solutions of various flexicates, to a bodily concentration of roughly 32 µg/ml, and incubated for five days. The control (PBS only injection) is shown in the centre.

Solutions of leading class Ia flexicates, [Fe2La-b3]Cl4 enantiomers, in PBS (10 µl)

were carefully injected into the open blood system of cohorts of G. mellonella

larvae. Larval masses varied slightly but were typically 250 mg, a value that was subsequently used to calculate treatment doses, to give a bodily concentration of 32- 64 µg/ml. After the initial injection, larvae (ten per condition), including negative controls injected with PBS only, were incubated over five days while being examined and scored daily, for survival. Figure 3.8 shows several example plates of

G. mellonella larvae at the endpoint of the experiment. It was found that the injection of the flexicates had no significant effect on larval survival. It would appear from this initial toxicity screen that the class Ia flexicates are not overtly toxic to multicellular eukaryotes, even at this relatively high dosage. This is in agreement with the previously observed low toxicity (LC50 > 400 µg/ml) of the class prototype,

3.5.2 Human cell line model

In order to investigate the cytotoxicity of the class Ia flexicates to mammalian cells, their ability to kill or otherwise damage immortalised human cells in vitro. Thiswas tested by determining the half-maximal lethal concentrations (IC50): defined as the

concentration of a therapeutic agent at which half of the maximal inhibition (or death) occurs, relative to an untreated control. This was conducted by Samantha Shepherd in the Phillips laboratory (University of Huddersfield) using a colorimetric MTT assay, which measures metabolic activity of a cell line via the reduction of the yellow tetrazole, MTT, to purple formazan; a process which can only occur if the cells present are alive and metabolically active. IC50 values are quoted for a specific

incubation time with the compound, since cells must be harvested in order to recover and measure formazan levels.

IC50 values (96 h incubation) were determined for all water-soluble class Ia

flexicates against ARPE-19 (ATCC CRL-2302), a non-cancerous retinal cell line. The values obtained are tabulated in Table 3.5. A rough measure of selectivity of the complexes for E. coli over non-cancerous cells, calculated as the molar ratio between the APRE-19 IC50 and the MIC against E. coli TOP10, is also included.

The IC50:MIC ratio provides an ad hoc index for prokaryote selectivity (therapeutic

index), however since IC50 and MIC values differ in how they are defined and

measured, it makes it somewhat difficult to draw firm conclusions regarding the potential for clinical applicability of a test compound using this index. Nevertheless, this metric is useful in comparing the relative prokaryote selectivity within compound libraries. For class Ia flexicates, it is obvious that xylyl-bridged compounds, [Fe2La-b3]Cl4 enantiomers, are much more selective for the prokaryote

organism (E. coli TOP10) over human APRE-19 cells, than other compounds of the class, reflective of their relatively low E. coli MICs and high ARPE-19 IC50 values.

This is again in line with the different behaviour observed of these lead compounds in the above in vitro assays, compared with other class Ia flexicates.

IC50 after 96 h (µM) Prokaryote Selectivity

Compound ARPE-19 𝐀𝐑𝐏𝐄𝟏𝟗𝐈𝐂𝟓𝟎 𝑬.𝒄𝒐𝒍𝒊 𝐓𝐎𝐏𝟏𝟎 𝐌𝐈𝐂 ΛFe-[Fe2La3]Cl4 13.90 ± 1.14 6.95 ΔFe-[Fe2La3]Cl4 18.69 ± 2.09 4.79 ΛFe-[Fe2Lb3]Cl4 6.39 ± 2.36 6.39 ΔFe-[Fe2Lb3]Cl4 22.44 ± 2.56 11.22 ΛFe-[Fe2Ld3]Cl4 0.17 ± 0.07 <0.01 ΔFe-[Fe2Ld3]Cl4 0.29 ± 0.12 0.01 ΛFe-[Fe2Le3]Cl4 0.89 ± 0.46 0.13 ΔFe-[Fe2Le3]Cl4 2.37 ± 0.46 0.18 ΛFe-[Fe2Lf3]Cl4 0.29 ± 0.12 0.01 ΔFe-[Fe2Lf3]Cl4 1.14 ± 0.31 0.17 ΛFe-[Fe2Lg3]Cl4 0.67 ± 0.25 0.03 ΔFe-[Fe2Lg3]Cl4 1.07 ± 0.32 0.08 ΛFe-[Fe2Lh3]Cl4 2.26 ± 1.07 0.09 ΔFe-[Fe2Lh3]Cl4 6.84 ± 1.57 0.26 ΛFe-[Fe2Li3]Cl4 1.98 ± 0.45 0.3 ΔFe-[Fe2Li3]Cl4 1.94 ± 0.56 0.29

Table 3.5: In vitro human cell line inhibition (IC50 values) and relative prokaryote selectivity of class Ia flexicates using non-cancerous ARPE-19 cells. Relative selectivity is given by the ratio of ARPE-19 IC50 to the E. coli TOP10 MIC in µM.

3.6 Summary

The work described in this chapter has demonstrated the range of in vitro biological activities displayed by class Ia flexicates, allowing the identification of ΛFe-

[Fe2Lb3]Cl4 as the lead compound (MIC vs.E. coli O157:H7 of 2 µg/ml, 1 µM), and

made progress towards understanding the underlying MOA. In general, the consistent potent activity of shorter xylyl-bridged flexicates, against Gram-negative Enterobacteriaceae (including against a panel of clinically relevant pathogenic examples), coupled with their relative selectivity for these species over Gram- positive S. aureus, Human ARPE-19 cells, and G. mellonella larvae, make these compounds strong candidates for further research and development in the field of antimicrobial chemotherapy.

Despite their activity against the Enterobacteriaceae, the lead compounds exhibited far less activity against certain other Gram-negative bacteria tested, namely the Pseudomonadales and a single carbapenem-resistant K. pneumoniae strain, KP02. This difference would appear to correlate with differences in the cell membrane composition and permeability (see section 3.4.1) suggesting the outer cell membrane is a key site of interaction for the lead class Ia flexicates, if not the major target site itself.

The MOA of the class Ia library is bacteriocidal in nature, and therefore concerns processes within the microbial cell that are crucial not only for growth, but for survival. However, the difference in cell death between cells that are actively dividing, and those in stationary phase, would appear to demonstrate a much stronger effect upon the actively replicating enterobacterial cell. It would appear that this MOA is specific in nature, since a range of activities is observed across both

complexes and species. Furthermore, despite the hypothesis of the MOA relating to a critical interaction between the lead class Ia flexicates and the enterobacterial cell membrane, it would appear that the MOA is not cytolytic in nature, at least when cells are in stationary phase.

As well as progression towards in vivo study (protection assays in mammalian models), future work on the antimicrobial activity of class Ia flexicates will inevitably involve further cycles of synthesis and screening to refine their potency and selectivity. However, the exact molecular interactions that give rise to the observed antimicrobial activity of the lead compound remain unknown; without this information, improvements to the activity of the lead compound will be somewhat serendipitous and therefore inefficient. Research towards deconvoluting the molecular targets, i.e. the mechanism of action, is the focus of the next chapter.