Investigating calcium channel blockers as antimalarials
The rise in resistance to current antimalarial drugs has led researchers to consider drug repositioning as a quicker alternative for drug development and discovery. Preliminary drug repositioning screens carried out at the University of Salford identified calcium channel blockers (CCBs) as potential antimalarial agents. A growing body of evidence has demonstrated the importance of calcium within the Plasmodium life cycle. Studies have shown CCBs and calmodulin inhibitors to exhibit antimalarial activity. The research carried out in this project aims to evaluate the antimalarial efficacy, safety profile, mode of action and drug interactivity of the commercially available CCB and calmodulin inhibitor fendiline, and a range of its synthetic analogues. Initial screening of fendiline alone and in combination with commercially available drugs was carried out using a SYBR Green (SG) plate reader assay. Both CalcuSyn-based combination studies and a chloroquine potentiation assay were carried out. This was succeeded by the synthesis of fendiline analogues, which were carried out via a two-step synthetic route starting with a palladium catalysed coupling reaction followed by a reductive amination. Both the antimalarial activity and the cytotoxicity of the synthesised compounds were evaluated which led to a lead candidate to be selected (the hydroxy fendiline analogue, 4c). Further investigations into the activity, stage specificity and the effect compound 4c has on the hERG channel was carried out to develop a preliminary understanding of the mode of action of the compound. Finally, optimisation experiments to develop a flow cytometry-based assay that would detect fluctuations in calcium levels within infected red blood cells (RBCs) were performed. The conducted research showed the commercially available fendiline to have activity towards the multi-drug resistant Plasmodium falciparum K1 strain within the micromolar range (IC50 = 3.74 ± 0.64 μM). CalcuSyn-based combinations studies showed fendiline to have either an antagonistic or additive effect with currently available drugs. Interestingly, fendiline was found to reverse chloroquine resistance, similar to verapamil, however at half the concentration required for verapamil. Furthermore, the range of synthesised fendiline analogues identified several compounds that exhibited more activity towards the P. falciparum infected RBCs. The 2’ hydroxyl fendiline analogue (4c) was 5.6-fold more potent than fendiline itself (IC50 = 0.67 ± 0.21 μM) on the P. falciparum K1 strain, with an almost one-hundred-fold difference between antimalarial activity and cytotoxicity. The compound was found to be slow acting that targets the schizont stages of the parasite blood stages. The hERG channel inhibition assay gave an IC50 of 4.03 ± 0.52 μM, which is within the range that most small compounds fall within (1-10 μM). Finally, the optimisation experiments showed the developed method was only sensitive to dramatic calcium changes within RBCs and not within the parasites themselves. Further work is required to improve the sensitivity of the assay. In conclusion, the hydroxy fendiline compound provides an interesting candidate to investigate further as a combinatory partner with other antimalarials, and as a scaffold to synthesise other potentially more potent fendiline analogues.
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