Out of next most prevalent categories namely: the antipsychotics (15%), the immune suppressants (5%) and the antibiotics (9%) the former was not taken forward due concerns about adverse effects. The latter two classes have already been widely reported in the literature for consideration and use in repositioning therapy for malaria respectively. Immunosuppressant compounds such as Cyclosporin A and rapamycin in particular have been studied extensively for their antimalarial potential (Bell et al., 1994; Bobbala et al., 2008). Unfortunately, their immunosuppressive properties are suggested to prevent repositioning of these compounds for malaria (Lotharius et al., 2014). Numerous antibiotics such as the tetracyclines and clindamycin, a licostamide antibiotic, have been used in combination therapy for malaria (Andrews et al., 2014), however, there are growing concerns that further exploitation of this important class of compounds will only accelerate the dwindling efficacy for its primary antimicrobial indication.
Of the remaining categories the interesting groups that offered potentially novel antimalarial scaffolds were analysed for tanimoto similarity with existing antimalarials and other group members. The three remaining LOPAC compounds: Emetine, SKF and SUH aligned well with the selected categories: namely, antiamoebic (antiparasitic), histamine receptor antagonist and serotonin receptor antagonist respectively and were therefore included for structural similarity analysis.
81 4.4.3 Tanimoto similarity of hit compounds
Various studies have employed similarity indexes to determine whether the chemical space occupied by novel antimalarial scaffold overlaps with the existing pharmacotherapies for malaria. In addition, structural similarity between novel chemical scaffolds can help inform lead optimisation of candidates. Compounds with differential potencies within the same cluster can be used to identify, and potentially modify, the structural components responsible for both off-target and on-target effects (Guiguemde et al., 2010 Plouffe et al., 2008).
In the current study the Tanimoto similarity index was used, firstly, to compare each MeSH category with a range of existing antimalarials, and secondly, to analyse the similarity of the compounds grouped within the same MeSH category. Compounds that were singly responsible for the identification of a particular MeSH category were grouped into ‘other’ and analysed for their similarity with known antimalarials only. Encouragingly, none of the novel scaffolds displayed a high degree of similarity with existing antimalarials (defined as > 0.85 (Guiguemde et al., 2010 Plouffe et al., 2008)). Although data was further validated through the distinct cluster formed by the quinoline antimalarials their relative similarity coefficients were not particularly high. This is somewhat expected as structural alterations have been made to reduce the non-target effects of quinine, the parent compound, to make the synthetic derivatives such as chloroquine and mefloquine more tolerable.
Overall, the similarity clustering between compounds within each MeSH category was not that informative, possibly due to the diversity of the library and the particularly small compound set analysed (n ~55). The only MeSH category to report a high level of similarity between the compounds was the calcium channel blockers, niguldipine and nicardipine. HCL (0.896). The similarity of the other compound clusters was clearly affected by the specificity of the MeSH category. For example, the serotonin and histamine receptor antagonist classifications contained different families of antagonist that target different receptor types (H1 and H2 or 5-HT1, 5-HT2 and 5-HT3 respectively). It is therefore perhaps not unexpected that compounds in these broad spectrum categories did not present a high degree of similarity to each other. Furthermore, although structure is related to activity, the reverse is not always so easily applicable. Indeed compounds that have the same function (activity) may bind to and inhibit different targets within a pathway for which the appropriate structure may be completely different (Plouffe et al., 2008).
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Indeed the angiotensin inhibitor telmisartan and spironolactone are known to act on the same pathway but are dissimilar in structure. Whereas, the high similarity of the calcium channel blockers would predict that they act on the same target.
In order to further refine the hit list for second-phase validation through dose response analysis, the most potent ENZO compounds from each category (propafenone, clemastine and telmisartan) were taken forward. Along with the three remaining LOPAC compounds that showed potential as ACT candidates. For the calcium channel blockers both compounds (nicardipine. HCL and niguldipine) were considered due to their high degree of structural similarity and the possibility of identifying structural activity relations. Although the preliminary screen indicated only a slight variation in potency, second-phase analysis may reveal a more profound difference. For the serotonin receptor scaffold, all compounds (fluoxetine, ondansetron and ketanserin) were taken forward for further validation. This cluster not only represented a relatively large proportion of hits (7%), but members of the class have previously been proposed as novel antimalarial candidates (Gamo et al., 2010). The dissimilarity between the compounds and the lack of a huge variation in potency precluded the selection of fewer representatives (56-73% inhibition). From the ‘other’ category the most potent compound a noradrenaline uptake inhibitor, nisoxetine. HCL, was also considered.
Once hit compounds have been validated, a more informative structural similarity analysis of the whole library would permit comparisons between active and inactive compounds. One foreseeable problem is that the relatively small library boasts a high level of diversity which may ultimately limit cluster size. However, the growing amount available data in the public domain, on preliminary screens, could be used for a larger scale in silico
analysis of compounds designated to promising MeSH categories (Guiguemde et al., 2012; Spangenberg et al., 2013). This would not only to increase cluster size, but also help build structural activity relations and instruct structural lead optimisation.
4.4.4 Secondary phase screening selected compounds
The second phase-screen of the ENZO library was in agreement with the preliminary screen. All of the selected compounds were validated as malarial inhibitors. The most potent compounds were the anti-histamine receptor antagonist, clemastine, and the anti- arrhythmic compound propafenone, both with estimated IC50‘s between 0.12-0.5µM (Table 4.2). This is consistent with the literature, as compounds from these classes have
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previously been identified as submicromolar inhibitors of malaria (Chong et al., 2006; Derbyshire et al., 2012; Plouffe et al., 2008; Weisman et al., 2006). For the other categories: namely, angiotensin receptor antagonist, calcium channel blockers, noradrenaline uptake inhibitors and serotonin receptor antagonist the IC50 estimates were between 1-10 µM (Table 4.2). In addition to the erythrocytic stage antimalarial activity presented here, Derbyshire and colleagues have shown that telmisartan is also inhibitive to liver stage parasites (Derbyshire et al., 2012). Unfortunately, the calcium channel blockers failed to show a distinct variation in potency hence would not be suitable for SAR investigations. The serotonin receptor antagonist, despite being the one of the most common classes, (although structurally diverse) were the least potent compounds in the second-phase screen. While these candidates are not ideal in their current formulations, they should be considered as novel antimalarial scaffolds for lead optimisation. To fit the criteria of an antimalarial candidate, compounds should display potent antimalarial activity, be effective against drug resistant strains, free of significant side effects and have good pharmacokinetic properties (Rottmann et al., 2010). Indeed, these candidate are known to be bioactive, have been screened against a multidrug resistant strain and fall within the criteria outlined by MMV that suggest and IC50 of < 1 µM as a good starting point for lead optimisation (Lotharius et al., 2014).
The wider dose range selected for the second-phase screen of the 3 selected LOPAC compounds permitted a clearer estimation of the IC50 against the multiple drug resistant strain K1. Despite the finding that the IC50 fell within the < 1 µM range, two of the compounds the histamine receptor antagonist SKF 95282 dimaleate, and the serotonin receptor antagonist S (-)-UH-301 hydrochloride displayed a large shift when compared with the IC50 obtained for 3D7 strain. Although multiple replicas were completed for SKF 95282 dimaleate no growth inhibition was observed at 200 nM, 200 x the previous reported IC50 value (1 nM) for 3D7 (Lucumi et al., 2010). For S (-)-UH-301 hydrochloride Lucumi et al., (2010) reported an IC50 value of 5 nM against 3D7, here we show that this level of inhibition is only achieved at a concentration of ~ 750 nM against K1 (150 x). Although Lucumi et al. (2010) used luciferase-based assay and the current study adopted SYBR green-based flow cytometry, the IC50 values determined for existing antimalarials were comparable between the two studies. Furthermore, as little as a 10 fold increase in IC50 value is often reported as clinically significant resistance (Gelb, 2007). Thus the 200 x and 150 x shift significantly limits these compounds for further
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antimalarial development. The most potent of the LOPAC compounds was Emetine dihydrochloride hydrate. Despite also showing an increase in the IC50, the potency against a multidrug resistant strain was well within guidelines of a good antimalarial candidate and even conformed to the more stringent EC50 < 100 nM potency cut-off outlined by Biamonte et al. (2013).
It was not within the remit of this study to investigate every hit obtained. Therefore based on potency, three compounds from the ENZO and LOPAC libraries were taken forward for the next phase of interrogation (accurate IC50 determination using the SG-FCM method). These included clemastine propafenone and emetine dihydrochloride hydrate. One compound from the serotonin class (ondansetron) was also progressed, not because of its potency against the malaria parasite, but for a secondary function as potential anti- emetic for combination with emetine. As its name suggested emetine induces strong emetic effects which would evidently cause serious compliance issues if it was deployed for the treatment of malaria. Combination with an anti-emetic may help nullify the adverse effects while bestowing the additional benefits of a combinatory regime, such drug synergy and a prolongation of resistance development.
4.4.5 Accurate IC50 determination of selected compounds
The IC50 obtained for 3 out of the 4 selected compounds was within the expected range, as predicted by the second-phase screens. Representatives of two classes of ENZO compounds, the anti-histamines and the anti-arryhtmics, have previously been reported to inhibit P.falciparum. The data presented here therefore further confirms their utility as antimalarial candidates. Clemastine and propafenone achieved an IC50 of 427 nM and 475 nM respectively, therefore further lead optimisation will be required. As Guiguemde et al., (2010) emphasised, it is not claimed that hit compounds identified from such phenotypic screens are clinical candidates in their current formulation but they are reasonable starting points.