Introduction

Diseases such as malaria, leishmaniasis and trypanosomiasis continue are the cause of suffering for many millions of people living in tropical and subtropical areas of the world. There is an urgent need to identify and evaluate novel chemical scaffolds to seed the drug discovery pipeline for these parasitic diseases to meet the challenges of emerging resistance to available drugs, risks of toxicity and the cost of these treatments. There has been a significant investment in international efforts in the screening of massive small-chemical libraries with several million compounds have been screened in phenotypic assays against malarial parasite, which has resulted in a solid pipeline of novel candidates in clinical and preclinical development (Kaiser et al., 2015; Preston et al., 2016; Burrows et al., 2013). In addition, the Drugs for Neglected Diseases initiative (DNDi) is a patient-needs driven, non- profit drug research and development (R&D) to provide new treatments for neglected diseases, notably leishmaniasis, sleeping sickness (human African trypanosomiasis, HAT) and Chagas’ disease (Don and Ioset, 2013).

In addition to massive libraries of synthetic compounds, activity screening also includes natural products, recognizing that they offer a potential source of new antiparasitic therapies. Natural products are derived from a wide array of organisms such as animals, fungi and the higher plants have been shown to contain secondary metabolites with a variety of underexplored chemical entities and pharmacological activities (Yamthe et al., 2017; Zulfiqar et al., 2017). Generally, natural products are more complex, when compared to

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synthetic molecules with the structures of natural products having greater numbers of carbon, oxygen and hydrogen atoms, as well as a generally high polarity and molecular weight. Recent efforts to elucidate the chemical structure and biological function of the active chemical structure within anti-parasitic natural product extracts have detected molecules with the potential to treat some Neglected Tropical Diseases (Cheuka et al., 2016). For instance, recently, the antiparasitic activity of alkamide (deca-2E,4E-dienoic acid 2- phenylethylamide isolated from Anacyclus pyrethrum roots) has been tested against L.

donovani, T. b. rhodesiense, T. cruzi and P. falciparum with EC50 of between 3 to 5µg/ml

across these diverse species (Althaus et al., 2017). Moreover, the anti-kinetoplastid activity of 472 natural products based library has been screened against L. donovani DD8, T. b. brucei and T. cruzi, and identified several compounds with novel activities (Zulfiqar et al., 2017). The utility of natural products as drugs is well established, and it has been estimated that approximately 50% of current registered drugs are derived from natural products or developed on the basis of natural compounds; such as camptothecin, lovastatin, maytansine, paclitaxel, reserpine and silibinin (Harvey et al., 2015; Pérez-Moreno et al. 2016; Ruiz- Torres et al., 2017). This is also perhaps best exemplified in malaria treatment, with artemisinin and quinine both good examples of drugs developed from a natural product (Ginsburg and Deharo, 2011). There are large numbers of studies that report phenotypic screens of antiparasitic activity from plant extracts chosen on the basis of ethnopharmacology reviews of the use of traditional medicines (Simoben et al., 2018; Zulfiqar et al., 2017; Pérez-Moreno et al., 2016; Harvey et al., 2015; Ibrahim et al., 2014). Although from this starting point, unless there is ethnopharmacological evidence of the use of a traditional medicine for the treatment of a parasitic disease (or the symptoms of that disease, e.g. fever), many other natural products produced by plants, marine invertebrates and fungi etc will not have been evaluated for their antiparasitic activity (Yang et al., 2011;

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Davis et al., 2011; Choomuenwai et al., 2015). Going some way to address this issue has been the creation of natural product libraries – a means to exploit the success of high throughput screening of synthetic compound libraries. A library of 96 compounds and 120 extracts from traditional Chinese medicines have been screened against P. falciparum (Nonaka et al., 2018), with the identification of new antiplasmodial activity in two medicinal plants. In Spain, a natural product extract collection (MEDINA) comprising 130000 extracts from soil bacteria and fungi has been prepared and a subset of 20000 extracts screened against P. falciparum resulting in the discovery of three new antiplasmodial compounds, albeit with moderate µM activities (Pérez-Moreno et al., 2016). Using the same MEDINA library, a second study screened a second subset of 5976 against the kinetoplastids T.cruzi,

L. donovani and T. brucei brucei, with 48 fractions selected for follow up studies (Annang et al., 2014). The Davis open access natural product-based library contains 472 compounds,

the majority of which are natural products that have been obtained from a diverse range of Australian natural sources. A similar kinetoplast screen to that described above for the MEDINA library, identified a single compound, lissoclinotoxin E, with low µM activity against all three parasites, although this compound showed low selectivity against T. brucei

brucei. Whilst these natural product screens have yet to provide a lead for development, they

do illustrate well how natural product libraries can facilitate a more efficient throughput in screening multiple parasites – a process that may lead to a scaffold that could be amenable to medicinal chemistry.

In this chapter, I report a similar multiple parasite screen of a proprietary library of purified natural products, the Phytopure library. PhytoQuest, a UK small to medium enterprise SME, has produced a library of approximately 1000 Molecules, isolated predominantly from temperate zone plants, this resource developed from work of the founder Professor Nash at the Royal Botanical Gardens and Institute of Grasslands and Environment

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(http://www.phytoquest.co.uk/). As such, the source plants are unlikely to be known in any literature of traditional medicines for parasitic tropical diseases given they are from temperate zone plants. The library encompasses a wide range of chemical classes, two thirds of which are novel, and the remaining third not commercially available. Critically, the library consists of isolated compounds, overcoming common issues with screening fractions of complex mixes where the active moiety is unknown and may be acting in synergy/antagonism with other unknown compounds. This library therefore represents a unique resource for lead discovery of high value chemicals from temperate zone plants against antimicrobial pathogens.

In this regard, 643 compounds within Phytopure library have been selected and provided to Keele University as part of a BBSRC High Value Chemicals from Plants initiative for screening against human parasitic diseases. These compounds have also been selected on the basis of their development potential: they have a high degree of functionality and physiochemical properties that meet Lipinski’s rules-of-five. The activity of these compounds were screened against intraerytrocytic Plasmodium falciparum, the blood- stream form of Trypanosoma brucei brucei and axenic Leishmania mexicana. The key aims of this work were to;

1. Screen and compare the growth inhibitory activity of the Phytopure library compounds against P. falciparum, T. brucei and L. mexicana.

2. Identify priorities for determination of their EC50

3. Provide initial toxicity data by determination of the compounds selectivity when compared to HepG2 cell lines, and the THP-1 cell line where appropriate.

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