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A Biosynthetic Platform for Antimalarial Drug Discovery bioRxiv preprint doi: https://doi.org/10.1101/814715; this version posted October 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A biosynthetic platform for antimalarial drug discovery 2 Mark D. Wilkinson1, Hung-En Lai2, Paul S. Freemont2 and Jake Baum3 3 1Department of Chemistry, Imperial College London, UK 4 2Department of Infectious Diseases, Faculty of Medicine, Imperial College London, UK 5 3Department of Life Sciences, Imperial College London, UK 6 7 Correspondence to: 8 Jake Baum ([email protected]), Department of Life Sciences, Imperial College 9 London, Exhibition Road, South Kensington, London SW7 2AZ, United Kingdom 10 Paul Freemont ([email protected]), Department of Infectious Diseases, Imperial 11 College London, Exhibition Road, South Kensington, London SW7 2AZ, United Kingdom bioRxiv preprint doi: https://doi.org/10.1101/814715; this version posted October 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 12 ABSTRACT 13 Advances in synthetic biology have enabled production of a variety of compounds using 14 bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring 15 indole pigment with antibiotic properties, can be biosynthetically engineered in Escherichia 16 coli expressing its non-native synthesis pathway. To explore whether this synthetic 17 biosynthesis platform could be used for drug discovery, here we have screened bacterially- 18 derived violacein against the main causative agent of human malaria, Plasmodium 19 falciparum. We show the antiparasitic activity of bacterially-derived violacein against the P. 20 falciparum 3D7 laboratory reference strain as well as drug-sensitive and resistant patient 21 isolates, confirming the potential utility of this drug as an antimalarial. We then screen a 22 biosynthetic series of violacein derivatives against P. falciparum growth. The demonstrated 23 varied activity of each derivative against asexual parasite growth points to potential for 24 further development of violacein as an antimalarial. Towards defining its mode of action, we 25 show that biosynthetic violacein affects the parasite actin cytoskeleton, resulting in an 26 accumulation of actin signal that is independent of actin polymerization. This activity points 27 to a target that modulates actin behaviour in the cell either in terms of its regulation or its 28 folding. More broadly, our data show that bacterial synthetic biosynthesis is a suitable 29 platform for antimalarial drug discovery with potential applications in high-throughput and 30 cost-effective drug screening with otherwise chemically-intractable natural products. 31 32 KEYWORDS 33 Violacein; drug discovery; synthetic biology; antimalarial 34 bioRxiv preprint doi: https://doi.org/10.1101/814715; this version posted October 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 35 INTRODUCTION 36 Malaria has a huge global health burden, with around half of the world’s population at risk 37 of contracting the disease which killed over 400 000 people in 20171. Malaria disease is 38 caused by apicomplexan parasites from the genus Plasmodium, with Plasmodium 39 falciparum causing the majority of deaths worldwide. The symptoms of malaria disease 40 develop during the asexual stages of the parasite life cycle, which occurs in the blood 41 stream. Here, the parasite undergoes multiple rounds of growth, replication and invasion of 42 red blood cells. Various drugs have been developed to target the asexual stages of the 43 parasite but, inevitably, resistance has evolved to every major front-line therapy for malaria 44 treatment including, most recently, artemisinin combined therapies (ACTs)2. Multi-drug 45 resistance to ACTs, focussed in the Greater Mekong Subregion of South East Asia, has been 46 reported both as delayed parasite clearance and, more worryingly, treatment failure3. The 47 challenges of emerging drug resistance combined with the cost associated with 48 development of new drugs make it essential to explore new ways to develop novel 49 antimalarial compounds. 50 51 Previous work identified violacein, a violet indolocarbazole pigment produced by bacteria 52 (Fig. 1a), as a potential antimalarial able to kill both asexual P. falciparum parasites in vitro 53 and protect against malaria infection in a mouse malaria model in vivo4–6. Violacein’s 54 antimalarial activity has therefore identified it as a potential for future drug development. 55 However, commercial violacein samples can only be obtained through laborious purification 56 from bacteria (Chromobacterium7,8 or Janthinobacterium9) because of the complexity of its 57 highly aromatic structure (Fig. 1a). Purification from these bacteria requires specialised 58 equipment and high-level biosafety equipment since these bacteria themselves can cause 59 deadly infections10. As such, commercially available violacein is extremely expensive. 60 Alternative strategies of violacein synthesis are being explored, in particular the use of 61 synthetic biology to engineer industrial bacterial species that can express non-native 62 violacein. Several groups including ours11 have been successful in implementing a five-gene 63 violacein biosynthetic pathway (vioABCDE) into Escherichia coli or other heterologous 64 hosts12–14, providing a route for robust, in-house and inexpensive compound production. 65 bioRxiv preprint doi: https://doi.org/10.1101/814715; this version posted October 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 66 We have previously extended the success of this biosynthetic pathway by generating 67 combinations of 68 new violacein and deoxyviolacein analogues. These combinations are 68 achieved by feeding various tryptophan substrates to recombinant E. coli expressing the 69 violacein biosynthetic pathway or via introduction of an in vivo chlorination step - the 70 tryptophan 7-halogenase RebH from the rebeccamycin biosynthetic pathway13,15–17. This 71 biosynthetic approach is able to produce large quantities of compound derivatives using 72 simple, cheap and non-hazardous bacteria compared to native producing strains in a 73 sustainable and flexible approach. 74 75 Here, we set out to explore whether use of this biosynthetic system could be developed as a 76 route to antimalarial compound production and testing, measuring the activity of 77 derivatives on the growth of P. falciparum sexual and asexual parasites. We have confirmed 78 the viability of the system, ensuring there is no background antiparasitic activity in bacterial 79 solvent extracts lacking violacein. We then tested the biosynthetic violacein extract from E. 80 coli and confirmed its half maximal inhibitory concentration (IC50) in agreement with a 81 commercial violacein standard and previous studies14. Finally, as well as using this approach 82 to explore the mode of action of violacein, we show that extracts representing a diverse 83 series of biosynthetically derived variants show varying effects on parasite growth, with 16 84 of the 28 compound mixtures inhibiting growth to a greater level than the parent violacein 85 molecule. Indeed, one purified compound, 7-chloroviolacein, exhibits a ~20% higher 86 inhibition activity than the underivatized violacein compound. The high-throughput 87 approach used in this study suggests that biosynthetic systems may therefore provide an, as 88 yet, untapped resource for screening complex compounds and optimising them for 89 antimalarial discovery. 90 bioRxiv preprint doi: https://doi.org/10.1101/814715; this version posted October 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 91 RESULTS 92 Violacein expressed using synthetic operons kills P. falciparum 3D7 parasites 93 Previous work has shown that violacein is able to kill asexual P. falciparum parasites in 94 vitro9,13 but noted erythrocytic rupture at concentrations above 10 µM and a cytotoxic IC50 95 of 2.5 µM. Taking this into consideration, we used concentrations of 2 µM violacein or less 96 to explore growth inhibition of P. falciparum asexual stages, noting no phenotypic effect on 97 erythrocyte morphology at the highest final concentration (Fig. S1). Our biosynthetic system 98 for violacein production requires chloramphenicol drug pressure, which is known to affect 99 parasite viability18. We first set out to ensure presence of this known antibiotic did not 100 affect parasite growth. Extract from bacteria lacking the violacein producing enzymes but 101 grown under chloramphenicol pressure (i.e. background) did not affect parasite viability 102 (Fig. S2). This gave us confidence that extracts from
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