Lead Optimisation of Dehydroemetine for Repositioned Use in Malaria

Home , Dehydroemetine

bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 Lead optimisation of dehydroemetine for repositioned use in

2 malaria

4 Priyanka Panwar1, Kepa K. Burusco2, Muna Abubaker1, Holly Matthews3, Andrey

5 Gutnov4, Elena Fernández-Álvaro5, Richard A. Bryce2, James Wilkinson1, Niroshini

6 Nirmalan1*

8 1Environment and Life Sciences, University of Salford, Greater Manchester, M5

9 4WT, UK.

10 2Division of Pharmacy and Optometry, School of Health Sciences, Faculty of

11 Biology, Medicine and Health, University of Manchester, Manchester Academic

12 Health Science Centre, Oxford Road, Manchester M13 9PL, UK.

13 3Keele University, Staffordshire, ST5 5BG, UK.

14 4Chiroblock GMBH, Andresenstrasse 1a, 06766 Wolfen, Germany.

15 5GlaxoSmithKline, Diseases of the Developing World Medicines Development

16 Campus, Severo Ochoa, 2. Tres Cantos, 28760, Spain.

20 *Niroshini Nirmalan

21 E-mail: [email protected]

22 Keywords: malaria, antimalarial drug interactions, SYBR Green flow-cytometry,

23 emetine, dehydroemetine, drug discovery, repositioning

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25 Abstract:

26 Drug repositioning offers an effective alternative to de novo drug design to

27 tackle the urgent need for novel anti-malarial treatments. The anti-amoebic compound,

28 emetine dihydrochloride, has been identified as a potent in-vitro inhibitor of the multi-

29 drug resistant strain K1 of Plasmodium falciparum (IC50: 47 nM + 2.1 nM). 2,3-

30 dehydroemetine, a synthetic analogue of emetine dihydrochloride has been claimed

31 to have less cardiotoxic effects than emetine. The structures of two diastereoisomers

32 of 2,3-dehydroemetine were modelled on the reported emetine binding site on cryo-

33 EM structure 3J7A and it was found that (-)-R,S-dehydroemetine mimicked the bound

34 pose of emetine more closely than (-)-S,S-dehydroisoemetine. (-)-R,S-

35 dehydroemetine was also found to be highly potent against the multi-drug resistant K1

36 strain of P. falciparum in comparison with (-)-S,S-dehydroisoemetine, which loses its

37 potency due to the change of configuration at C-1´. In addition to its effect on the

38 asexual erythrocytic stages of P. falciparum, the compounds exhibited gametocidal

39 properties with no cross-resistance against any of the multi-drug resistant strains

40 tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic

41 antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride, and (-)-

42 R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and

43 displayed atovoquone-like activity on the mitochondrial membrane potential.

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48 Author Summary:

49 Malaria is one of the oldest diseases on earth and has taken many lives. With

50 nearly half of the world’s population at risk, the spread of drug resistance and

51 insecticide resistance poses a big threat to malaria control measures. With resistance

52 emerging to the artemisinin combination therapy (ACT), the world is in dire need of a

53 new antimalarial. Due to long time-lines for de novo drug discovery, repositioning of

54 drugs provides a viable alternative way. Here, we followed up on the previous

55 repositioning work done in our lab and tested a known anti-amoebic compound for

56 repositioned use in malaria. We found, the synthetic analogue of emetine,

57 dehydroemetine is highly potent against the K1 multi-drug resistant strain and shows

58 gametocidal activity with no cross-resistance. Our study provides a rationale for further

59 optimisation of dehydroemetine for use in complicated cases of malaria.

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69 Introduction:

70 Malaria presents a huge burden on the economic development of endemic

71 countries (1). In 2016, there were 216 million malaria cases reported globally, with an

72 estimated 445,000 deaths occurring mostly amongst african children (57). The 20th

73 century witnessed the development of a range of antimalarials including quinine

74 alternatives such as mepacrine, chloroquine and primaquine, antifolates such as

75 sulphadoxine and pyrimethamine, and artemisinin (2) (3) (4) (5) (6) (7). However, the

76 emergence of resistance against all known classes of anti-malarial drugs including

77 artemisinin chemotherapy, has warranted research into the development of new drugs

78 with novel targets against the parasite (8) (9). Drug discovery is hindered by long

79 development timelines, high attrition rates and soaring R&D costs (10). Antimalarial

80 chemotherapy has predominantly relied on compounds based on natural products

81 (11). Emetine dihydrochloride hydrate, a natural product alkaloid derived from

82 Psychotria ipecacuanha has been widely used for 4 decades as a frontline anti-

83 amoebic drug, despite its emetic and cardiotoxic side effects. It was superseded by

84 the introduction of a safer drug metronidazole by the 1970s (12). Repositioning

85 screens carried out at the University of Salford, UK, identified emetine dihydrochloride

86 to have potent, nanomolar in vitro anti-malarial efficacy in the multi-drug resistant K1

87 Plasmodium falciparum parasite strain. The comparatively significant difference in in

88 vitro anti-protozoan efficacies (IC50 47 + 2.1 nM in P. falciparum as compared to IC50

89 26.8 + 1.27 µM in Entamoeba histolytica) dictate that the safety profile for its

90 repositioned use as an antimalarial could be different (13) (14). The pleiotropic natural

91 product drug has been reported to have anti-viral and anti-cancer properties, including

92 recent reports of interrupting viral replication and cell entry for Zika and Ebola viruses

93 (15). The 40S ribosomal subunit of the eukaryotic 80S ribosome was recently reported

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94 to be the site of action of emetine (16). The definition of the target binding site for

95 emetine enables a chimeric approach to refine using rational design, a drug

96 discovered through repositioning.

97 The (R) configuration at C-1´ and the presence of a secondary nitrogen at

98 position 2´ are important for emetine’s biological activity (Fig 1).

99 Indeed, the (S) configuration at C-1´ (isoemetine) or the substitution of the

100 secondary amine result in loss of activity. Even if the asymmetry at carbons 2 and 3

101 are lost with unsaturation at position 2-3 (2,3-dehydroemetine) the biological activity

102 is retained (17). In 1980, a study was conducted on the cross-resistance of emetine-

103 resistant mutants of Chinese hamster ovary cells to related compounds such as (-)-

104 cryptopleurine (phenanthroquinolizidine-type alkaloids), (-)-tylocrebrine

105 (phenanthroindolizidine type), and (-)-emetine, (-)-tubulosine, (-)-cephaeline, and (-)-

106 dehydroemetine (benzoiosoquinoline alkaloids). They concluded that the planar

107 structure of the molecule, with two aromatic rings was made slightly electronegative

108 by hydroxyl or methoxyl groups (18). The distance between the two aromatic rings and

109 the angle between the nucleophilic element, such as nitrogen, and the rings were

110 essential for biological activity.

111 Emetine in its crude form has been in use since 1658 for the treatment of

112 dysentery and was first brought to Europe from Brazil by Piso (19). Large doses of

113 emetine in powdered ipecacuanha form were introduced in Mauritius in 1858 and the

114 annual death rate from severe dysentery was reduced from 10-18% to 2% (20).

115 Generalised muscle weakness, vomiting and cardiotoxicity were the side-effects with

116 prolonged use of emetine, as in the treatment of hepatic amoebiasis (21) (22).The

117 synthetic analogue of emetine, 2,3-dehydroemetine, was introduced in 1959 (23). It

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118 was found to be less emetic than emetine and could be given orally as a resinate. 2,3-

119 Dehydroemetine was also comparatively safer than emetine when given parenterally

120 at similar doses. At high doses, the electrocardiographic changes were similar but

121 were less marked and of shorter duration (24). Experimental results have also shown

122 that changes observed in heart conduction, contractility, automaticity and ECG

123 abnormalities by emetine and 2,3-dehydroemetine could be due to their effect on

124 membrane permeability to Na+, K+ and, Ca++ ions (25). 2,3-Dehydroemetine has

125 been claimed to have a less cardio-toxic effects than emetine in a study that assessed

126 the effect of chloroquine in combination with emetine hydrochloride and 2,3-

127 dehydroemetine for the treatment of amoebic liver abscesses (26).

128 A study was conducted to observe the distribution and excretion of 2,3-

129 dehydroemetine and emetine in guinea pigs (27). It was found that 91.5% of 2,3-

130 dehydroemetine compared to 67% of emetine was excreted 72 hours after stoppage

131 of doses. 2,3-Dehydroemetine was eliminated more rapidly from the heart than the

132 liver and reverse was found to be true for emetine. This could be one of the reasons

133 for reduced cardiotoxicity observed in treatment with 2,3-dehydroemetine, as emetine

134 binds more tightly to cardiomyocytes even though it is not specifically concentrated in

135 them. Less toxic analogues have not been analysed for antimalarial potency,

136 therefore, based on the anecdotal evidence, two diastereoisomers of dehydroemetine,

137 (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine were synthesised. Molecular

138 modelling tools (28) (29) were used to predict the activity of the diastereoisomers and

139 their potency against the multi-drug resistant K1 strain of P. falciparum was tested.

140 It was observed that 2,3-dehydroemetine and emetine were potent inhibitors of

141 protein synthesis in HeLa cells (17). At a concentration of 5 x 10-8 M, emetine was able

142 to effectively inhibit protein synthesis by 50% in HeLa cells but had no effect on the

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143 incorporation of amino acids into the mitochondrial fraction even at 1.4 x 10-4 M (30).

144 On cultured chicken embryo cardiac cells, emetine led to cessation or progressively

145 decreased rate of beating in atrial and ventricular cells depending on the

146 concentration. In the study washing did not reverse the effects but the addition of

147 NADH (10-5 M) resulted in prolonged reversal lasting 30 to 45 minutes whereas intense

148 brief reversal of effects was observed by addition of ATP (10-6 M) indicating that at

149 higher concentrations, emetine causes physical disruption of the plasma membrane

150 of the cells and at a lower concentration, it suppresses the process of formation of high

151 energy phosphate compounds (31). It was thought that the site of inhibition was at the

152 enzymatic oxidation of substrates mediated by nicotinamide adenine dinucleotide

153 (NAD). In this study we tested the activity of emetine dihydrochloride and its two

154 synthetic analogues against hERG potassium channel. Drugs inhibiting the hERG

155 channel can prolong the QT interval and cause a dangerous cardiac arrhythmia,

156 Torsades de pointes (32). The three compounds were also tested for any effect on

157 mitochondrial membrane potential.

158 Parasites are transmitted from the mammalian hosts to mosquito vectors

159 through mature Plasmodium gametocytes and hence the reinfection cycle could be

160 broken with the use of transmission blocking anti-malarials (33). Cross-resistance and

161 gametocidal activity of emetine dihyrochloride and its synthesised analogues were

162 tested by GSK in a bid to determine the potential of these compounds as transmission

163 blocking drugs.

164 The preference of combinatorial regimes over monotherapy for the treatment

165 of malaria has affected the drug discovery pipeline in a crucial way (34). More often

166 than not, instead of developing two new drugs for combination therapy, newer drug

167 candidates are tested for synergistic activities with existing anti-malarial treatments

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168 (35). Combination therapy not only helps in dose reduction and better therapeutic

169 efficacy, it is also useful in delaying the emergence of resistance and widens the shelf-

170 life of the existing cohort of drugs (36).

171 Chou and Talalay developed a method based on the argument that the issue

172 of synergy is more physiochemical rather than statistical in nature and employed the

173 mass-action law principle to derive a median-effect equation where additivity could be

174 defined using the resulting combination index (CI = 1) with antagonism and synergism

175 defined as >1 and <1 respectively. Calcusyn and Compusyn are two software

176 programmes based on the complex algorithms for median-effect analysis to allow

177 automation and eliminate subjectivity during data analysis (37). We have previously

178 demonstrated the use of Calcusyn as a reliable method to define antimalarial drug

179 interactivity for combinatorial regimes (38).

180 Hence, to reduce dose-dependent side effects, combinatorial partner drugs

181 showing synergistic activity were sought. (-)-R,S-Dehydroemetine was taken forward

182 for drug interaction studies as it was found to be highly potent against the K1 strain of

183 P. falciparum. Atovaquone and proguanil were first evaluated to test the efficacy of the

184 method as the two drugs are known to exhibit synergism (39). The methodology was

185 then applied to evaluate combinatorial partner drugs for the potent anti-malarial

186 candidate (-)-R,S-dehydroemetine. Pre-determined IC50 values for each compound

187 were used to select the constant-ratio for each combination.

188

189

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190 Results:

191 Molecular modelling of ligand interactions with P. falciparum 80S ribosome:

192 In order to explore the structural basis of the relative inhibitory activity of (-)-

193 R,S-dehydroemetine and (-)-S,S-dehydroisoemetine, we predicted and compared

194 their molecular interaction with P. falciparum 80S ribosome using computational

195 docking. As the receptor structure, we use the recently solved cryo-EM structure of

196 the 80S ribosome of P. falciparum, determined to a resolution of 3.2 Å (16). The

197 electron density corresponding to the bound emetine is located in the E-site of the

198 ribosomal small subunit, ie. Pf40S; this E-site, a binding site that pactamycin also

199 recognises in the bacterial 30S subunit, is at the interface between 18S rRNA helices

200 23, 24, 45 and the C-terminus of protein uS11.

201 On closer examination of the published cryo-EM structure of the complex, the

202 ligand structure originally modelled into the density, unfortunately, corresponds to the

203 non-natural enantiomer of emetine, (1S,2R,3S,11bR)-emetine. This modelled

204 structure was not the ligand employed in the associated cryo-EM experiments (16).

205 Therefore, we docked emetine, i.e. the (1R,2S,3R,11bS) structure, into the identified

206 Pf40S binding site using MOE-Dock [Chemical Computing Group Inc., 1010

207 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2016.]. The

208 docked emetine geometry maps well into the electron density envelope (shown at a

209 contour level 0.1542e/A^3 (3.50rmsd) in Fig 2a); the bound pose broadly follows the

210 twisted U-shape conformation of the observed electron density, resembling some

211 aspects of the previously modelled non-natural emetine enantiomer geometry (Fig 2b).

212 In this U-shape, an intermolecular T-shaped -stacking interaction is observed

213 between the two cyclic systems of emetine, ie. benzo[a]quinolizine rings A/B/C and

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214 isoquinoline rings D/E (Fig 1, Fig 2a). The docked emetine also forges a number of

215 comparable interactions with the Pf40S subunit as its modelled enantiomer (Fig 2b),

216 with a key -stacking interaction between the A/B/C rings of emetine with the purine

217 ring of Gua973 of h23 (16). The location of the emetine secondary and tertiary amines

218 are broadly similar (within 1 - 2 Å), allowing hydrogen bonding interactions with a

219 backbone oxygen atom of Ura2061 (h45) and the 2’-hydroxyl group of Ura1068 (h24)

220 respectively (Fig 2b). The tertiary amine also forms a salt bridge interaction with the

221 carboxylate side chain of C-terminal residue Leu151 of uS11 (Fig 2b). This interaction

222 was not highlighted in the cryo-EM study and is a consequence of modelling into the

223 E-site the natural emetine geometry.

224 Subsequently, the two diastereomers of emetine, (-)-R,S-dehydroemetine and

225 (-)-S,S-dehydroisoemetine, were docked in turn into the emetine-binding region of the

226 Pf40S subunit. We found that the preferred docked pose of the (-)-R,S-

227 dehydroemetine adopts the familiar U-shaped conformation, superimposing rather

228 closely onto the bound pose of emetine (Fig 3a). The docking scores are

229 correspondingly similar for emetine, with a London dG value of -7.2 kcal/mol, and (-)-

230 R,S-dehydroemetine, which has a dG score of -7.3 kcal/mol. As observed for emetine,

231 the (-)-R,S-dehydroemetine forms the  stacking interaction with the Gua973

232 pyrimidine ring and polar interactions with Ura2061, Ura1068 and Leu151 (Fig 3b).

233 However, (-)-S,S-dehydroisoemetine docks into the binding site with a lower dG

234 score of -6.5 kcal/mol and does not overlay in conformation so readily with emetine or

235 the (-)-R,S-dehydroemetine (Fig 3a). The particular stereochemical configuration of (-

236 )-S,S-dehydroisoemetine appears to result in its secondary amine being more distant

237 from the E-site residues of Pf40S. Consequently, this amine N…O Ura2061 distance

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238 extends by 0.8 Å in proceeding from R to S isomer (Fig 3b). The tertiary amine

239 interaction with Ura1068 is maintained, however, as is the interaction with the terminal

240 carboxylate of Leu151 (Fig 3b). The  stacking interaction with Gua973 is also present,

241 but at a slightly larger distance between planes, increased by ~0.7 Å. Fig 4 (a) and (b)

242 shows the predicted binding site residues for (-)-R,S-dehydroemetine and (-)-S,S-

243 dehydroisoemetine.

244 Dose-response experiments to test activity against the K1 strain of P.

245 falciparum:

246 Based on the anecdotal evidence, two synthetic analogues of emetine

247 dihydrochloride, (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine were

248 synthesised. Experiments to test drug efficacy were set up and (-)-R,S-

249 dehydroemetine was tested in two fold serial dilutions from 12.5 nM to 200 nM. A dose

250 response experiment was set up on ring stage of the K1 strain of P. falciparum to be

251 read at 72 hours. IC50 of (-)-R,S-dehydroemetine was observed to be 69.58 + 2.62 nM.

252 Results are shown in Fig 5. (-)-S,S-dehydroisoemetine was tested in two fold serial

253 dilutions from 0.625 µM to 10 µM. IC50 was observed to be 1.85 + 0.2 µM

254 concentration. Results are shown in Fig 5.

255 IC50 speed assay for (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine:

256 Accurate determination of the parasite killing rate in response to treatment is

257 crucial in the development of drugs against P. falciparum as chemotherapy remains

258 the primary element in the control of malaria. The speed of action of compounds on

259 the viability of parasites is difficult to measure through traditional techniques (40). It is

260 important to identify new drugs with rapid parasite killing kinetics early in the drug

261 development process. Besides rapid relief of symptoms, a fast-acting drug also helps

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262 to curtail the mutations causing the development of new mechanisms of drug

263 resistance. In 2012, Tres Cantos developed a labour intensive low-throughput assay

264 taking up to 28 days to determine in vitro the parasite reduction ratio (PRR) and the

265 presence or absence of a lag phase in response to a drug (41). The method used in

266 this study to differentiate between fast and slow acting compounds gives initial results

267 in 4 to 7 days and is able to detect static or cidal effects of compounds (42).

268 The IC50 speed assay was performed for (-)-R,S-dehydroemetine and (-)-S,S-

269 dehydroisoemetine (with concentrations of 25 nM, 125 nM, 625 nM, 3125 nM and

270 15625 nM and 0.5 µM, 2.5 µM, 12.5 µM, 62.5 µM and 312.5 µM, respectively) using

271 unsynchronised cultures of P. falciparum. The ratio of 24 hours IC50 values to 72 hours

272 IC50 values for (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine could not be

273 determined as IC50 could not be reached within 24 hours. Both compounds only

274 achieved 50% inhibition at the previously defined IC50 values after 48 hours of

275 exposure, indicating that the isomers have delayed action against the multi-drug

276 resistant K1 strain of P. falciparum. The results are shown in Fig 6.

277 Stage-Specificity assay:

278 The two isomers of 2,3-dehydroemetine were tested on synchronous cultures

279 to determine the stage specificity of the compounds by measuring the concentration-

280 dependent growth of schizonts and rings following incubation with the two compounds.

281 It was observed that (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine

282 affect both the ring and trophozoite/schizont stages of the parasite. The isomers were

283 found to be more active in the late trophozoite/schizont stages which is consistent with

284 the proposed protein synthesis target of the parent compound. The lower potency

285 against ring stage parasites may also in part explain the lag-phase observed during

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286 the speed assay, where the IC50 was not reached after 24 hours of exposure against

287 unsynchronised cultures.

288 The IC50 speed assay suggests that both the compounds have a slow action

289 against unsynchronised cultures. However, the stage specificity assay shows a slow

290 activity (~40% action) against the rings even at high concentration and fast activity on

291 late trophozoites/schizonts (~80% action) (Fig 7). By correlating the data obtained

292 from the two assays, both (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine

293 should have a slow action against the rings and fast action against the late

294 trophozoites/schizonts stages.

295 MTT assay for cell cytotoxicity against HepG2 cells:

296 MTT assay for cell cytotoxicity was performed for (-)-R,S-dehydroemetine and

297 (-)-S,S-dehydroisoemetine. Emetine and cisplatin were used as control drugs (Fig 8).

298 The plates were read at 24 hours (43).

299 Emetine was tested in three-fold serial dilutions from 24.7 nM to 2000 nM with

300 IC50 observed to be 229.4 + 18.5 nM. (-)-R,S-dehydroemetine was tested in two-fold

301 serial dilution from 11.72 nM to 750 nM with IC50 observed to be 204.2 + 1.35 nM. (-)-

302 S,S-dehydroisoemetine was tested in two fold serial dilutions from 0.6 µM to 12 µM

303 with IC50 observed to be 1.442 + 0.086 µM. Cisplatin was tested in two fold serial

304 dilutions from 0.78 µM to 25 µM and with IC50 observed to be 12.43 + 0.95 µM. The

305 selectivity index, calculated as IC50 HepG2 / IC50 parasites (44), of (-)-R,S-

306 dehydroemetine was approximately 3 and (-)-S,S-dehydroisoemetine were observed

307 to be approximately equal to 1.

308 Determination of cross-resistance through hypoxanthine incorporation assay:

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309 This assay relies on the parasite incorporation of labelled hypoxanthine that is

310 proportional to P. falciparum growth. In vitro cross-resistance of the compounds is

311 measured as an IC50 ratio between the IC50 value for the corresponding P. falciparum

312 strain versus the IC50 value for 3D7A. Every replicate from an MDR strain involved a

313 simultaneous determination using a 3D7A replicate to avoid any artefact linked to

314 experimental conditions. Table 1 shows the IC50 values of emetine dihydrochloride, (-

315 )-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine in P. falciparum sensitive strain

316 (3D7A) and two Pf resistant strains (Dd2 and W2). The ratios of in vitro cross-

317 resistance of emetine dihydrochloride and (-)-R,S-dehydroemetine in both resistant

318 strains (Dd2 and W2) using 3D7A strain as reference were found to be 1.

319 Table 1: Analysis of results to determine cross-resistance through

320 hypoxanthine incorporation assay

3D7A Dd2 W2 Compounds Pf IC50 Pf IC50 Pf IC50 ratio ratio st dev st dev st dev (µM) (µM) (µM) Dd2/3D7A W2/3D7a

Emetine 0.234 0.022 0.269 0.025 0.181 0 1 1 dihydrochloride

(-)-R,S- 0.146 0.003 0.177 0.014 0.168 0.005 1 1 dehydroemetine

(-)-S,S- 1.031 0.041 ND ND dehydroisoemetine

321

322 In-vitro IC50 values of emetine dihydrochloride, (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine

323 in P. falciparum sensitive strain (3D7A) and Pf resistant strains (Dd2 and W2). It also shows the ratios

324 of in vitro cross-resistance of emetine dihydrochloride and (-)-R,S-dehydroemetine in both resistant

325 strains (Dd2 and W2) using 3D7A strain as reference.

326

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327 The results showed that the inhibitory potency observed for emetine

328 dihydrochloride and (-)-R,S-dehydroemetine in both multidrug resistant strains (Dd2

329 and W2) is similar to the sensitive strain 3D7A. These results suggest that there is no

330 cross-resistance with any of the MDR strains tested.

331 In vitro IC50 against P. falciparum male and female activated gametes:

332 This bioassay assesses the malaria transmission blocking potential of

333 compounds by estimating their ability to prevent male mature gametocytes to progress

334 to male microgametes or/and to inhibit female gamete activation, as indicators of

335 gametocyte functionality. The activation of male gametocytes into mature

336 microgametes is evaluated by the process of exflagellation (extrusion of rapidly waving

337 flagellum-like microgametes from the infected erythrocyte). The activation of female

338 gametocytes is evaluated based on the specific expression of the Pfs25 protein at the

339 surface of the female activated gametes (45). Male P. falciparum gametocytes

340 exflagellate when activated, causing the movement of the surrounding RBCs in the

341 media. Detecting these changes in cells position, we were able to detect activated

342 male gametes. Female P. falciparum gametocytes round up when activated and the

343 Pfs25 protein distributes widely in the membrane of the gamete. Using a monoclonal

344 antibody against this protein we were able to specifically detect activated female

345 gametes. Dual Gamete Formation assay was performed and the results are shown in

346 table 2.

347

348

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349 Table 2: In-vitro IC50 values of (-)-R,S-dehydroemetine and (-)-S,S-

350 dehydroisoemetine against male and female gametocytes in P. falciparum NF54

351 strain

P. falciparum Dual Gamete Formation Assay Male Female Compounds Average St dev Average St dev (-)-R,S-dehydroemetine 0.43 0.02 1.04 0.02 (-)-S,S-dehydroisoemetine >10 >10 352

353

354 hERG channel inhibition assay:

355 Dose related cardiovascular side-effects have been observed following

356 treatment of amoebiasis with emetine which included ECG changes such as T-wave

357 inversion, prolongation of QT interval and to some degree increased width of QRS

358 complex and PR interval. Hypotension, tachycardia and precordial pain were also

359 observed (25). Stoppage of treatment resulted in complete recovery of cardiovascular

360 functions. Cardiac microscopic examination revealed a separation of muscle fibres

361 and destruction of myocardial fibres but an absence of inflammatory cells leading to

362 the interpretation that myocarditis is toxic rather than inflammatory in origin. In another

363 study on thirty two patients, pain was noted at injection site along with ECG

364 abnormalities, myalgia, muscle weakness and increased levels of serum creatinine

365 phosphatase (46). In a study on guinea pigs by Schwartz and Herrero (1965), it was

366 observed that dehydroemetine was excreted faster than emetine. It was also

367 postulated that reduced cardiotioxity of dehydroemetine could be due to decreased

368 tissue affinity to the heart in comparison to emetine (25).

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369 In a resting cardiac cell, the concentration of K+ is high intracellularly, which

370 creates a chemical gradient for K+ to diffuse out of the cells. A subunit of the rapid

371 delayed rectifier potassium ion channel is involved in the cardiac repolarisation (47).

372 It is encoded by the hERG gene (Human ether-a-go-go related gene). Since emetine

373 is known to affect the movements of Na+, K+ and Ca2+ ions, 2,3-dehydroemetine is

374 also thought to affect ion permeability. The study aims to establish if hERG channel

375 inhibition is responsible for the cardiotoxicity observed with emetine therapy in a bid

376 to propose analogues with reduced side effects.

377 Data Analysis of hERG channel inhibition assay:

378 For each replicate the hERG response is calculated using the following

379 equation:

Post ‒ compound current (nA) 380 % hERG response = x 100 Pre ‒ compound current (nA)

381 The % hERG response was plotted against concentration for the test compound

382 and, where concentration-dependent inhibition was observed, the data were fitted to

383 the following equation and an IC50 value calculated:

ymax ‒ ymin 384 y = s + ymin IC50 1 + ( 푥 )

385 Where: y = hERG response, ymax = mean vehicle control response, x = concentration,

386 IC50 = concentration required to inhibit current by 50% and s = Hill slope

387

388

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389 Table 3: hERG channel inhibition assay results for (-)-R,S-dehydroemetine and

390 (-)-S,S-dehydroisoemetine

Cyprotex hERG channel inhibition (IC50 determination) % Mean inhibition Compounds 0 0.08 0.4 10 0.016 µM 2 µM 50 µM IC (µM) µM µM µM µM 50 (-)-R,S-dehydroemetine 0 7.79 6.34 12.7 7.21 39.4 61.4 19.3 (-)-S,S-dehydroisoemetine 0 8.13 4.61 9.33 36 70.7 96.9 2.99 391

392

393 (-)-R,S-dehydroemetine has an IC50 of 19.3 µM for hERG channel whereas (-)-

394 S,S-dehydroisoemetine has an IC50 of 2.99 µM. The IC50 of quinidine which was used

395 as a positive control is 1.99 µM. Thus it was found that (-)-R,S-dehydroemetine is not

396 a potent hERG channel inhibitor, but (-)-S,S-dehydroisoemetine is a potent inhibitor.

397 Staining with rhodamine123 and draq5 for fluorescence microscopy and

398 measurement of mitochondrial membrane potential:

399 Changes in mitochondrial membrane potential was measured using

400 rhodamine123, a cationic fluorescent dye (48). It was visualised on fluorescence

401 microscopy that rhodamine123 localises with the cytoplasm of the parasites. Draq5 is

402 a far-red fluorescent DNA dye which is cell permeable and was used to identify the

403 parasites. SYBRgreen was not preferred for this experiment as it emits in the same

404 channel as rhodamine123 and would result in the overlap of emission signals.

405 Changes in mitochondrial membrane potential can be observed using

406 rhodamine123 (excitation wavelength: 511 nm and emission wavelength: 534 nm), a

407 membrane permeable fluorescent dye which accumulates by electrostatic attraction in

408 the mitochondria because of its negative transmembrane potential. A change in the

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409 dye’s concentration in the mitochondria is caused by a depolarisation event and can

410 be visualised as a shift in fluorescent intensity of rhodamine123 (49). Draq5,

411 (excitation wavelength: 647 nm and emission wavelength: >665 nm) a DNA dye was

412 used to distinguish the parasites on flow-cytometry in APC-Cy7-A channel.

413 Atovaquone, a known mitochondrial inhibitor was used as a control. A decrease in

414 florescent intensity measured in FITC-A channel indicates a loss of mitochondrial

415 membrane potential. A shift in fluorescent intensity was observed after treatment with

416 all four compounds. Emetine, (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine

417 showed a shift in fluorescent intensity of rhodamine123 in a direction similar to

418 atovaquone indicating a possible mitochondrial effect (Fig 9).

419 Validation of the CalcuSyn assay for malaria:

420 CalcuSyn method has been validated in our laboratory as a useful tool anti-

421 malarial drug interaction analysis (38). To establish the robustness of the method the

422 known synergistic drug combination, atovaquone and proguanil, was used. Triplicate

423 samples were analysed by SG-plate-reader after 72 hours. Calclusyn predicted strong

424 synergism between the two drugs which is in accordance with the published literature.

425 Following validation of the Calcusyn software employing Chou-Talalay method

426 for drug interaction analysis using the atovaquone-proguanil combination, the

427 interaction between (-)-R,S-dehydroemetine-atovaquone and (-)-R,S-dehydroemetine

428 - proguanil were studied. The doses used for each compound were based on the

429 known ED50 values which served as the midpoint for a two-fold, constant-ratio dose

430 series as shown in table 4.

431

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432 Table 4: The dose series used for the combination of existing antimalarials (AM)

433 with (-)-R,S-dehydroemetine

Level (-)-R,S-dehydroemetine (nM) Atovaquone (nM) Proguanil (uM)

A 12.5 0.5 1.75

B 25 1 3.5

C 50 2 7

D 100 4 14

E 200 8 28

F 400 16 56

G 800 32 112

Combination ratio with (-)- -- 1:25 140:1 R,S-dehydroemetine

434

435

436 CalcuSyn-based analysis of the atovaquone - proguanil combination:

437 The Calcusyn based analysis of the drug interactivity between atovaquone and

438 proguanil using a constant-ratio combination of 1:7000 dose was carried out. The

439 output included a dose-effect curve and a median-effect plot in addition to the

440 Combination Index (CI) and an isobologram plot, to figuratively depict the compound’s

441 potency and conformity to the mass action law (Fig 10). Specifically, CI = 0.20, 0.34

442 and 0.57 at the ED50, ED75 and ED90 levels, respectively were obtained inferring strong

443 synergism at ED50 and ED75, and synergism at ED90. Good correlations coefficients of

444 the median-effect plot were reported for atovaquone (r = 0.99), proguanil (r = 0.89),

445 and the combination (r = 0.93), inferring good conformity to the mass-action law as

446 shown in table 5.

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447

448 Table 5: CalcuSyn based analysis of drug interaction between atovaquone and

449 proguanil

Drug CI Values at

ED50 ED75 ED90 Dm m r

Atovaquone N/A N/A N/A 6.5262 0.88422 0.99782

Proguanil N/A N/A N/A 27771 1.06144 0.89349

Combination 0.2088 0.34424 0.57296 0.51518 0.68303 0.93063 (1:7000)

450

451 CalcuSyn-based analysis of the (-)-R,S-dehydroemetine - atovaquone

452 combination:

453 The Calcusyn based analysis of the drug interactivity between (-)-R,S-

454 dehydroemetine and atovaquone using a constant-ratio combination of 25:1 dose was

455 done (Fig 11). Specifically, CI = 0.88, 0.88 and 0.89 at the ED50, ED75 and ED90 levels,

456 respectively were obtained inferring mild synergism at all measurement points. Good

457 correlations coefficients of the median-effect plot were reported for atovaquone (r =

458 0.94), (-)-R,S-dehydroemetine (r = 0.94), and the combination (r = 0.96), inferring good

459 conformity to the mass-action law as shown in table 6.

460

461

462

463

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464 Table 6: Calcusyn based analysis of drug interaction between atovaquone and

465 (-)-R,S-dehydroemetine

Drug CI Values at

ED50 ED75 ED90 Dm m r

Atovaquone N/A N/A N/A 8.56439 1.51904 0.94197

(-)-R,S-dehydroemetine N/A N/A N/A 85.98414 2.41228 0.94623

Combination (1:25) 0.88215 0.88286 0.89502 2.16472 2.08897 0.96644

466

467 CalcuSyn-based analysis of the (-)-R,S-dehydroemetine - proguanil

468 combination:

469 The CalcuSyn based analysis of the drug interactivity between (-)-R,S-

470 dehydroemetine and proguanil using a constant-ratio combination of 140:1 dose was

471 done (Fig 12). Specifically, CI = 0.67, 1.04 and 1.62 at the ED50, ED75 and ED90 levels,

472 respectively were obtained implying synergism at ED50, near–additive effect at ED75

473 and antagonism at ED90. Good correlation coefficients of the median-effect plot were

474 obtained for proguanil (r = 0.90), (-)-R,S-dehydroemetine (r = 0.86), and the

475 combination (r = 0.95), suggesting good conformity to the mass-action law as shown

476 in table 7.

477

478

479

480

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481 Table 7: CalcuSyn based analysis of drug interaction between proguanil and (-

482 )-R,S-dehydroemetine

Drug CI Values at

ED50 ED75 ED90 Dm m r

Proguanil N/A N/A N/A 20.931 1.61801 0.90662

(-)-R,S-dehydroemetine N/A N/A N/A 232.4649 0.81711 0.86213

Combination ratio (140:1) 0.67736 1.04995 1.62774 14.16876 0.98301 0.95879

483

484 After 72 hours of incubation, the (-)-R,S-dehydroemetine and atovaquone

485 interaction was classified as synergistic at all inhibitory levels analysed. For the (-)-

486 R,S-dehydroemetine and proguanil combination the ED50 level of inhibition was

487 classified as synergism, the ED75 as nearly additive and ED90 as antagonism.

488 Discussion:

489 In conclusion, we report here the nanomolar antimalarial efficacy of the synthetic

490 emetine analogue(-)-R,S-dehydroemetine in the multi drug resistant K1 strain of

491 Plasmodium falciparum (IC50 of 69.58 + 2.62 nM). The clinical use of emetine

492 dihydrochloride as an anti-amoebic drug was superseded by the better tolerated

493 synthetic analogue 2,3-dehydroemetine in the 1070s. The isomer (-)-S,S-

494 dehydroisoemetine was found to be less potent with an IC50 of 1.85 + 0.2 µM.

495 Molecular modelling suggests that this greater potency of (-)-R,S-dehydroemetine is

496 linked to its ability to mimic more fully the interactions of emetine with the E-site of

497 Pf40S subunit, compared to the (-)-S,S-dehydroisoemetine isomer. The improvements

498 in the dose-dependent cardiotoxicity previously reported for 2,3-dehydroemetine in

499 comparison to emetine dihydrochloride was linked to decreased affinity to cardiac

500 myocytes and increased clearance from the body (27). We propose that the very

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501 significant differential in vitro activities of emetine dihydrochloride in Entamoeba

502 histolytica (IC50 26.8 + 1.27 µM) and Plasmodium falciparum (IC50 47 + 2.1 nM) (13)

503 (14) would mean that the dose related toxicity profile for its re-positioned use will be

504 different.

505 Analysis done by GSK showed that in asexual blood stages, the compounds exhibited

506 no cross-resistance issues. Mosquito vector dynamics, the number of people with

507 peripheral gametocytemia in the population, and the infectiousness of circulating

508 gametocytes to mosquitoes determines the transmission of malaria. Interrupting

509 transmission is an important aspect of preventing malaria in endemic areas and there

510 has been renewed interest in compounds preventing the formation of gametocytes.

511 (-)-R,S-Dehydroemetine was found to be gametocidal. It displayed activity against

512 both male and female gametes. Thus it also has the potential to block the transmission

513 of malaria.

514 The selectivity indices calculated using MTT assays in HepG2 cell lines was similar

515 for emetine and its analogues. It is important to note that the low selectivity index was

516 expected given the documented anticancer properties of emetine (50). This could

517 explain the low IC50 obtained on Hep G2 lines as these are rapidly multiplying cancer

518 cell lines. The selective advantage of (-)-R,S-dehydroemetine for cardiotoxic effects is

519 linked to its faster elimination from body, hence the appropriate toxicity investigation

520 to define this would be the use of in vivo animal models. It is imperative to progress

521 this work in this direction. It was also clearly established that the cardiac toxicity for (-

522 )-R,S-dehydroemetine was not effected through the hERG channel. Our observations

523 suggest that emetine and its two synthetic analogues have atovaquone-like activity

524 upon measurement of changes in the mitochondrial membrane potential at IC50

525 concentrations, indicating multi-modal mechanisms of action. The failure of

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526 monotherapy has been unambiguously demonstrated in malaria, hence, the forceful

527 insistence by the WHO for the use of artemisinin-based combination therapy (ACT) as

528 a policy standard. In addition to more potent therapeutic efficacies, other benefits of

529 combination regimes include decreased toxicity, favourable synergistic interactions

530 and most significantly, the potential to impede or delay the onset of resistance. Drug

531 interaction analyses presented here emphasise a route to further dose reduction to

532 minimise toxicity. (-)-R,S-Dehydroemetine was found to exhibit synergistic activity with

533 atovaquone and proguanil.

534 Emetine and its analogues, constitute a pleiotropic group of natural product-derived

535 compounds which could potentially enhance the depleted antimalarial

536 armamentarium, should a catastrophic gap in the drug market occur as a result of the

537 spread of resistance to frontline anti-malarials. The work presented here provides

538 strong justification for further optimisation, particularly for use in a hospital setting in

539 cerebral malaria, where appropriate monitoring could obviate progression of the

540 reversible cardiotoxicity previously reported. Its hepatic concentration ability could

541 indeed have favourable consequences for the treatment of Plasmodium vivax

542 infections. The history of antimalarial chemotherapy has been largely reliant on natural

543 product-derived leads. We provide here a significant body of evidence to progress and

544 further optimised a previously overlooked, potent and affordable natural product

545 compound.

546 Materials and Methods:

547 Culture of Plasmodium falciparum

548 RPMI 1640 media containing 25 mM HEPES and 0.3 g/L L-glutamine (Gibco,

549 Life Technologies, UK) supplemented with sterile filtered 2.5 g Albumax (Sigma, UK),

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550 2.5 ml hypoxanthine (Sigma, UK), 2.5 ml 40% glucose (Dextrose Anhydrous, Fisher

551 Scientific, UK) and 0.5 ml gentamycin (Sigma, UK), were used for the culture of

552 erythrocytic stage, strain K1, P. falciparum parasites (gifted by Prof John Hyde,

553 University of Manchester, UK, original source: Thai-K1 clone), under a 5% CO2 5% O2

o 554 and 90% N2 gas mixture (BOC Limited, UK) at 37 C. All routine culture methods were

555 consistent with those employed by (51).

556 O+ human blood (purchased from NHS Blood Bank, Manchester, UK) was used

557 routinely to maintain the parasites. Continuous cultures were maintained at 5%

558 haematocrit. Since synchronous parasite development is observed in natural hosts

559 and this synchrony is lost quite rapidly in in-vitro cultures, sorbitol was used to keep

560 the parasites in tight synchrony. In brief, the parasitized blood pellet was re-suspended

561 1:10 in 5 % sorbitol (prepared in distilled water and filtered using 0.22 µm porosity

562 Millipore filter), incubated at room temperature for 5 minutes and then centrifuged at

563 3000 rpm for 5 minutes. The supernatant was removed and complete media was used

564 to wash the pellet 3 times before setting up a new culture.

565 Synthesis of (-)-2,3-Dehydroemetine:

566 (-)-2,3-Dehydroemetine was synthesised as per the established literature

567 (Brossi 1967, Patent No. 3,311633 [16]). The synthesis was outsourced to Chiroblock

568 GMBH, Germany. Previously published patent literature by chemists at Hoffmann-La

569 Roche did not include modern analytical data such as mass spectrometry or NMR.

570 The methodology adopted for the synthesis (-)-R,S-dehydroemetine 7 used in this

571 study followed the published patent with minor modifications as depicted in (see Fig

572 13) below.

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573 The two compounds synthesised were (-)-R,S-dehydroemetine, 7, and (-)-S,S-

574 dehydroisoemetine, 8. Briefly, the Mannich base 1 was quaternized and reacted with

575 the imine 2 giving piperidinone 3 as an approximately 5:1 mixture of diastereomers

576 which were not separated. Knoevenagel condensation with malononitrile led to

577 unsaturated dinitrile 4 in 69% yield. Hydrolysis and decarboxylation, with concomitant

578 alekene isomerization, gave 5 after methylation. Resolution with (+)-dibenzoyl-D-

579 tartaric acid gave homochiral (-)-5. Hydrolysis and amidation with homoveratrylamine

580 followed by Bischler–Napieralski cyclization led to dihydroisoquinoline derivative 6,

581 also known as 2-dehydro-O-methyl psychotine. The cyclic imine was reduced with

582 sodium borohydride in methanol to give a 1:1 mixture of the desired 2,3-

583 dehydroemetine 7 and 2-dehydroisoemetine dihydrobromide 8, separated by

584 fractional crystallization. The purity of (-)-R,S-dehydroemetine obtained through this

585 method was 85% with the balance consisting of the diastereomer, 8.

586 Molecular modelling:

587 In preparation for predicting the bound pose of emetine, (-)-R,S-

588 dehydroemetine and (-)-S,S-dehydroisoemetine, the cryo-EM structure of P.

589 falciparum 80S ribosome bound to emetine dihydrochloride was obtained from the

590 Protein Data Bank (PDB code 3J7A) (16). We note that this cryo-EM structure features

591 the enantiomer of emetine rather than emetine itself; therefore, an initial 3D structure

592 of emetine was obtained from the OMEGA-generated conformer in the Pubchem

593 database (55). Subsequently emetine derivatives (-)-R,S-dehydroemetine and (-)-S,S-

594 dehydroisoemetine were constructed using the software package MOE (56). Prior to

595 in silico docking, crystallographic water molecules and a magnesium ion were

596 removed (the latter was within 13 Å of the active site and was found to unduly influence

597 docking solutions). Hydrogens were assigned consistent with physiological pH.

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598 Docking of ligands to the Pf40S subunit was performed using MOE-Dock[Chemical

599 Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada,

600 H3A 2R7, 2016]. The binding site region was defined using the cryo-EM ligand

601 location. Ligand placement used the Triangle Matcher protocol. Internal flexibility was

602 permitted for ligands but not ribosome. Poses were scored via the London dG scoring

603 function. While this docking protocol was able to reproduce well the cryo-EM pose of

604 the emetine stereoisomer bound to the Pf40S subunit (S1 Figure, Supporting

605 Information), please see comment later regarding this ligand’s structure.

606 Drug preparation:

607 Emetine dihydrochloride was obtained from Sigma Aldrich, UK. The

608 dehydroemetine analogues were synthesised as per the established literature

609 (Scheme 1, Brossi 1967, Patent No. 3,311633 [16]). Derivatives were validated using

610 NMR, HPLC and FTIR. The synthesis process was outsourced to Chiroblock GMBH,

611 Germany. Drug stock solutions were prepared in DMSO in accordance with

612 manufacturer instructions and primary stock concentration of 5 mM was aliquoted and

613 stored at -20oC until further use. Serial dilutions of the working solution were made

614 using complete media for the experiments.

615 Drug dosing for IC50 determination:

616 Refined dose ranges were selected for emetine dihydrochloride (12.5 nM – 200 nM),

617 (-)-R,S-dehydroemetine (12.5 nM - 200 nM), and (-)-S,S-dehydroisoemetine (0.625

618 µM - 10 µM) to permit accurate IC50 calculation against the K1 strain of P. falciparum.

619 Ring stage parasites were diluted to 0.5-1% parasitaemia, 2.5% haematocrit (in a 96

620 well plate format, 200 µl final well volume) and treated for 72 hours. Dose response

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621 parasitaemia was determined using the SYBR Green flow cytometer method

622 previously optimised in the laboratory (14).

623 SYBR Green staining of erythrocytic stage P. falciparum for flow cytometry:

624 Following drug efficacy experiments, 150 µl from the control and drug-treated

625 wells on a 96 well plate were transferred to a microcentrifuge tube and samples were

626 washed once with PBS. The supernatant was removed and samples were incubated

627 in the dark at room temperature for 20 minutes following addition of 1 ml of 5 x SYBR

628 Green solution (prepared by adding 5 µl of 10,000 x SG to 10 ml PBS). After staining,

629 the samples were centrifuged for 1 minute at 14,000 rpm and the supernatant was

630 discarded. Samples were re-suspended in 250 µl of 0.37% formaldehyde fixation

631 solution prepared by diluting 36.5% formaldehyde (Sigma, UK) with PBS to a final

632 concentration of 0.37%. The samples were placed in the fridge and incubated at 4℃

633 for 15 minutes. Subsequently, PBS was used to wash the samples 3 times and finally

634 the samples were suspended in 1 ml PBS. Parasitaemia was determined by SYBR

635 green fluorescence using the FITC channel of the BD FACsVerse flow cytometer

636 system (Blue laser, excitation laser line 488 nm EXmax 494 nm/Emmax 520 nM) and cell

637 size (forward scatter, FSC-A). Fifty thousand events were recorded and in three

638 replicates for each. Fluorescent events in drug-treated samples were compared with

639 infected and uninfected blood counterparts and gated accordingly to obtain the

640 percentage parasitaemia.

641 IC50 speed assay:

642 The assay was used to determine the speed of action of the emetine analogues.

643 Unsynchronised cultures of K1 strain P. falciparum was used and parasites were

644 grown in presence of (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine for three

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645 incubation periods of 24 hours, 48 hours and 72 hours. The assays were analysed by

646 determining SYBR green fluorescence as described above in the methodology.

647 Stage-specificity assay:

648 Parasite cultures with ≥ 80% trophozoites and ≥ 80% rings were obtained by

649 synchronisation with 5% Sorbitol (42). The cultures were synchronised twice, at 0

650 hours and 31 hours to obtain young rings which were up to 3 hours old. To obtain early

651 schizont stages, the culture was synchronised twice with the second synchronisation

652 being 6 to 8 hours after the first. Each synchronous stage was incubated for 24 hours

653 at 37 °C on a 96-well microtitre plate with a two-fold serial dilution of the drugs ranging

654 from 1.6 to 100 fold the IC50 of each drug. The plates were washed 4 times after the

655 incubation in order to dilute the drug concentration by > 1000 fold. The plates were

656 incubated for another 24 hours at 37°C following which SYBR green staining was used

657 to read the plates as described above.

658 MTT Assay for testing cell cytotoxicity:

659 HepG2 cells (University of Salford stocks purchased from American Type

660 Culture Collection (ATCC), USA), were grown in media consisting RPMI 1640, 2 mM

661 L-Glutamine, HEPES, 10% foetal bovine serum, 1% non-essential amino acids and

662 1% penicillin-streptomycin. Cells were calculated on a haemocytometer to make a new

663 cell-media solution to a concentration of 5000 cells/100 µl and 100 µl of this new cell

664 solution was added into each well of the 96 well plate. After 24 hours incubation, 100

665 µl of the drug prepared in the media were added to the wells. This was repeated in

666 triplicate and the plate was incubated for a further 24 hours (52). After the incubation

667 period, 50 µl of MTT solution was added to each well and incubated for 3 hours. The

30 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

668 liquid in each well was aspirated carefully and 200 µl of DMSO was added to each

669 well and results were read on the Ascent plate reader.

670 Derivation of dose-response curves and IC50 values:

671 The infected blood controls were set at 100% and percentage parasitaemia was

672 calculated for drug treated samples relative to the infected control. For IC50, GraphPad

673 prism 5.0 was used to further process the data. The largest value in the data set was

674 set to correspond to 100% and the smallest value to 0% by normalising the data. IC50

675 values were calculated using nonlinear regression (Graphpad prism 5.0) by using log-

676 transformed drug concentrations plotted against the dose response. The log(inhibitor)

677 vs. Normalised response-Variable slope option was used for IC50 calculation. Emetine

678 dihydrochloride was used as a control drug to validate the method.

679 Determination of cross-resistance through IC50 determination in multidrug

680 resistant P. falciparum strains (carried out at Tres Cantos, GSK):

681 The three strains used for the assay were 3D7A, Dd2 and W2 based on their

682 resistance profiles. 3D7A is chloroquine–sensitive, Dd2 is resistant to chloroquine,

683 mefloquin and pyrimethamine (53) and W2 is resistant to chloroquine, quinine,

684 pyrimethamine, cycloguanil, and sulfadoxine (54) (from the Malaria Research and

685 Reference Reagent Resource Center MR4). A culture of parasitized red blood cells

686 (RBC) of the corresponding strain, (0.5% parasitemia, 2% haematocrit) in RPMI-1640,

687 5% albumax and 5 µM hypoxanthine was exposed to 9 dilutions (3-fold serial dilutions)

688 of the compound starting at 5 µM. 100 µl of culture volume was plated in 96-well flat

689 bottom microtiter plates + 0.5 µl drug (stock x200 in DMSO). Plates were incubated

690 for 24 h at 37 ºC, 5% CO2, 5% O2, 90% N2. Next, 3H-hypoxanthine was added and

691 plates were incubated for another 24 h period. After that, parasites were harvested on

31 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

692 a glass fiber filter using a TOMTEC Cell harvester 96. Filters were dried and melt-on

693 scintillator sheets were used to determine the incorporation of 3Hhypoxanthine.

694 Radioactivity was measured using a microbeta counter. Data are normalized using the

695 incorporation of the positive control, (parasitized red blood cells without drug). IC50

696 values were determined using Grafit 7 program (41).

697 Determination of transmission blocking potential through in vitro inhibition of

698 gamete activation

699 Asexual cultures of P. falciparum NF54 strain parasites were used to seed

700 gametocyte cultures at 0.5% parasitemia, 4% hematocrit in 50 ml total volume under

701 3% O2/5% CO2/92% N2 gas. Culture medium (RPMI 25 mM HEPES, 50 mg/l

702 hypoxanthine, 2 g/l NaHCO3 without L-glutamine + 5% human serum and 5%

703 Albumax) was replaced daily for 14 days. At day 14, the concentration of non-purified

704 cultures was adjusted to plate 700.000 total cells per well in each 384-well plate. The

705 test drugs were then added in two-fold serial dilutions starting at 10 µM (10 µM –

706 0.00976 µM) and incubated 48 h at 37 ºC (3% 02, 5% CO2, 92% N2). DMSO was used

707 as the negative control and Thiostrepton as the positive control. Activation was

708 performed with ookinete medium (Same RPMI base used for culture but supplemented

709 with xanthurenic acid 50 µM) supplemented with the antibody anti-Pfs25-Cy3 at a final

710 concentration of 1/2000 (from 1 mg/ml stock). Plates were analyzed to detect

711 exflagellation centres. "Triggered" cultures were then incubated (protected from light)

712 at 26 °C for 24 h (in a thermoregulated incubator). Then, plates were analyzed to

713 detect female activated gametes. Activation of male gametes was detected based on

714 light changes provoked by flagella movements which caused movement of

715 surrounding cells. A 10-frame video was taken and then analyzed to determine these

716 changes in cell position based on pixels change. Then, the script determined where

32 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

717 exflagellation centers were located, based also on size and intensity of light changes.

718 Activation of female gametes was based on detection of fluorescent Cy3-Anti Pfs25

719 antibody (as the primary parameter) followed by a selection of events according to

720 their size, roundness and the intensity of the fluorescence. Both measurements were

721 performed using an automated inverted microscope Ti-E Nikon using JOBS software.

722 Analysis of images and videos was performed with the ICY program. The IC50 value

723 was determined using Microsoft Excel and Graphpad.

724 hERG Channel Inhibition (IC50 Determination) assay Protocol:

725 The experiment to test the potential of emetine, (-)-R,S-dehydroemetine and (-

726 )-S,S-dehydroisoemetine to inhibit hERG channel was outsourced to Cyprotex, UK.

727 100 µl of a 20 mM concentration of all three compounds in DMSO was provided to

728 Cyprotex. Compound dilutions were prepared by diluting a DMSO solution (default 10

729 mM) of the test compound using a factor 5 dilution scheme into DMSO, followed by

730 dilution into extracellular buffer such that the final concentrations tested are typically

731 0.008, 0.04, 0.2, 1, 5, 25 µM (final DMSO concentration 0.25%). Chinese Hamster

732 Ovary cells expressing the hERG potassium channel were dispensed into 384-well

733 planar arrays and hERG tail-currents were measured by whole-cell voltage-clamping.

734 Amphotericin B was used as a perforating agent is circulated underneath the

735 PatchPlateTM to gain electrical access to the cell. The pre-compound hERG current

736 was measured. Emetine, (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine in a

737 range of concentrations were then added to the cells and a second recording of the

738 hERG current was made. The test compound was left in contact with the cells for

739 300 sec before recording currents. Quinidine, an established hERG inhibitor, was

740 included as a positive control, and vehicle control (0.25% DMSO) as the negative

741 control. All buffers, cell suspensions and drug compound solutions were at room

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742 temperature. The percent change in hERG current was measured and used to

743 calculate an IC50 value.

744 Each concentration was tested in 4 replicate wells on the PatchPlateTM

745 (maximum of 24 data points). It was ensured that only acceptable cells were used to

746 assess hERG inhibition by applying filters. The cell must maintain a seal resistance

747 of greater than 50 MOhm and a pre-compound current of at least 0.1 nA, and ensure

748 cell stability between pre-and post-compound measurements.

749 Staining with rhodamine123 and draq5 for fluorescence microscopy and

750 mitochondrial membrane potential disruption:

751 Rhodamine123 and draq5 were used to observe the effect of emetine and its

752 two synthetic analogues on mitochondrial membrane potential. Rhodamine123 is a

753 mitochondrial specific dye which emits in FITC channel on flow-cytometry whereas

754 draq5 stains the DNA and emits in APC-Cy7-A channel. The two dyes were chosen to

755 perform the experiment as there is very minimal overlap in their emission signals.

756 Synchronised culture of P. falciparum K1 strain at trophozoite stage was incubated

757 with 1 ml of 200 nM Rhodamine123 for 1 hour, followed by a further incubation with

758 100 µl of 10 µM Draq5 for 20 mins. Smears prepared after the incubation were

759 immediately viewed under the fluorescence microscope at 100 x magnification to

760 visualise localisation of the dyes inside the parasite.

761 To test the effect of emetine and its analogues on mitochondria, synchronised

762 cultures of P. falciparum K1 strain at trophozoite stage were incubated at 2.5%

763 haematocrit (in a 96 well plate format, 200 µl final well volume) for 2 hours with IC50

764 concentrations of atovaquone, emetine hydrochloride, (-)-R,S-dehydroemetine and (-

765 )-S,S-dehydroisoemetine. Compounds were then washed away by centrifuging the

34 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

766 cultures and a pellet was prepared for each drug concentration. Pellets were then

767 incubated with 1 ml of 200 nM rhodamine123 for 1 hour, followed by a further

768 incubation with 100 µl of 5 µM draq5 for 20 mins. After a wash with PBS, the

769 experiment was read with flow-cytometer on FITC channel. Loss of mitochondrial

770 membrane potential was indicated by a decrease in fluorescent intensity.

771 Drug interaction analysis for (-)-R,S-dehydroemetine:

772 Primary stock solutions were prepared as mentioned above. For experimental

773 set up the primary stock solutions were further diluted with complete medium to give

774 final test concentrations. A dose range of 0.125–8 x the ED50 was made by a two-

775 fold serial dilution for atovaquone, proguanil, and (-)-R,S-dehydroemetine. For

776 atovaquone, the doses ranged from 0.25nM to 16nM for combination with proguanil,

777 and 0.5 nM to 32 nM for combination with (-)-R,S-dehydroemetine. For proguanil the

778 doses ranged from 1.75 µM to 112 µM. For (-)-R,S-dehydroemetine the doses

779 ranged from 12.5 nM to 800 nM. At each level, the compounds were co-administered

780 for example ED50 of atovaquone was combined with the ED50 of (-)-R,S-

781 dehydroemetine and 2 x ED50 atovaquone was combined with 2 x ED50 (-)-R,S-

782 dehydroemetine and so forth. Parasites were treated at ring stage and incubated for

783 72 hours in a 96 well plate format. The SYBR Green Plate reader method was used

784 to determine drug susceptibility. The data was analysed for the median-effect using

785 CalcuSyn software (Biosoft) by converting triplicate data to an averaged percentage.

786 The CalcuSyn software generates the CI over a range of fa levels at different growth

787 inhibition percentages. The interpretation of CI was done in accordance with table 8

788 (37).

789

35 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

790 Table 8: Classification of synergism or antagonism using CI values generated

791 by Chou-Talalay method (Source: Calcusyn manual, Biosoft, 2006)

Range of CI Symbol Description <0.1 +++++ Very strong synergism 0.1-0..3 ++++ Strong synergism 0.3-0.7 +++ Synergism 0.7-0.85 ++ Moderate synergism 0.85-0.90 + Slight synergism 0.90-1.10 + Nearly additive 1.10-1.20 - Slight antagonism 1.20-1.45 -- Moderate antagonism 1.45-3.3 --- Antagonism 3.3-10 ---- Strong antagonism >10 ----- Very strong antagonism 792

793 Ethics Statement:

794 For routine malaria culture, anonymised whole blood packs deemed unfit/outdated for

795 clinical use were purchased from the NHS Blood Bank at Plymouth Grove,

796 Manchester, UK. For experiments carried out in GlaxoSmithKline, Diseases of the

797 Developing World Medicines Development Campus, Tres Cantos, Spain, the human

798 biological samples were sourced ethically and their research use was in accord with

799 the terms of the informed consents under an IRB/EC approved protocol.

800

801

802

803

804

805

806

36 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

807 Figures:

808 Fig 1: Structure of emetine hydrochloride.

809 Fig 2 (a) Overlay of docked pose of emetine (green) with its enantiomer present in the

810 cryo-EM structure (maroon); observed electron density envelope is also shown

811 (wireframe surface with contour level 0.1542e/A^3 (3.50rmsd)); (b) Interactions of

812 docked emetine (green) with Pf40S residues and comparison with previously modelled

813 interactions of its enantiomer (maroon) (16)

814 Fig 3 (a) Overlay of docked poses of (-)-R,S-dehydroemetine (red) and (-)-S,S-

815 dehydroisoemetine (cyan) with emetine (blue). (b) Interactions of (-)-R,S-

816 dehydroemetine (red) and (-)-S,S-dehydroisoemetine (cyan) with the Pf40S binding

817 site. Distances (dotted lines) in Å.

818 Fig 4 (a) Docking through MOE showing the binding site residues for (-)-R,S-

819 dehydroemetine molecule. (b) Docking through MOE showing the binding site

820 residues for (-)-S,S-dehydroisoemetine molecule.

821 Fig 5: (a) Dose-response experiment for (-)-R,S-dehydroemetine read at 72 hours (b)

822 Dose-response experiment for (-)-S,S-dehydroisoemetine read at 72 hours

823 Fig 6: IC50 speed assay using unsynchronised cultures of Plasmodium

824 falciparum.

825 IC50 could not be reached within 24 hours. R, S-2,3-dehydroemetine and (-)-S,S-

826 dehydroisoemetine reached the IC50 by 48 hours. (a) IC50 speed assay for (-)-R,S-

827 dehydroemetine with concentration (25, 125, 625, 3125 and 15625 nM), (b) (-)-S,S-

828 dehydroisoemetine with concentration (0.5, 2.5, 12.5, 62.5 and 312.5 µM)

37 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

829 Fig 7: Stage specificity assay using synchronised cultures of Plasmodium

830 falciparum.

831 The decrease in growth of trophozoites/schizonts was observed to be more marked in

832 comparison to rings thus explaining the slow action observed in the IC50 speed assay.

833 (a) Stage specificity assay for (-)-R,S-dehydroemetine, (b) Stage specificity assay for

834 (-)-S,S-dehydroisoemetine

835 Fig 8: 48 hour MTT assay

836 (a) 48 hour MTT assay for emetine, (b) 48 hour MTT assay for cisplatin, (c) 48 hour

837 MTT assay for (-)-R,S-dehydroemetine and (d) 48 hour MTT assay for (-)-S,S-

838 dehydroisoemetine

839 Fig 9: Microscopy and flow-cytometry results for effect of atovaquone, emetine,

840 (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine on mitochondrial

841 membrane potential

842 (a) P. falciparum K1 strain trophozoites stained with 10 µM draq5 and 200 nM

843 rhodamine123 visualised under fluorescence microscope at 100x magnification, (b)

844 and (c) Changes in mitochondrial membrane potential observed on treatment with IC50

845 of atovaquone, emetine, (-)-R,S-dehydroemetine and (-)-S,S-dehydroisoemetine

846 Fig 10: CalcuSyn based median effect plot, dose-effect curve and isobologram for

847 atovaquone and proguanil

848 Fig 11: Calcusyn based median effect plot, dose-effect curve and isobologram for

849 atovaquone and (-)-R,S-dehydroemetine

38 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.

850 Fig 12: Calcusyn based median effect plot, dose-effect curve and isobologram for

851 proguanil and (-)-R,S-dehydroemetine

852 Fig 13: Synthesis of 2, 3-dehydroemetine.

853 Conditions: a) i. iodomethane, ethanol, rt, 24 h, ii. 2, KOAc, reflux, 3 h, 72%; b)

854 malononitrile, ammonium acetate/acetic acid, toluene, reflux, 2.5 h, 69%; c) i. 20%

855 HCl, reflux, 5 h, ii. methanolic HCl, rt, 18 h, 45%; d. (+)-dibenzoyl tartrate, methanol,

856 two recrystallizations, 25%; e) i. 3M HCl, reflux, 90 min, ii. 3,4-

857 dimethoxyphenethylamine, xylenes, reflux, 18 h, iii. POCl3, benzene, reflux, 1 h, 28%;

858 f) NaBH4, methanol, rt, 1 h, 81% of a 1:1 diastereomeric mixture.

859 7: (S)-2-((R)-6,7-Dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-3-ethyl-9,10-

860 dimethoxy-1,6,7,11b-tetrahydro-4H-pyrido[2,1-a]isoquinoline * dihydrobromide

861 8: (S)-2-((S)-6,7-Dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-3-ethyl-9,10-

862 dimethoxy-1,6,7,11b-tetrahydro-4H-pyrido[2,1-a]isoquinoline * dihydrobromide

863

864 Acknowledgement:

865 We would like to acknowledge the receipt of Pump Priming Award from the University

866 of Salford, Manchester.

867

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1002 57. Who., 2017. World malaria report 2017. World Health Organization.

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1024 S1 Figure: Overlay of docked pose of emetine (pink) and its cryo-EM geometry (green).

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43 bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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. bioRxiv preprint doi: https://doi.org/10.1101/538413; this version posted February 1, 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.