key: cord-0328175-gvvwflmc authors: Partridge, Frederick A.; Poulton, Beth C.; Lake, Milly A.I.; Lees, Rebecca A.; Mann, Harry-Jack; Lycett, Gareth J.; Sattelle, David B title: Actions of camptothecin derivatives on larvae and adults of the arboviral vector Aedes aegypti date: 2021-09-06 journal: bioRxiv DOI: 10.1101/2021.09.06.458863 sha: 73dfb025476a624fb3172485eb33fed83ff3cc25 doc_id: 328175 cord_uid: gvvwflmc Mosquito-borne viruses including dengue, Zika and Chikungunya viruses as well as parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti (New Orleans strain) were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. Of the 400 compounds screened, we identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that significantly reduced Ae. aegypti larval motility compared to DMSO controls. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50s of 25.5 ± 5.0 μM and 22.3 ± 5.4 μM respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to Ae. aegypti adults at 100 μM and 10 μM and completely blocked egg laying when fed at 100 μM. Camptothecin and its derivatives, inhibitors of topoisomerase I, have known activity against several agricultural pests and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries. Both humans and mosquitoes express the highly conserved topoisomerase I target, however, the design of derivatives with differing pharmacokinetic properties may offer a promising route towards the development of insect-specificity of this chemistry. where the virus, spread via the mosquito Aedes aegypti, was introduced into the Americas and then 55 spread rapidly, infecting perhaps 500,000 people, underscores vividly the pandemic potential of vector-56 borne diseases (Musso et al., 2019) . 57 58 An important method for reducing the impact of vector-borne diseases is to target the vector. In the case 59 of malaria, the incidence of clinical disease fell by 40% between 2000 and 2015, and it has been 60 estimated that over half of this reduction was due to insecticide-treated nets (ITNs) that target the 61 disease-transmitting Anopheles adult mosquitoes (Bhatt et al., 2015) . However, ITNs have limitations, 62 in particular the growing resistance to pyrethroids and other insecticides (Hemingway et al., 2016) . 63 ITNs are less useful for the control of pathogens spread by Ae. aegypti, which include chikungunya, 64 dengue, yellow fever and Zika, as the mosquitoes prefer to feed outdoors at dawn and dusk. Larval 65 source management is also important for vector control. This targets the larval stages of mosquitoes, 66 which in the case of Ae. aegypti develop primarily in artificial, aquatic, urban environments, such as 67 used tyres, drains, and sewers, with the aim of reducing the prevalence of the adult vector. Application 68 of mosquito larvicides is an important component of larval source management. The major classes of 69 larvicides in current use are chemical insecticides, typically organophosphates, such as temephos, the 70 sodium channel targeting pyrethroids, bacterial larvicides such as Bacillus thuringiensis toxin, which 71 impact gut cell membrane permeability, and insect growth regulators, like diflubenzuron, which block 72 development of the insect. 73 However, resistance to current larvicides is a problem, with, for example, Ae. aegypti resistance to 74 temephos, the major organophosphate larvicide, widespread in Brazil (Valle et al., 2019) . And whilst 75 pyrethroid use in water sources is now prohibited because of toxicity to fish (Farag et al., 2021) , 76 pyrethroids used in agriculture are known to leach into aquatic ecosystems. Even at the low 77 concentrations observed, early larval exposure is thought to exacerbate the development of pyrethroid 78 resistance in adults (Churcher et al., 2016) in areas where mosquito control is needed (Diabate et al., 79 2002 ) and that such larval stressors can impact the adult immune response (Hauser and Koella, 2020) . where the median movement score at 2 h and/or 24 h was < 40% of the same wells at 0 h. 118 The secondary screen was carried out on two occasions, each time with five independent assay plates 119 (n = 10). The 24-h time point was analysed. For each assay plate, the median movement score of the 120 negative and positive control replicate wells was calculated and used for subsequent analysis. The effect 121 of compound treatment was determined using a one-way ANOVA test, and the identity of active 122 compounds was then determined by Dunnett's test, in comparison with the DMSO negative control. 123 Rubitecan, and the related compounds camptothecin and topotecan, were then re-tested using solid 124 material at an assay concentration of 100 µM. Camptothecin (208925), rubitecan (9-nitrocamptothecin, 125 R3655) and topotecan hydrochloride (T2705) were obtained from Merck Life Science. 126 Concentration response curves were fitted using the R package drc (Ritz et al., 2015) . 127 Adult treatment assays 128 10 mM camptothecin stock was made in DMSO. Blood containing 100 µM and 10 µM camptothecin, 129 1% and 0.1% DMSO (as solvent controls respectively) and no additions (no DMSO control) was fed to 130 3 pools of 10 New Orleans adult (5-7 days old) females for each compound-concentration using a 131 hemotek system. Adults were allowed 20 minutes to feed and any unfed adults were removed. Adults 132 were maintained in paper cups supplied ad libitum with 10% sugar solution on cotton wool. 133 At 4 days post blood feeding, the surviving individuals (mortality recorded -0-96 h) were transferred 134 to a 5 mL bijou tube with a 2.5cm Whatman paper no.3 disk soaked in water pushed to the bottom to 135 form a slight concave shape with a small pool of water for egg laying. Females were held in these tubes 136 for 24 h to permit laying after which females were removed (mortality recorded -96-120 h). 137 Lids were removed from tubes which were batch covered with netting to permit drying of the filter 138 paper and eggs but preventing undesired egg laying by other mosquitoes. 7 days later 2 mL of yeast 139 suspension (1 yeast tablet dissolved/suspended in 500 mL water) was added to each tube to stimulate 140 larval hatching and netting was replaced. 2 days later the number of larvae hatched, and number of eggs 141 laid were counted. 142 Differences in lethality, the number of eggs and larvae, and the percentage of laid eggs that hatched 143 were assessed with a Tukey HSD post hoc test in R. 144 145 The actions on Ae. aegypti larval motility of each of the 400 compounds in the Pandemic Response Box 147 was measured using the INVAPP system at 0, 2 and 24-hour timepoints. Fig 1A shows the effects of 148 each compound tested, as well as DMSO-only and deltamethrin controls, on motility at 2h and 24 h. 149 Deltamethrin at 10 µM effectively paralyses the larvae, but some compounds in the library showed 150 some reduction in larval motility. The data for all 400 compounds in the MMV Pandemic Response 151 Box are provided in the S1 Table. Fourteen compounds, highlighted in blue in Fig 1A, that reduced 152 motility at 2h and/or 24 h to less than 40% of controls were selected as candidate hits and taken forward 153 to a secondary screen. These compounds were retested at 10 µM in a secondary screen (Fig 1B) The structure of rubitecan is shown in Fig 1C. Rubitecan is a topoisomerase inhibitor, originally 159 developed as a potential therapy for various cancers (Clark, 2006) . 160 Having pursued these initial studies using library material stored as DMSO stocks, it was important to 171 confirm the activity of rubitecan in the larval motility assay using solutions freshly prepared from solid 172 material. Rubitecan is a synthetic derivative of camptothecin (Fig 2B) , an alkaloid isolated from 173 Camptotheca acuminata, a tree native to China. We also wanted to determine the activity of 174 camptothecin itself, as well as topotecan (Fig 2C) , another camptothecin derivative, which is approved 175 for the treatment of cervical, ovarian and small cell lung cancers. These compounds were tested in the 176 same 24-hour treatment larval motility assay at 100 µM. These results are shown in Fig 2A. A one-way 177 ANOVA showed a significant effect of treatment, F(3,16)=22.0 p=6.32e-06. Dunnett's test was then 178 used to compare each treatment with the DMSO-only control. Camptothecin (P = 8.5e-6), rubitecan (P 179 = 8.3e-6) and topotecan (P = 0.00087) all showed a significant difference in motility compared to the 180 control, although the effect on motility was less in the case of topotecan. Examples of mosquito 181 morphology and movement in wells treated with each compound are presented in the S1 movie. A time-182 lapse montage is also shown in Fig 2D. in Fig 4B and C. There was a significant difference in lethality between treatment groups as determined 228 by one-way ANOVA (F(4,10) = 40.72, p = 3.7x10 -6 ). A Tukey post hoc test (95% CI ± 25.7) revealed 229 significant increased mortality in females fed 100μM camptothecin compared to those fed no compound 230 (+80.0%, p = 9.8x10 -6 ), 1 % DMSO (+76.7%, p = 1.45x10 -5 ) and 10 μM camptothecin (+41.2%, p = 231 0.0026) and in those fed 10 μM camptothecin compared to those fed no compound (+38.8%, p = 0.004) 232 and 0.01% DMSO (+39.4%, p = 0.0036). 233 We also measured the number of eggs laid per adult mosquito (Fig 4D) , the number of larvae that 234 hatched per adult mosquito (Fig 4E) , and the proportion of eggs that hatched for each treated adult 235 mosquito (Fig 4F) . Significant differences in the number of eggs laid were also observed between 236 treatment groups using a one-way ANOVA (F(4,131) = 12.52, p = 1.16x10 -8 ). Females exposed to 100 237 μM camptothecin did not lay any eggs. A Tukey post hoc (95% CI ± 22.19) indicated significant 238 differences in the number of eggs laid by females fed 100 μM camptothecin and females fed no 239 compound (-53.9 eggs, p < 1x10 -7 ), 1% DMSO (-48.13 eggs, p = 2x10 -7 ) and 10 μM camptothecin (-240 40.1 eggs, p = 1.81 x10 -5 ). 241 The number of larvae that hatched also differed significantly by treatment using a one-way ANOVA 242 (F(4,131) = 9.846, p = 5.38x10 -7 ) but the significant reductions observed with a post hoc Tukey (95%CI 243 ± 18.9) were between females fed 100 μM camptothecin and females fed no compound (-38.7 larvae, 244 p = 9x10 -7 ), 1% DMSO (-37.4 larvae, p = 2.3x10 -6 ) and 10 μM camptothecin (-23.7 larvae, p = 245 0.00632). 246 No significant differences in egg laying and number of larvae hatched were observed between females 247 fed 10 μM camptothecin and controls and no significant differences in larval hatch percentage between 248 treatments were detected using a one-way ANOVA (F(3,87) residues that contact the drug in a topotecan / human topoisomerase I crystal structure (Staker et al., 282 2002) conserved across insects (Fig 5) . This likely limits our ability to make more insect-specific 283 derivative molecules by exploiting differences in target binding. 284 However, camptothecin itself has not found use in humans due to problems with pharmacokinetics. 285 These problems include limited water solubility, rapid ring opening in plasma, where the active lactone 286 is converted to an inactive carboxylate in plasma, and variable bioavailability, preventing oral dosing 287 of camptothecin (Gupta et al., 2000; Herben et al., 1998) . The route to clinical approval involved the 288 development of water-soluble analogues of camptothecin, of which the first to be approved was 289 topotecan, for intravenous administration, in 1996. Topotecan has appreciable oral bioavailability, 290 around 35-40% (Herben et al., 1996) , and ten-fold greater stability as the active lactone form in human 291 blood compared to camptothecin (Burke and Bom, 2000) . Topotecan was approved for oral 292 administration in 2007. Interestingly, in our mosquito assay, topotecan was much less active than 293 camptothecin. This suggests that there is potential for identification of camptothecin derivatives that 294 have acceptable safety profiles by exploiting pharmacokinetic differences between target insects and 295 people, such as drug access or metabolism. It should also be noted that organophosphates, currently 296 important mosquito larvicides, have a poor safety record. Organophosphate poisoning, either due to 297 occupational exposure or self-harm, kills an estimated 200,000 people each year (Eddleston and 298 Chowdhury, 2016) . This underscores the need to develop safer effective mosquito larvicides. 299 Use as an adulticide 306 Females fed 100 µM and 10 µM camptothecin in a blood meal demonstrated significantly increased 307 mortality (90% and 48.8% respectively) across the experiment compared to controls. Absolute blocking 308 of egg laying was observed in females fed 100 µM camptothecin but no effect on egg laying, larval 309 hatching or hatch percentage was observed for those fed 10 µM and many laid eggs prior to death during 310 the experiment. This suggests that a concentration between 10 and 100 µM camptothecin would be 311 required to impact the reproductive ability of Ae. aegypti. 312 TOP1 analogues are found in all eukaryotes and appear to be an essential enzyme during development 313 in a wide variety of animals. During the process of DNA replication and transcription TOP1 is 314 responsible for relaxing supercoiled DNA (Li et al., 2017) . Knock outs of TOP1 are embryonically 315 lethal in Mus musculus (Morham et al., 1996) and Drosophila melanogaster (Zhang, CX et al, 2000) . 316 TOP1 has been demonstrated to be essential for larval and pupal growth, oogenesis and embryogenesis 317 in D. melanogaster (Zhang et al., 2000) . Larvae are developing and undergoing more growth, cell 318 replication and differentiation than in adults, which explains the greater susceptibility of larval stages 319 that we observed. 320 Is it feasible to propose the use of a camptothecin derivative to target adult mosquitoes? We note the 321 high concentrations of camptothecin (10 or 100 µM) that needed to be delivered in a blood meal to 322 impact adult survival. Ivermectin is capable of killing Anopheles mosquitoes after they bite a human 323 host who has taken the drug (Smit et al., 2019) . However the concentration of ivermectin required for 324 lethality is in the low nanomolar range (Dreyer et al., 2018) , and it is well tolerated and is used widely 325 for mass drug administration (MDA) control of helminths in areas where mosquitoes and malaria are a 326 problem (Richards, 2017) . We have already discussed the potential anti-viral use of camptothecin 327 derivatives. Clearly compounds with improved potency against mosquitoes, as well as a much-328 improved safety profile, would need to be developed to be useful as anti-viral agents in humans with 329 the additional benefit of controlling blood-feeding insect vectors. 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Clinical 389 pharmacokinetics of camptothecin topoisomerase I inhibitors Clinical Pharmacokinetics of 391 Topotecan An antagonist of the retinoid X receptor reduces the viability of Trichuris muris in vitro Camptothecin (CPT) and its derivatives are known to target 396 topoisomerase I (Top1) as their mechanism of action: did we miss something in CPT analogue 397 molecular targets for treating human disease such as cancer? 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ACS Infect Topoisomerase 1 inhibition suppresses inflammatory genes and 436 protects from death by inflammation Upon entering an age of global ivermectin-based integrated mass drug 438 administration for neglected tropical diseases and malaria Dose-Response Analysis Using R Vector biology meets disease control: using basic research to 442 fight vector-borne diseases Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial) An Integrated Systems Biology Approach Identifies the Proteasome 449 as A Critical Host Machinery for ZIKV and DENV Replication The 452 mechanism of topoisomerase I poisoning by a camptothecin analog Vertical Transmission of Zika Virus in Aedes aegypti Mosquitoes Six Laws of Open Source Drug Discovery Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017 Open Source Drug Discovery with the Malaria Box 462 Compound Collection for Neglected Diseases and Beyond Unpacking the Pathogen Box-An Open Source Tool for Fighting Neglected 464 Tropical Disease Antiviral screen identifies EV71 inhibitors and reveals 466 camptothecin-target, DNA topoisomerase 1 as a novel EV71 host factor Essential functions of DNA 469 topoisomerase I in Drosophila melanogaster Effects of camptothecin and 471 hydroxycamptothecin on insect cell lines Sf21 and IOZCAS-Spex-II We thank Medicines for Malaria Venture for designing and supplying the Pandemic Response Box. 335 336