Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti

Abstract

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and 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 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. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC₅₀ 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 A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are 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.

Keywords: Aedes; camptothecin; insecticide; mosquito; rubitecan; vector.

Figure 1

Screening 400-compound MMV Pandemic Response Box chemical library in an A. aegypti larval motility assay led to identification of hit compound rubitecan. (a) Primary screen. Each point is effect of one compound on motility at 2 h and 24 h, normalized to motility at 0 h timepoint. n = 3. DMSO-only and deltamethrin were negative and positive control compounds, respectively. Blue points indicate 14 compounds that were selected as candidate hit compounds. (b) Secondary screen, showing effects of each compound on motility after 24 h. n = 10. * indicates p < 0.05 compared to that of DMSO-only control (Dunnett’s test). Dots indicate individual outlying datapoints beyond the boxplot whiskers. (c) Structure of rubitecan.

Figure 2

Retesting actions on larval motility of rubitecan and related compounds prepared freshly from solid material. (a) Retesting in A. aegypti larval motility assay of rubitecan prepared from solid material and testing of two related compounds, camptothecin, and topotecan, also prepared from solid material. Compounds were screened at 100 µM. Black dots indicate the within-batch median movement score for each of n = 5 biological replicates (batches of independently hatched mosquito larvae). Blue bars indicate between-batch median. A one-way ANOVA showed a significant effect of compound treatment F(3,16) = 22.0 p = 6.32 × 10⁻⁶. A posthoc Dunnett’s test was then used to compare compound treatments with the DMSO-only control. *** indicates p < 0.001. (b) Structure of camptothecin. (c) Structure of topotecan. (d) Time-lapse montage of representative assay wells. This is presented as video in Video S1.

Figure 3

Concentration dependence of actions on larval motility of (a) camptothecin and (b) rubitecan. Curve fitted using the 4-factor log-logistic model. n = 3. Each point shows the measured movement score for a single well of mosquito larvae treated with the indicated concentration. Dotted lines indicate EC₅₀.

Figure 4

Assessing effects of camptothecin on adult A. aegypti. (a) Diagrammatic representation of methodological process. (b) Effect of camptothecin concentration on percentage lethality across entire experiment—each point represents percentage lethality in each experimental replicate (n = 3). (c) Effect of camptothecin concentration on percentage of total females which died indicating period during experiment when death occurred (red = 0–96 h, yellow = 96–120 h) or survived to end of the experiment (grey). (d) Effect of camptothecin concentration on number of eggs laid per individual mosquito. Each point represents number of eggs laid by an individual female (n = 30 per condition). (e) Effect of camptothecin concentration on number of larvae hatched per individual mosquito. Each point represents number of larvae hatched from an individual female (n = 30 per condition). (f) Effect of camptothecin concentration on hatch percentage. Each point represents the proportion of eggs which hatched for each individual female (n = 30 per condition). No females laid eggs and so no hatch percentage could be calculated where (n/a) is noted. Vertical blue lines indicate mean and significance as determined using a Tukey posthoc assessment is indicated as follows on (b,d–f) (**** < 0.0001, *** < 0.001, ** < 0.01, ’absence of bracket’ > 0.05).

Figure 5

Conservation of camptothecin binding site in topoisomerase I between insects and vertebrates. Residues of TOP1 that make direct contact with topotecan in a crystal structure [38] are highlighted above alignment.