UC Santa Cruz

Each year, mosquito-borne diseases such as Malaria, Dengue, and Zika continue to affect millions of people worldwide. New approaches for mosquito vector control are imperative as current insecticides are not only losing efficacy towards mosquitoes due to rising resistance but also bioaccumulate, resulting in adverse ecological effects due to their non-selectivity amongst target and non-target organisms. The major challenges to developing new control agents are ensuring human safety, limiting ecological off-target effects, and confronting resistance. To address these, novel and efficient high throughput screening platforms are being used to identify selective mosquitocidal agents that exhibit novel mechanisms of action.

Our library of marine microbial-derived natural products was screened in a novel mosquito cell-based assay to identify metabolites lethal to mosquito cells but inactive against other insect cell lines such as Drosophila melanogaster (Kc and S2R+) and Spodoptera frugiperda (SF9). One compound, a novel boron-containing macrolide, “NP-34,” was identified as a selective mosquito toxin as it led to approximately 80% Anopheles gambiae cell death at a concentration of 50 nM whereas toxicity to D. melanogaster and S. frugiperda cell lines was negligible with less than 10% cell death at the same concentration. This selectivity was further observed when tested against larval and adult stage mosquitoes at concentrations like that of permethrin treatment. Additionally, the compound has impressive activity against cells, larvae, and mosquitoes from permethrin-resistant strains of Aedes aegypti. Preliminary data with RNAseq and genomic data of Culex quinquefasciatus resistant mutants has revealed potential markers of resistance within the vector, which can likewise shed light onto NP-34’s mechanism of action.

Based on the cellular and subsequent mosquito and mouse studies, we believe this unique macrolide is a promising candidate for the development of an eco-friendly mosquitocide. As we sought to isolate more NP-34 and analogs to continue biological testing to find the best in vivo potency, there were extensive challenges including validating the presence of boron in analogs and issues in fermentation leading to diminishing yields of material. To aid in screening and validation, a pulse sequence was applied to 11B NMR to increase the sensitivity of the experiment by eliminating external probe noise that was hindering data acquisition. This allowed for detection of low levels of boron rapidly from crude microbial extracts. The feasibility of this experiment was confirmed with the biologically relevant boronic compounds Aplasmomycin and autoinducer-2. Additional methods for identification explored included LC- and ICP-MS.

Once the 11B NMR was optimized as a screening method, the experiment was used to determine the best conditions for fermentation and isolation of NP-34 and other co-eluting analogs. Improvements including shortened fermentation time, swapping of chromatographic techniques, and stripping of all acid buffer, resulted in a 12 time increase in NP-34 yield compared to when yields started diminishing. Along the course of optimization, various analogs have been isolated and characterized for biological testing, while other analogs have been generated with late-stage functionalization. Some of these efforts included installing clickable handles for affinity chromatography that were unsuccessful. To help start understanding the biology and reactivity of NP-34 and similar analogs, material has been crystalized to get confirmational information. This is all in effort to understand and confirm NP-34’s selective mosquitocidal activity, to be able to carry it forward for development as an eco-friendly insecticide.