Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito.

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background in which there is not a clear orthologue in Vectorbase and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (6), chloropyriphos (6.6) and fenthion (3.2) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies.

A drug repurposing screen for whipworms informed by comparative genomics.

Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 µM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug development against whipworms.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Invertebrate neurones, genomes, phenotypic and target-based screening; their contributions to the search for new chemical leads and new molecular targets for the control of pests, parasites and disease vectors.

Insect-borne diseases of humans, animals and plants can be devastating. The direct damage to crops by insect and nematode pests can also severely reduce crop yields and threaten harvests. Parasitic nematodes can impair human health and the health of farm livestock. Effective control for all such pests, vectors and pathogens is required as the economic and health burden can be substantial. Insecticides, nematicides and anthelmintics have been at the forefront of control and will remain important in the immediate future, even as we explore new and more sustainable methods to maintain the necessary disease control and the growth in food supply. Many important chemicals deployed for the control of invertebrate disease vectors and pathogens of humans, agricultural crops and farm livestock are active on ion channels, resulting in rapid actions. Understanding their modes of action has been accelerated by studies on the physiology of identifiable invertebrate excitable cells. Nematode and insect genetic model organisms and comparative genomics have contributed to defining the molecular targets of insecticides and anthelmintics, facilitating target-based screening. Automated phenotyping, which allows high-throughput screening of chemical libraries for new and re-purposed compounds, has been increasingly deployed in the search for new molecules of interest. With a growing world population to be fed and a 20-49% loss of global harvest to pests, we need to maintain control of the pests, parasites and pathogens that threaten global food supply and global health.

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

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.

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

Structural Requirements for Dihydrobenzoxazepinone Anthelmintics: Actions against Medically Important and Model Parasites: Trichuris muris, Brugia malayi, Heligmosomoides polygyrus, and Schistosoma mansoni.

Nine hundred million people are infected with the soil-transmitted helminths Ascaris lumbricoides (roundworm), hookworm, and Trichuris trichiura (whipworm). However, low single-dose cure rates of the benzimidazole drugs, the mainstay of preventative chemotherapy for whipworm, together with parasite drug resistance, mean that current approaches may not be able to eliminate morbidity from trichuriasis. We are seeking to develop new anthelmintic drugs specifically with activity against whipworm as a priority and previously identified a hit series of dihydrobenzoxazepinone (DHB) compounds that block motility of ex vivo Trichuris muris. Here, we report a systematic investigation of the structure-activity relationship of the anthelmintic activity of DHB compounds. We synthesized 47 analogues, which allowed us to define features of the molecules essential for anthelmintic action as well as broadening the chemotype by identification of dihydrobenzoquinolinones (DBQs) with anthelmintic activity. We investigated the activity of these compounds against other parasitic nematodes, identifying DHB compounds with activity against Brugia malayi and Heligmosomoides polygyrus. We also demonstrated activity of DHB compounds against the trematode Schistosoma mansoni, a parasite that causes schistosomiasis. These results demonstrate the potential of DHB and DBQ compounds for further development as broad-spectrum anthelmintics.

Turning a Drug Target into a Drug Candidate: A New Paradigm for Neurological Drug Discovery?

The conventional paradigm for developing new treatments for disease mainly involves either the discovery of new drug targets, or finding new, improved drugs for old targets. However, an ion channel found only in invertebrates offers the potential of a completely new paradigm in which an established drug target can be re-engineered to serve as a new candidate therapeutic agent. The L-glutamate-gated chloride channels (GluCls) of invertebrates are absent from vertebrate genomes, offering the opportunity to introduce this exogenous, inhibitory, L-glutamate receptor into vertebrate neuronal circuits either as a tool with which to study neural networks, or a candidate therapy. Epileptic seizures can involve L-glutamate-induced hyper-excitation and toxicity. Variant GluCls, with their inhibitory responses to L-glutamate, when engineered into human neurons, might counter the excitotoxic effects of excess L-glutamate. In reviewing recent studies on model organisms, it appears that this approach might offer a new paradigm for the development of candidate therapeutics for epilepsy.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Anthelmintic drug discovery: target identification, screening methods and the role of open science.

Helminths, including cestodes, nematodes and trematodes, are a huge global health burden, infecting hundreds of millions of people. In many cases, existing drugs such as benzimidazoles, diethylcarbamazine, ivermectin and praziquantel are insufficiently efficacious, contraindicated in some populations, or at risk of the development of resistance, thereby impeding progress towards World Health Organization goals to control or eliminate these neglected tropical diseases. However, there has been limited recent progress in developing new drugs for these diseases due to lack of commercial attractiveness, leading to the introduction of novel, more efficient models for drug innovation that attempt to reduce the cost of research and development. Open science aims to achieve this by encouraging collaboration and the sharing of data and resources between organisations. In this review we discuss how open science has been applied to anthelmintic drug discovery. Open resources, including genomic information from many parasites, are enabling the identification of targets for new antiparasitic agents. Phenotypic screening remains important, and there has been much progress in open-source systems for compound screening with parasites, including motility assays but also high content assays with more detailed investigation of helminth physiology. Distributed open science compound screening programs, such as the Medicines for Malaria Venture Pathogen Box, have been successful at facilitating screening in diverse assays against many different parasite pathogens and models. Of the compounds identified so far in these screens, tolfenpyrad, a repurposed insecticide, shows significant promise and there has been much progress in creating more potent and selective derivatives. This work exemplifies how open science approaches can catalyse drug discovery against neglected diseases.

Actions on mammalian and insect nicotinic acetylcholine receptors of harmonine-containing alkaloid extracts from the harlequin ladybird Harmonia axyridis.

The harlequin ladybird, Harmonia axyridis (H. axyridis), possesses a strong chemical defence that has contributed to its invasive success. Ladybird beetle defensive chemicals, secreted in response to stress and also found on the coating of laid eggs, are rich in alkaloids that are thought to be responsible for this beetle's toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists, hence we have explored the actions of alkaloids of the ladybird H. axyridis on both mammalian and insect nAChRs. Electrophysiological studies on native and functionally expressed recombinant nAChRs were used to establish whether an alkaloid extract from H. axyridis (HAE) targeted nAChRs and whether any selectivity exists for insect over mammalian receptors of this type. HAE was found to be an inhibitor of all nAChRs tested with the voltage-dependence of inhibition and the effect on ACh EC50 differing between nAChR subtypes. Our finding that an HAE fraction consisting almost entirely of harmonine had a strong inhibitory effect points to this alkaloid as a key component of nAChR inhibitory actions. Comparison of HAE inhibition between the mammalian and insect nAChRs investigated indicates some preference for the insect nAChR supporting the view that investigation of ladybird alkaloids shows promise as a method for identifying natural product leads for future insecticide development.

Whipworm and roundworm infections.

Trichuriasis and ascariasis are neglected tropical diseases caused by the gastrointestinal dwelling nematodes Trichuris trichiura (a whipworm) and Ascaris lumbricoides (a roundworm), respectively. Both parasites are staggeringly prevalent, particularly in tropical and subtropical areas, and are associated with substantial morbidity. Infection is initiated by ingestion of infective eggs, which hatch in the intestine. Thereafter, T. trichiura larvae moult within intestinal epithelial cells, with adult worms embedded in a partially intracellular niche in the large intestine, whereas A. lumbricoides larvae penetrate the gut mucosa and migrate through the liver and lungs before returning to the lumen of the small intestine, where adult worms dwell. Both species elicit type 2 anti-parasite immunity. Diagnosis is typically based on clinical presentation (gastrointestinal symptoms and inflammation) and the detection of eggs or parasite DNA in the faeces. Prevention and treatment strategies rely on periodic mass drug administration (generally with albendazole or mebendazole) to at-risk populations and improvements in water, sanitation and hygiene. The effectiveness of drug treatment is very high for A. lumbricoides infections, whereas cure rates for T. trichiura infections are low. Novel anthelminthic drugs are needed, together with vaccine development and tools for diagnosis and assessment of parasite control in the field.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

C. elegans expressing D76N ß2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis.

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of ß2-microglobulin (D76N ß2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N ß2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N ß2-m expressing worms. We also demonstrated the specificity of the ß2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when ß2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.

Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin.

A possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca2+-signalling, which may contribute to the pathology associated with another serpinopathy, α1-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca2+, its responses to thapsigargin (TG), an ER Ca2+-ATPase blocker, and store-operated Ca2+-entry (SOCE). Our fura2 based Ca2+ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.

Insect toxins - selective pharmacological tools and drug/chemical leads.

Insect toxins comprise a diverse array of chemicals ranging from small molecules, polyamines and peptide toxins. Many target nervous system and neuromuscular ion channels and so rapidly affect the behaviour of animals to which the toxin is applied or injected. Other modes of action have also been identified. Wasps, bees, flies, beetles and ants generate a rich arsenal of channel-active toxins, some of which offer selective pharmacological probes that target particular ion channels, while others act on more than one type of channel. Philanthotoxins from the digger wasp have been fruitful in adding to our understanding of ligand-gated ion channels both in the nervous system and at neuromuscular junctions. Fire ants produce the toxic alkaloid solenopsin, a molecule which has stimulated attempts to generate synthetic compounds with insecticidal activity. Apamin from bee venom targets calcium-activated potassium channels, which can in turn influence the release of neuropeptides. Melittin, another bee venom component, is a membrane-acting peptide. The saliva of the assassin bug contains toxins that target the voltage-gated calcium channels of their insect prey. Certain beetles produce diamphotoxin, a haemolytic peptide toxin with traditional use as an arrow poison and others generate leptinotarsin with similar properties. Mastoparan is a powerful peptide toxin present in the venom of wasps. Its toxic actions can be engineered out leaving a potent antimicrobial molecule of interest. In this short review we describe the actions of selected insect toxins and evaluate their potential as neuroactive pharmacological tools, candidate lead molecules for insect control and therapeutic candidates with potential antimicrobial, antiviral and anti-cancer applications.

2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm.

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle.

The fungal alkaloid Okaramine-B activates an L-glutamate-gated chloride channel from Ixodes scapularis, a tick vector of Lyme disease.

A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400 nA currents in response to 100 µM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ±â€¯0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 µM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 µM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ±â€¯0.04) and the phenylpyrazole fipronil (pIC50 6.90 ±â€¯0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ±â€¯0.43) and may serve as a candidate lead compound for the development of new acaricides.