Neurotoxicity and Mode of Action of N,N-Diethyl-Meta-Toluamide (DEET)

Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD₅₀ ca. 1.5 µg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC₅₀ values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6–12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC₅₀: 120 µM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na⁺ and K⁺ channels in patch clamped rat cortical neurons, with IC₅₀ values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans.     

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

Background

Chemicals are used on bed nets in order to prevent infected bites and to kill aggressive malaria vectors. Because pyrethroid resistance has become widespread in the main malaria vectors, research for alternative active ingredients becomes urgent. Mixing a repellent and a non-pyrethroid insecticide seemed to be a promising tool as mixtures in the laboratory showed the same features as pyrethroids.

Methodology/Principal Findings

We present here the results of two trials run against free-flying Anopheles gambiae populations comparing the effects of two insect repellents (either DEET or KBR 3023, also known as icaridin) and an organophosphate insecticide at low-doses (pirimiphos-methyl, PM) used alone and in combination on bed nets. We showed that mixtures of PM and the repellents induced higher exophily, blood feeding inhibition and mortality among wild susceptible and resistant malaria vectors than compounds used alone. Nevertheless the synergistic interactions are only involved in the high mortality induced by the two mixtures.

Conclusion

These field trials argue in favour of the strategy of mixing repellent and organophosphate on bed nets to better control resistant malaria vectors.     

The mysterious multi-modal repellency of DEET

DEET is the most effective insect repellent available and has been widely used for more than half a century. Here, I review what is known about the olfactory and contact mechanisms of DEET repellency. For mosquitoes, DEET has at least two molecular targets: Odorant Receptors (ORs) mediate the effect of DEET at a distance, while unknown chemoreceptors mediate repellency upon contact. Additionally, the ionotropic receptor Ir40a has recently been identified as a putative DEET chemosensor in Drosophila. The mechanism of how DEET manipulates these molecular targets to induce insect avoidance in the vapor phase is also contested. Two hypotheses are the most likely: DEET activates an innate olfactory neural circuit leading to avoidance of hosts (smell and avoid hypothesis) or DEET has no behavioral effect on its own, but instead acts cooperatively with host odors to drive repellency (confusant hypothesis). Resolving this mystery will inform the search for a new generation of insect repellents.     

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.     

An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.     

Different Repellents for Aedes aegypti against Blood-Feeding and Oviposition

Methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA) and butyl anthranilate (BA) were previously shown to repel Aedes aegypti mosquitoes from landing on human skin. However, the effect of these compounds on the orientation of flying mosquitoes in a choice situation and their effect on mosquito oviposition are not yet known. Here, we used a modified Y-tube olfactometer to test the effect of these compounds on the orientation of Aedes aegypti flying towards skin odor (human fingers), and we tested their effect on Aedes aegypti oviposition choice in a cage assay. In both behavioral situations we compared the effect to the well-documented repellent N,N-diethyl-meta-toluamide (DEET). MDA, EA, and DEET inhibited Aedes aegypti from flying towards skin odor while BA had no such effect. Conversely, MDA had no effect on oviposition while EA, BA, and DEET deterred oviposition, with the strongest effect observed for BA. Thus, we confirm that EA and DEET are generally repellent, while MDA is repellent only in a host-seeking context, and BA is deterrent only in an oviposition context. These compounds appear of potential use in mosquito control programs.     

The mysterious multi-modal repellency of DEET

DEET is the most effective insect repellent available and has been widely used for more than half a century. Here, I review what is known about the olfactory and contact mechanisms of DEET repellency. For mosquitoes, DEET has at least two molecular targets: Odorant Receptors (ORs) mediate the effect of DEET at a distance, while unknown chemoreceptors mediate repellency upon contact. Additionally, the ionotropic receptor Ir40a has recently been identified as a putative DEET chemosensor in Drosophila. The mechanism of how DEET manipulates these molecular targets to induce insect avoidance in the vapor phase is also contested. Two hypotheses are the most likely: DEET activates an innate olfactory neural circuit leading to avoidance of hosts (smell and avoid hypothesis) or DEET has no behavioral effect on its own, but instead acts cooperatively with host odors to drive repellency (confusant hypothesis). Resolving this mystery will inform the search for a new generation of insect repellents.     

Mechanistic Insights into Allosteric Structure-Function Relationships at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M₁ muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M₁ mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M₁ mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400^7.35 in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M₁ mAChR-targeting PAMs.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

Generic insect repellent detector from the fruit fly Drosophila melanogaster

Background

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Methodology

Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.

Conclusions

For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.     

Laboratory Evaluation of Controlled-release Repellent Treated Pulp Fabric on Human Volunteers Against Mosquito Vectors

Three controlled‐release personal‐use pulp fabric impregnated insect repellent formulations of 5% N, N‐diethyl‐3‐methylbenzamide (DEET), and 10% and 15% neem oils were evaluated in an environmental chamber on volunteers for their repellent efficacy against three mosquito species, Culex pipiens pallens, Aedes aegypti and Ochlerotatus togoi. The 5% DEET formulation showed significant repellency in pulp fabric (5 mm in width) against Culex pipiens pallens and Aedes aegypti (P < 0.05), providing an average repellency of 88.0%, 66.3%, and 46.8% of Cx. pipiens pallens, Ae. Aegypti and O. togoi bites, respectively, during the 6 hours of exposure period. Against night‐biting mosquitoes Cx. pipiens pallens, the DEET formulation provided mostly complete protection for at least 4 hours after the application. In pulp fabric of 10 mm in width, the 5% deet formulation showed significantly the highest repellency among the repellents against O. togoi (P<0.05), providing an average repellency of 52.3% during the 6 hours of exposure period. However, the pulp fabrics treated with 10% and 15% neem oil were less effective than 5% DEET against three mosquito species. This study demonstrated the potential of 5% DEET as pulp fabric repellent against both day‐ and night‐biting mosquitoes.     

三色迷彩油添加避蚊胺对白纹伊蚊驱避效果的实验室评价

参照中华人民共和国国家标准GB/T 17322.10-1998和《军队特需药品研发勤务技术指导原则(试行)》规定的测试方法,测试了避蚊胺和水溶性二氧化钛处理迷彩油(CFP)对白纹伊蚊Aedes albopictus的驱避效果,为研发我军专用的防蚊虫迷彩油提供依据。结果表明:在3种颜色迷彩油中加入DEET后,不影响DEET的驱蚊效果;添加不同浓度DEET对蚊虫的驱避效果差异显著,20%DEET和25%DEET+迷彩油对白纹伊蚊的平均防护时间均达到或超过10 h,达到《军队特需药品研发勤务技术指导原则(试行)》规定的标准,15%DEET+迷彩油平均防护时间不足10 h,未达《军队特需药品研发勤务技术指导原则(试行)》规定的标准。研究表明:将适量DEET与迷彩油混合涂抹于皮肤可能作为演习或战时的伪装和防蚊的一种有效的综合保护措施。     

Field Evaluation of Picaridin Repellents Reveals Differences in Repellent Sensitivity between Southeast Asian Vectors of Malaria and Arboviruses

Scaling up of insecticide treated nets has contributed to a substantial malaria decline. However, some malaria vectors, and most arbovirus vectors, bite outdoors and in the early evening. Therefore, topically applied insect repellents may provide crucial additional protection against mosquito-borne pathogens. Among topical repellents, DEET is the most commonly used, followed by others such as picaridin. The protective efficacy of two formulated picaridin repellents against mosquito bites, including arbovirus and malaria vectors, was evaluated in a field study in Cambodia. Over a period of two years, human landing collections were performed on repellent treated persons, with rotation to account for the effect of collection place, time and individual collector. Based on a total of 4996 mosquitoes collected on negative control persons, the overall five hour protection rate was 97.4% [95%CI: 97.1–97.8%], not decreasing over time. Picaridin 20% performed equally well as DEET 20% and better than picaridin 10%. Repellents performed better against Mansonia and Culex spp. as compared to aedines and anophelines. A lower performance was observed against Aedes albopictus as compared to Aedes aegypti, and against Anopheles barbirostris as compared to several vector species. Parity rates were higher in vectors collected on repellent treated person as compared to control persons. As such, field evaluation shows that repellents can provide additional personal protection against early and outdoor biting malaria and arbovirus vectors, with excellent protection up to five hours after application. The heterogeneity in repellent sensitivity between mosquito genera and vector species could however impact the efficacy of repellents in public health programs. Considering its excellent performance and potential to protect against early and outdoor biting vectors, as well as its higher acceptability as compared to DEET, picaridin is an appropriate product to evaluate the epidemiological impact of large scale use of topical repellents on arthropod borne diseases.     

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca²⁺-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.     

Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

Background

DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).

Methodology/Principal Findings

Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(−)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs.

Conclusions/Significance

These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.     

Mosquito repellents in frog skin

The search for novel insect repellents has been driven by health concerns over established synthetic compounds such as diethyl-m-toluamide (DEET). Given the diversity of compounds known from frog skin and records of mosquito bite and ectoparasite infestation, the presence of mosquito repellents in frogs seemed plausible. We investigated frog skin secretions to confirm the existence of mosquito repellent properties. Litoria caerulea secretions were assessed for mosquito repellency by topical application on mice. The secretions provided protection against host-seeking Culex annulirostris mosquitoes. Olfactometer tests using aqueous washes of skin secretions from L. caerulea and four other frog species were conducted to determine whether volatile components were responsible for repellency. Volatiles from Litoria rubella and Uperoleia mjobergi secretions were repellent to C. annulirostris, albeit not as repellent as a DEET control. The demonstration of endogenous insect repellents in amphibians is novel, and demonstrates that many aspects of frog chemical ecology remain unexplored.     

Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor

The M₃ muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure­-activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M₃ mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M₃ mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M₁, M₂, and M₄ mAChRs, and moderate selectivity over the M₅ mAChR, indicating that compound 9 is an M₃-preferring M₃/M₅ dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M₃ mAChR for further investigation of its pharmacological function both in vitro and in vivo.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

Sodium channel activation underlies transfluthrin repellency in Aedes aegypti

BackgroundVolatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial.Methodology/Principal findingsHere, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency.Conclusions/SignificanceThese results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.