The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases

Abstract

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

Fig 1. DEET potentiates the carbamate-induced anticholinesterase effect in insect DUM neurons.

A) Dorsal view camera lucida drawing of typical DUM neuron morphology revealed by anterograde cobalt staining performed on a soma located along the midline of the cockroach terminal abdominal ganglion (TAG) of the nerve cord. A, anterior; P, posterior; scale bar 120μm. B) Light micrograph of the whole cell patch-clamp technique adapted on the isolated DUM neuron cell body obtained after enzymatic digestion and mechanical dissociation of the TAG. C) Anticholinesterase effects of the carbamate, propoxur, the anticholinesterase compound BW284c51 and the repellent DEET on the duration of the ACh-induced inward currents (measured at 50% of the maximum current amplitudes) obtained in whole-cell voltage-clamp at a steady-state holding potential of -50 mV. D) Comparative bar graph summarizing the anticholinesterase effect of the specific inhibitor BW284c51 (100nM) and the carbamate, propoxur (prop) (100nM) measured on the duration of the ACh-induced inward currents (measured at 50% of the maximum current amplitudes) obtained in whole-cell voltage-clamp at a steady-state holding potential of -50 mV. E) Concentration-dependent inhibition of the residual AChE activity determined spectrophotometrically induced by propoxur and expressed as percentage of initial activity (i. e., without propoxur). The curve represents the best fit to the data points according to the Hill equation yielding the corresponding IC₅₀ (i.e., the concentration of propoxur that produces 50% inhibition of the AChE enzymatic activity) as illustrated in the comparative bar graph shown in inset. This indicates that isolated DUM neurons express functional AChE. F) Bar graph summarizing the unexpected concentration-dependent effect of DEET on the ACh-induced inward current duration. At low concentration (10nM), DEET produces a more important anticholinesterase effect than those observed with higher concentrations (i.e., 100nM and 1μM). By contrast, DEET (1μM) do not produce any effect on the carbachol(CCh)-induced current. G) Comparative bar graph illustrating the anticholinesterase effects of DEET (10nM) and propoxur (100nM) tested alone and in combination (DEET/propoxur). Pretreatment of DUM neuron with low concentration of DEET (10nM), for 15 minutes, strongly potentiates the propoxur-induced anticholinesterase effect. H) Comparative bar graph showing that synergistic effect between DEET and propoxur is only observed at low concentration of DEET (i. e., 10nM) and not with higher concentration (i. e., 1μM). I) Semi-logarithmic concentration-response curves for the anticholinesterase effect induced by propoxur applied alone and in the presence of 10nM DEET. The sigmoid curves represent the best fit to the mean data points according to the Hill equation yielding the corresponding IC₅₀ of 2.10^-8M and 6.10^-8M estimated for DEET and propoxur applied in combination and for propoxur applied alone, respectively. Number of experiments varies from 10 to 16 cells. Data are means ± S.E.M. ** and ***, values significantly different, p < 0.01 and p<0.001, respectively; ns, not significant (p > 0.05).

Fig 2. Synergism between DEET and propoxur occurs through a positive allosteric-like modulation of insect M1/M3 muscarinic receptors and intracellular calcium-dependent signaling pathways.

A) Bath application of 10nM DEET increases intracellular free calcium concentration ([Ca²⁺]_i) in Fura-2 loaded DUM neurons (inset 2). Note that, under control condition, calcium spark-like events are detected (inset 1). B) Pretreatment with 1μM atropine, a specific antagonist of muscarinic receptors (mAChRs), completely blocks the enhancement of [Ca²⁺]_i produced by 10nM DEET, indicating the involvement of mAChRs. C) Bar graph summarizing the inhibitory effect of M1 and M3 mAChR antagonists pirenzepine (PZP) and 4-DAMP, respectively, on the synergism between DEET and propoxur. D) Bar graph illustrating that TMB-8 (100μM) also completely blocks the synergism between DEET and propoxur. E,F) Characterization of the intracellular calcium-dependent molecular events involved in the synergistic action of DEET on the propoxur-induced anticholinesterase effect. Intracellular application of 0.5mM W7, the calmodulin inhibitor and 50nM calmodulin (CaM) inhibit the positive potentiating effect of DEET on the toxic activity of propoxur. By contrast, KN-62 (10μM), which binds to CaM kinase II and blocks its activation by calmodulin, does not produce any effect (E). If pretreatment with 10μM of U73122, an inhibitor of PI-PLC known to regulate AChE activity, partially counteracts the effect of 0.5mM W7, application of the PI-PLC activator, m-3M3FBS (10μM) produces similar inhibition of the synergism between DEET and propoxur to that of observed with W7 tested alone (F). G) Modulation of the maximum amplitude of muscarine-elicited currents versus the concentration of DEET applied. The limited window of DEET concentration within which a maximum response potentiating effect is observed, is around 10nM (G). For higher DEET concentrations, the sensitizing effect is counteracted and eventually outweighed by an inhibitory action of DEET. Inset illustrates the semi-logarithmic dose-response curve for the muscarine-induced current applied by pressure ejection. Arrow indicates that the maximum current amplitude is obtained for pressure ejection duration of 500ms. H) DEET induces a transient concentration-dependent [Ca²⁺]i rise in Fura-2-loaded DUM neuron cell bodies. The changes in [Ca²⁺]_i response amplitudes and the window of concentrations for DEET action were very similar with those illustrated in 2G. (I) Bath application of MT-7 (30nM), which is known to bind on M1 mAChR allosteric site, partially reversed the inhibitory effect observed for high concentration of DEET. Number of experiments varies from 8 to 13 cells. Data are means ± S.E.M. *, ** and ***, values significantly different, p < 0.05, p < 0.01 and p < 0.001, respectively. ns, not significant (p > 0.05). Scale bar: 20μm.

Fig 3. Synergism between DEET and propoxur is effective in vivo, in female mosquitoes Aedes aegypti.

A) Mortality rates relative to the increasing concentrations of DEET (ng of active ingredient (a.i.) / mg female) applied on the thorax of females Aedes aegypti in the presence/absence of propoxur (red triangles represent the mortality rates induced by increasing doses of DEET alone and blue diamonds represented the increase of mortality rates when increasing doses of DEET were combined with propoxur at LD₁₀). B) Variation of the estimate of DEET/propoxur interaction term in our general linear model relative to the applied doses of DEET. When the interaction term is significantly above 0 (non overlapping of 95%CI), the interaction between DEET and propoxur is synergistic; when the interaction term is significatly below 0, the interaction is antagonistic. C) Proposed model summarizing the essential components of the intracellular signaling pathway that may explain the synergism between DEET and propoxur in insect cell (see text for details). AChE, acetylcholinesterase; CaM, calmodulin; PI-PLC, phosphatidylinositol (PI)-specific phospholipase C; IP3, inositol 1,4,5-triphosphate; IP3R, receptor; mAChR, muscarinic ACh receptor.

Fig 4. DEET interacts with mammal M1 and M3 muscarinic receptors.

The functional properties of DEET are investigated using CHO cells expressing human M1 (hM1) and M3 (hM3) mAChR subtypes. A) Dose-dependent inhibition of [³H]-NMS binding to M1 and M3 human muscarinic ACh receptor (mAChR) subtypes by DEET. The results are expressed as the ratio of the specific [³H]-NMS binding measured with (B) or without DEET (Bo). B-C) Signals acquired for calcium fluorescence after the addition at 20 sec of carbamylcholine (CCh) (300nM) and DEET (1mM) on CHO-hM1 cells (n = 3). DEET inhibition of the Ca²⁺ mobilization after pretreatment of the cells with increasing concentrations of DEET (3nM to 3 mM), followed by a sub-maximal concentration of CCh (100 nM) (C). D-E) in silico Docking of DEET into human M1 and rat M3 mAChRs. The two binding regions (allosteric and orthosteric sites) of DEET molecules (green) in human M1 mAChR are represented (D). In red are shown residues of a M1 mAChR monomer interacting with MT-7 loops previously indentified. Analogous interaction residues from the second M1 mAChR monomer are indicated in blue (D) close to the hypothetical M1 mAChR allosteric site occupied by DEET. The in silico docking results to rat M3 mAChR crystal structure 4DAJ (E) also show that DEET binds on two distinct sites (allosteric and orthosteric sites). Ten DEET poses from each group located in both sites are shown in green. The following colour coding for transmembrane helices used: TM1—orange, TM2—green, TM3—dark blue, TM4—yellow, TM5—red, TM6—magenta, TM7—light blue. ECL, extracellular loop.

Fig 5. Orthosteric and allosteric binding sites of mammal M1 and M3 muscarinic receptors.

In all panels, the M1 and M3 mAChRs orthosteric and allosteric binding sites are shown with the ligand DEET in green. Residues observed in vicinity of DEET in M1 mAChR orthosteric site (A) and allosteric region (B) and in M3 mAChR orthosteric site (C) and allosteric site (D) are shown in licorice representations. The following colour coding for transmembrane helices used: TM1—orange, TM2—green, TM3—dark blue, TM4—yellow, TM5—red, TM6—magenta, TM7—light blue.