Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to variable subunits heteromeric olfactory receptors. Previous studies have shown, however, that specific odor ORco, common subunit receptor heteromers, may allosterically alter function and profoundly affect subsequent behavioral responses. Using an insect cell–based screening platform, we identified characterized several antagonists coreceptor African malaria vector Anopheles gambiae (AgamORco) a small collection natural volatile organic compounds. Because some were previously shown strongly repel Culex mosquitoes, examined bioactivities against Aedes, third major genus Culicidae family. The tested inhibited Ae. aegypti ORco ex vivo repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures elicited higher repellency than single antagonists, competition assays suggested this enhanced repellence is due antagonist interaction with distinct sites. Our results also suggest mosquito additive rather synergistic effects combinations on function. Taken together, these findings provide novel insights concerning molecular aspects Moreover, our demonstrate simple assay be used for identification allosteric modifiers olfactory-driven capable providing personal protection multiple mosquito-borne infectious diseases. Insect receptors (ORs) ligand-gated ion channels expressed neurons inside sensilla (1Sato K. Pellegrino M. Nakagawa T. Vosshall L.B. Touhara channels.Nature. 2008; 452: 1002-1006Crossref PubMed Scopus (699) Google Scholar, 2Smart R. Kiely A. Beale Vargas E. Carraher C. Kralicek A.V. Christie D.L. Chen Newcomb R.D. Warr C.G. Drosophila seven transmembrane domain proteins can signal independently heterotrimeric G proteins.Insect Biochem. Mol. Biol. 38: 770-780Crossref (207) 3Wicher D. Schafer Bauernfeind Stensmyr M.C. Heller Heinemann S.H. Hansson B.S. both cyclic-nucleotide-activated cation 1007-1011Crossref (603) Scholar). Together odorant-binding (OBPs) (4Vogt R.G. Riddiford L.M. Pheromone inactivation moth antennae.Nature. 1981; 293: 161-163Crossref (830) Scholar) odorant-degrading enzymes (5Vogt Prestwich G.D. Kinetic properties sex pheromone-degrading enzyme: sensillar esterase Antheraea polyphemus.Proc. Natl. Acad. Sci. U. S. 1985; 82: 8827-8831Crossref (280) produced accessory cells (6Justice R.W. Biessmann H. Walter M.F. Dimitratos S.D. Woods D.F. Genomics spawns approaches control.BioEssays. 2003; 25: 1011-1020Crossref (27) 7Leal W.S. Odorant reception insects: roles receptors, proteins, degrading enzymes.Annu. Rev. Entomol. 2013; 58: 373-391Crossref (787) 8McIver S.B. Sensilla (Diptera: Culicidae).J. Med. 1982; 19: 489-535Crossref (159) secreted lymph surrounding neurons, ORs constitute gateway pathway associated important survival reproduction (9Suh Bohbot J. Zwiebel L.J. Peripheral signaling insects.Curr. Opin. 2014; 6: 86-92Crossref (114) complexes consist obligatory highly conserved subunit, (10Benton Sachse Michnick S.W. Atypical membrane topology vivo.PLoS 2006; 4: e20Crossref (656) 11Jones W.D. Nguyen T.A.T. Kloss B. Lee K.J. Functional conservation gene across 250 million years evolution.Curr. 2005; 15: R119-R121Abstract Full Text PDF (201) 12Larsson Domingos A.I. Jones Chiappe M.E. Amrein Or83b encodes broadly essential olfaction.Neuron. 2004; 43: 703-714Abstract (843) 13Neuhaus E.M. Gisselmann G. Zhang W. Dooley Störtkuhl Hatt heterodimerization system melanogaster.Nat. Neurosci. 8: 15-17Crossref (231) 14Vosshall A unified nomenclature coreceptor.Chem. Senses. 2011; 36: 497-498Crossref (205) Scholar), one many ligand-binding ORx (12Larsson 15Vosshall Wong A.M. Axel An sensory map fly brain.Cell. 2000; 102: 147-159Abstract (760) as yet undetermined molar ratios. act either agonists or ORx-specific manner (16Carey A.F. Wang Su C.Y. Carlson J.R. gambiae.Nature. 2010; 464: 66-71Crossref (352) 17Hallem E.A. Nicole Fox Olfaction: human-sweat odorant.Nature. 427: 212-213Crossref (154) 18Nakagawa Sato Amino acid residues contributing complex.PLoS One. 2012; 7: e32372Crossref (97) 19Nichols A.S. Luetje C.W. Subunit contributions function: channel block recognition.Chem. 781-790Crossref (58) 20Nichols Transmembrane segment 3 melanogaster 85b contributes ligand-receptor interactions.J. Chem. 285: 11854-11862Abstract (59) 21Tsitoura P. Andronopoulou Tsikou Agalou Papakonstantinou M.P. Kotzia G.A. Labropoulou V. Swevers L. Georgoussi Z. Iatrou Expression lepidopteran cells.PLoS 5: e15428Crossref (40) 22Wang Carey Molecular basis coding gambiae.Proc. 107: 4418-4423Crossref (250) In cell cultures, homomeric formed activated (OAs) such VUAA1 OrcoRAM2 (23Bohbot J.D. Dickens J.C. modulation: ternary paradigm mode action repellents.Neuropharmacology. 62: 2086-2095Crossref (48) 24Chen Identification new co-receptor subunit.PLoS 7e36784Crossref 25Jones P.L. Pask G.M. Rinker D.C. agonism channels.Proc. 108: 8821-8825Crossref (175) previous studies, utilized OBPs (AgamOBPs; (26Andronopoulou Douris Specific interactions among gambiae.Insect 797-811Crossref 27Biessmann Q.K. Le Microarray-based survey subset putative genes 14: 575-589Crossref (80) 28Biessmann Isolation cDNA clones encoding odourant from antennae malaria-transmitting mosquito, 2002; 11: 123-132Crossref (90) 29Li Z.X. Pickett J.A. Field Zhou J.J. expression malaria-carrying arabiensis.Arch. Physiol. 175-189Crossref (57) 30Xu P.X. Smith D.P. family atypical 12: 549-560Crossref (156) 31Zhou He X.L. yellow fever Aedes aegypti: genome annotation comparative analyses.Insect 17: 147-163Crossref (129) Scholar)) tools discovery compounds (VOCs) modifying olfaction-mediated (32Biessmann M.R. Eliopoulos Guerin P.M. Justice Krober Marinotti O. Tsitoura protein 1 (AgamOBP1) mediates indole recognition female mosquitoes.PLoS 5e9471Crossref (155) 33Kröber Koussis Bourquin Konstantopoulou Awolola T.S. Dani F.R. Qiao Pelosi Odorant-binding protein-based spatial repellents 2018; 96: 36-50Crossref (11) This effort resulted strong repellent activities (33Kröber suggesting existence phylogenetically mechanisms outputs mosquitoes. Further revealed most potent acted inhibitors AgamOR blocked odorant-specific responses interacting directly AgamORco (34Tsitoura Inhibition repellents.J. 2015; 290: 7961-7972Abstract (20) addition, An. ORx/Orco functional terms potency efficacy two orders magnitude presence OA (35Tsitoura Positive modulation agonists.Front. Cell 2016; 10: 275Crossref (10) These induction conformational rearrangements ligand-bound ORx/ORco caused resulting inward currents into receptor-expressing cells. view given demonstrated importance functionality OR heteromers OR-dependent (36DeGennaro McBride C.S. Seeholzer Dennis E.J. Goldman Jasinskiene N. James A.A. Orco mutant lose preference humans not DEET.Nature. 498: 487-491Crossref (212) 37Franco T.A. Oliveira D.S. Moreira Leal Melo A.C.A. Silencing RproOrco affects physiology behavior Chagas disease Rhodnius prolixus.Insect 69: 82-90Crossref (28) 38Koutroumpa F.A. Monsempes François De Cian Royer Concordet J.P. Jacquin-Joly Heritable editing CRISPR/Cas9 induces anosmia crop pest moth.Sci. Rep. 29620Crossref (51) 39Liu Q. Liu Zeng Hao Huang Y. Deletion Bombyx mori (BmOrco) impairs sensitivity silkworms.Insect 2017; 86: 58-67Crossref (36) 40Soffan Antony Abdelazim Shukla Witjaksono Aldosari S.A. Aldawood RferOrco reduces response pheromones red palm weevil, Rhynchophorus ferrugineus.PLoS 11e0162203Crossref (16) 41Trible Olivos-Cisneros McKenzie S.K. Saragosti Chang N.C. Matthews B.J. Oxley P.R. Kronauer D.J.C. mutagenesis causes loss antennal lobe glomeruli impaired social ants.Cell. 170: 727-735.e710Abstract (106) 42Yan Opachaloemphan Mancini Yang Gallitto Mlejnek Leibholz Haight Ghaninia Huo Perry Slone X. Traficante Penick C.A. et al.An engineered orco mutation produces aberrant defective neural development 736-747.e739Abstract (95) 43Zhang Gao Dendroctonus armandi leads EAG declining host volatiles.Sci. 23136Crossref (23) employed toward rapid detection potential AgamORco. relies stable constitutively expressing luminescence-emitting calcium biosensor reporter protein. Here, report VOCs plant, arthropod, bacterial origins modulators AgamORco-specific antagonists. Considering high degree phylogenetic its relevance, which was inhibiting activity repelling at least genera, Culex, whether active Aedes. Two significant inhibition mediated Xenopus laevis oocytes (AaegORco). Examination bioactivity well binary thereof, available laboratory populations albopictus avoidance behavior. Some anosmia-like similar equivalent doses N,N-diethyl-3-methylbenzamide (DEET). Antagonist point simultaneous OA-binding site more alternative sites other plausible cause observed mixtures. platform study exploits property homomers form cultured 35Tsitoura photoprotein Photina detects entry Ca2+ ions upon activation. protocol, performed 96-well format, involved sequential addition compound N-(4-ethylphenyl)-2-{[4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl]thio}acetamide, Receptor Activator Molecule, ORcoRAM2, OA, 100 ?M concentrations, transformed (Fig. 1). Similarly working scheme presented earlier general principle agonist/antagonist has been ethanol solvent devoid ORco-binding would allow influx cellular luminescence agonist, whereas acting prevent, partially completely, emission secondary agonist. same allows agonists. case, expected hence emission, no known agonist after dissipation first burst, owing temporary For hits, set arbitrarily maximum 60% normal i.e., 40% response. case agonists, primary screened greater relative obtained 2). examination 50 (Table S1) control isopropyl cinnamate (IPC) (compound II; (44Christophers S.R. Mosquito repellents; being work inquiry, Cambridge, 1943-5.J. Hyg. (Lond). 1947; 45: 176-231Crossref (21) initial screen five hits antagonistic carvacrol (CRV) cumin alcohol (CA) (compounds I III, respectively), effective spp inhibit extent determined time contrast, relevant information existed three #4 (linalyl acetate [LA]), #39 ((2E,4E)-2,4-octadienal [OCT]), #45 ((1S)-3-carene [CAR]) 2 Table S1). No inducing found VOC collection. Although #5, 8, 10, 13, 14, 17, 18 produced, notable suggestive agonist-like behavior, ranged between 20% 30% considerably lower minimum limit noted, further. quantitative assessment undertaken determining OA-dependent increasing concentrations. As depicted dose–response curves Figure 3, all antagonize dose-dependent IC50 values ranging 23 83 ?M. To confirm cross-species CRV OCT 3), Across treatments, consistent increase VUAA1-induced second administrations 4A). reason, level 4B) calculated normalizing amplitude stimulation, average current last stimulations. neither water-injected oocyte controls S1), reduced VUAA1-activated 4A) approximately 85%, respectively 4B), accordance cell-based results, where inhibitor compared 3). subsequently aggressive species, albopictus. Repellency evaluated reduction number landings exposed portion human hand. widely DEET (45Mccabe E.T. Barthel W.F. Gertler S.I. Hall repellents. III. N,N-diethylamides.J. Org. 1954; 493-498Crossref (122) IPC 46Hall Travis B.V. H.A. Repellent Composition. . U.S. Patent No. 2,390,249A, Washington, DC: Trademark Office.1945Google standards. bioassays (see S2 quantifications landing numbers) showed that, highest dose (0.2 ?l/cm2, 1–1.4 ?mole/cm2), significantly counts 5A At 0.04 ?l/cm2 (210–280 nmole/cm2), strongest OCT, displayed comparable DEET, CA noticeably 5B lowest (0.01 52–70 display weaker eliciting 5C 1).Table 1Repellence indices hand assaysCompound name (abbreviation)StructureMolecular weight?L per cm2nmole cm2Mean % repellencyDEET Pr(<[t])N,N-Diethyl-3-methylbenzamide (DEET)191.30.21042100 ± 00.04208100 00.015296.7 2.6Carvacrol (CRV)150.20.2130099.8 0.70.377370.0426099.1 20.239940.016567.8 96.9E-16Cumin (CA)150.20.2130099.8 0.70.042760.0426078 71.3E-060.016563.2 11.81.3E-14Isopropyl (IPC)190.20.2107096 1.60.048350.04214--0.0154--Linalyl (LA)196.30.292072.3 15.60.000910.04184--0.0146--(2E,4E)-2,4-Octadienal (OCT)124.20.21410100 0-0.0428097.9 2.70.063090.017013.8 12.88.6E-25(1S)-3-Carene (CAR)136.20.2127089.8 4.30.001810.04254--0.0164--All tests carried out over period 5 min. Dichloromethane experiments involving 0.2, 0.04, 0.01 S2. Statistically differences, control, those Pr(<[0.05]).-, examined. Open table tab All Pr(<[0.05]). -, 1) bioassay mixtures, using different doses, medium low 5, B–C, respectively, Tables S2).Table 2Low onesCompound?L each Pr(<[t])Single Pr(<[t])DEET0.015296.7 2.6CRV0.016567.8 9.06.9E-16CA0.016563.2 11.81.3E-14OCT0.017013.8 12.88.6E-25CRV+CA0.00532.5 + 32.593.1 6.80.02971CRVCA1.8E-053.7E-05CRV+OCT0.00532.5 3584.2 6.61.3E-09CRVOCT0.000873.9E-09CA+OCT0.00532.5 3576.5 6.98.0E-14CAOCT0.024238.3E-08CRV+CA+OCT0.003321.7 21.7 23.391.1 5.20.00019CRVCAOCT4.3E-059.6E-054.5E-09All numbers Pr(<[0.05]).CA, alcohol; CRV, carvacrol; N,N-diethyl-3-methylbenzamide; (2E,4E)-2,4-octadienal. e