The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.
Beddington J (2010) Food security: contributions from science to a new and greener revolution. Phil Trans R Soc B: Biological Sciences 365: 61–71.
[3]
Elbert A, Nauen R, McCaffery A (2008) IRAC, Insecticide Resistance and Mode of Action Classification of Insecticides. Weinheim: Wiley-VCH Verlag GmbH. pp. 753–771.
[4]
Hemingway J, Field L, Vontas J (2002) An Overview of Insecticide Resistance. Science 298: 96–97.
[5]
Oyarzún MP, Quiroz A, Birkett MA (2008) Insecticide resistance in the horn fly: alternative control strategies. Med Vet Entomol 22: 188–202.
[6]
Acevedo G, Zapater M, Toloza A (2009) Insecticide resistance of house fly, Musca domestica (L.) from Argentina. Parasitol Res 105: 489–493.
[7]
Schafer WR (2002) Genetic analysis of nicotinic signaling in worms and flies. Journal of Neurobiol 53: 535–541.
[8]
Vardy E, Arkin IT, Gottschalk KE, Kaback HR, Schuldiner S (2004) Structural conservation in the major facilitator superfamily as revealed by comparative modeling. Protein Science 13: 1832–1840.
[9]
Eiden LE (1998) The Cholinergic Gene Locus. Journal of Neurochemistry 70: 2227–2240.
[10]
Kaufman PE, Nunez SC, Mann RS, Geden CJ, Scharf M (2010) Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies. Pest Manag Sci 66: 290–294.
[11]
Millar N, Denholm I (2007) Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invert Neurosci 7: 53–66.
[12]
Patchett AA, Nargund RP, Tata JR, Chen MH, Barakat KJ, et al. (1995) Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue. Proc Natl Acad Sci U S A 92: 7001–7005.
[13]
Bondensgaard K, Ankersen M, Thogersen H, Hansen BS, Wulff BS, et al. (2004) Recognition of Privileged Structures by G-Protein Coupled Receptors. J Med Chem 47: 888–899.
[14]
Hughes DJ, Worthington PA, Russel CA, Clarke ED, Peace JE, et al. (2003) Spiroindolinepiperidine Derivatives. WO/2003/ 106457:
[15]
Cheng Y, Chapman KT (1997) Solid phase synthesis of spiroindoline. Tet Lett 38: 1497–1500.
[16]
Maligres PE, Houpis I, Rossen K, Molina A, Sager J, et al. (1997) Synthesis of the orally active spiroindoline-based growth hormone secretagogue, MK-677. Tetrahedron 53: 10983–10992.
[17]
Cassayre J, Molleyres L-P, Maienfisch P, Cederbaum F (2005) Spiroindoline Derivatives Having Insecticidal Properties. WO/2005/ 058897:
[18]
Jeschke P (2010) The unique role of halogen substituents in the design of modern agrochemicals. Pest Manag Sci 66: 10–27.
[19]
Thompson GD, Dutton R, Sparks TC (2000) Spinosad – a case study: an example from a natural products discovery programme. Pest Manag Sci 56: 696–702.
[20]
Rand JB, Russell RL (1984) Choline acetyltransferase-deficient mutants of the nematode caenorhabditis elegans. Genetics 106: 227–248.
[21]
Nguyen M, Alfonso A, Johnson CD, Rand JB (1995) Caenorhabditis elegans Mutants Resistant to Inhibitors of Acetylcholinesterase. Genetics 140: 527–535.
[22]
Alfonso A, Grundahl K, Duerr JS, Han HP, Rand JB (1993) The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter. Science 261: 617–619.
[23]
Rand JB (1989) Genetic Analysis of the cha-1-unc-17 Gene Complex in Caenorhabditis. Genetics 122: 73–80.
[24]
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.
[25]
Clarkson ED, Rogers GA, Parsons SM (1992) Binding and active transport of large analogues of acetylcholine by cholinergic synaptic vesicles in vitro. J Neurochem 59: 695–700.
[26]
Henry J-P, Scherman D (1989) Radioligands of the vesicular monoamine transporter and their use as markers of monoamine storage vesicles. Biochem Pharmacol 38: 2395–2404.
[27]
Keiser MJ, Setola V, Irwin JJ, Laggner C, Abbas AI, et al. (2009) Predicting new molecular targets for known drugs. Nature 462: 175–181.
[28]
Fliri AF, Loging WT, Thadeio PF, Volkmann RA (2005) Biological spectra analysis: Linking biological activity profiles to molecular structure. Proc Natl Acad Sci U S A 102: 261–266.
[29]
Evans BE, Rittle KE, Bock MG, DiPardo RM, Freidinger RM, et al. (1988) Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists. J Med Chem 31: 2235–2246.
[30]
Ojeda AM, Kolmakova NG, Parsons SM (2004) Acetylcholine binding site in the vesicular acetylcholine transporter. Biochemistry 43: 11163–11174.
[31]
Kim M-H, Lu M, Lim E-J, Chai Y-G, Hersh LB (1999) Mutational Analysis of Aspartate Residues in the Transmembrane Regions and Cytoplasmic Loops of Rat Vesicular Acetylcholine Transporter. J Biol Chem 274: 673–680.
[32]
Bravo DT, Kolmakova NG, Parsons SM (2005) Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter. Biochemistry 44: 7955–7966.
[33]
Zhu H, Duerr JS, Varoqui H, McManus JR, Rand JB, et al. (2001) Analysis of point mutants in the Caenorhabditis elegans vesicular acetylcholine transporter reveals domains involved in substrate translocation. J Biol Chem 276: 41580–41587.
[34]
Khare P, White AR, Parsons SM (2009) Multiple Protonation States of Vesicular Acetylcholine Transporter Detected by Binding of [3H]Vesamicol. Biochemistry 48: 8965–8975.
[35]
Horti AG, Gao Y, Kuwabara H, Dannals RF (2010) Development of radioligands with optimized imaging properties for quantification of nicotinic acetylcholine receptors by positron emission tomography. Life Sci 86: 575–584.
[36]
Lind RJ, Hardick DJ, Blagbrough IS, Potter BVL, Wolstenholme AJ, et al. (2001) [3H]-Methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors. Insect Biochem Mol Biol 31: 533–542.
[37]
Varoqui H, Erickson JD, Susan GA (1998) Functional identification of vesicular monoamine and acetylcholine transporters. Methods Enzymol 296: 84–99.
[38]
Anderson P, Henry FE, Diane CS (1995) Mutagenesis. Methods Cell Biol 48: 31–58.
[39]
Griswold CM, Roebuck J, Andersen RO, Stam LF, Spana EP, et al. (2002) A toolkit for transformation and mutagenesis in Drosophila using piggyBac. Dros Inf Serv 85: 129–132.
[40]
Brittain RT, Levy GP, Tyers MB (1969) Observations on the neuromuscular blocking action of 2-(4-phenylpiperidino)-cyclohexanol (AH 5183). Br J Pharmacol 36: 173–174.
