WO2017106914A1 - Method of screening - Google Patents

Abstract

The present disclosure relates to methods of screening for a compound which modifies mosquito behaviour. More specifically, the present invention relates to 5 methods of screening for a compound which modifies the ability of particular thio compounds to bind and/or activate mosquitoes or a mosquito neuron receptor. The present disclosure further provides methods and uses for identified compounds.

Description

METHOD OF SCREENING

FIELD OF THE INVENTION

The present disclosure relates to methods of screening for a compound which modifies mosquito behaviour. More specifically, the present invention relates to methods of screening for a compound which modifies the ability of particular thio compounds to bind and/or activate mosquitoes or a mosquito neuron receptor. The present disclosure further provides methods and uses for the identified compounds. BACKGROUND OF THE INVENTION

Mosquitoes are vectors for disease in human beings and animals. These insects carry diseases such as malaria, heartworm, dengue fever, encephalitis, yellow fever and West Nile virus, causing millions of human deaths around the world every year.

Mosquitoes in the genus Anopheles are the principle vectors of malaria. Malaria is a devastating infectious disease caused by Plasmodium spp. that results in approximately 660,000 million deaths per year.

Plasmodium falciparum, the cause of the most virulent form of malaria, has developed resistance to currently used drugs. This in turn has led to an increase in the incidence of malaria and to fewer drugs for both treatment and prophylaxis of the disease.

Aedes aegypti is the main vector of the viruses that cause Yellow fever and Dengue. Other viruses, the causal agents of various types of encephalitis, are also carried by Aedes spp. mosquitoes. Wuchereria bancrofti and Brugia malayi, parasitic roundworms that cause filariasis, are usually spread by mosquitoes in the genera Culex, Mansonia, and Anopheles. Thus, chemical attractants and repellants remain an important tool for reducing rates of infection and spread of disease.

Female mosquitoes choose their mammalian hosts based in part on complex chemical cues. Some of these signals, such as carbon dioxide, have been well characterized on a molecular level. For example, carbon dioxide is not only a potent mosquito stimulant but also augments mosquito feeding behaviors and modulates attraction to other human body odors. Thus, carbon dioxide has been used previously as an effective tool against infection. However, Anopheles gambiae strains that lack functional C0₂ receptors are still capable of locating human hosts, indicating that additional chemical signals also drive host preference. Further, logistical issues can make the provision of carbon dioxide difficult, in particular in low resource areas. There is a therefore a need to identify compounds for modifying the behaviour of mosquitoes.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that thio compounds present in

Plasmodium sp. infected subjects, and related compounds, can be used to modify the behaviour of mosquitoes. Thus, these thio compounds can be used to identify additional compounds that can be used to modify the behaviour of mosquitoes.

Accordingly, in a first aspect, the present invention provides a method of screening for a compound which modifies mosquito behaviour, the method comprising:

(a) contacting a mosquito neuron receptor, or a variant thereof, with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3 -methyl thio-propanol, (E)-l-methylthio- 1-propene, (Z)-l-methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof,

(b) determining if the candidate compound modifies the ability of the thio compound to bind and/or activate the receptor.

Various candidate compounds can be screened using the methods of the present disclosure. In an example, the candidate compound is a thio compound. For example, the thio compound may be a thioether. In an example, the thioether is a compound of Formula 1 :

R-L R2

S

Formula 1

wherein Rl and R2 are independently selected from an optionally substituted Ci_

₆alkyl and an optionally substituted C₂_₆alkenyl. In an example, Rl and R2 are independently selected from an unsubstituted Ci_-6alkyl, an unsubstituted C₂-₆alkenyl and a C].₆alkyl substituted with a hydroxyl group.

Various mosquito neuron receptors can also be used to screen candidate compounds according to the present disclosure. For example, the receptor may be a mosquito CpA or CpC neuron receptor, or a variant thereof. In another example, the receptor is a mosquito odorant (Or) or a gustatory (Gr) receptor, or a variant thereof. In another example, the receptor is selected from the group consisting of mosquito Or28, Gr22, Gr23, Gr24, or a variant thereof.

In another example, the receptor comprises two or more different subunits and: i) one of the subunits is mosquito Or28 or a variant thereof, and a further subunit is mosquito Or7 or a variant thereof, or

ii) one of the subunits is mosquito Gr22 or a variant thereof, and a further subunit(s) is mosquito Gr23 and/or Gr24, or a variant of one or both thereof.

In another example, the receptor is from an Anopheles spp., Aedes spp., Culex spp., Culiseta spp. or Haemagogus spp.. For example, the receptor may be from an Anopheles spp.. In an example, the Anopheles spp. is An. arabiensis, An. funestus, An. gambiae, An. moucheti, An. nili, An. stephensi, An. bellator, An. Cruzii or An. farauti.

In an example, the receptor comprises an amino acid sequence as set forth in any one of SEQ ID NO's 1 to 24, or a sequence at least 90% identical to an amino acid sequence as set forth in any one or more of SEQ ID NO's 1 to 24.

Such receptors can be provided in an in-vitro assay system. For example, the receptor, or variant thereof, may be present in a cell. In an example, the cell is Xenopus oocyte, HEK293, Sf9, S2, HeLa, Bm5, high five or yeast cell. In another example, the receptor, or variant thereof, is detectably labelled.

Various assays can be used for determining if the candidate compound modifies the ability of the thio compound to bind and/or activate the receptor. For example, binding of the candidate compound to the receptor is determined via gel electrophoresis, ELISA, immunoblot, or surface plasmon resonance. In another example, receptor activation is determined by electrophysiology.

In an embodiment, the methods of the present disclosure may comprise further steps. For example, the methods may further comprise testing the ability of the candidate compound to modify the behaviour of a mosquito. In another example, the methods may further comprise selecting a compound which

i) activates a mosquito,

ii) activates and attracts a mosquito,

iii) activates and repels a mosquito,

iv) does not activate, or reduces activation of, a mosquito in the presence of human subject(s), or

v) does not activate, or reduces activation of, a mosquito in the presence of human subject(s) with a Plasmodium infection. In these examples, the mosquito may be female.

In an embodiment, the methods of the present disclosure encompass methods of screening that involve contacting candidate compounds directly with mosquitoes. Thus, in another embodiment, the present disclosure relates to a method of screening for a compound which modifies mosquito behaviour, the method comprising: (a) contacting a mosquito with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio- propane, 3-methylthio-propanol, (E)-l -methyl thio- 1-propene, (Z)-l-methylthio-l- propene, a derivative thereof, or a mixture of two or more thereof,

(b) determining if the candidate compound modifies the effect of the thio compound on the behaviour of the mosquito. Such a method may also comprise additional steps. For example, the method may further comprise selecting a compound which

i) activates the mosquito,

ii) activates and attracts the mosquito,

iii) activates and repels the mosquito,

iv) does not activate, or reduces activation of, the mosquito in the presence of human subject(s), or

v) does not activate, or reduces activation of, the mosquito in the presence of human subject(s) with a Plasmodium infection. In another example, the method may further comprise selecting a compound which reduces the ability of the thio compound to activate the mosquito.

In an embodiment, compounds identified using the methods of the present disclosure can be used to modify mosquito behaviour. Thus, in another embodiment the present disclosure relates to a method of modifying mosquito behaviour, the method comprising releasing an identified candidate compound which modifies mosquito behaviour. In an example, a compound is released by evaporation, diffusion, atomization or dispersion. In another example, the present disclosure relates to the use of a compound identified by the methods of the present disclosure for modifying the behaviour of mosquitoes. Such methods and uses are applicable to various mosquitoes. For example, the mosquito can be an Anopheles spp., Aedes spp., Culex spp., Culiseta spp., Haemagogus spp. or a combination of two or more thereof. For example, the mosquito can be Anopheles spp.. Exemplary Anopheles spp. include An. arabiensis, An. funestus, An. gambiae, An. moucheti, An. nili, An. stephensi, An. bellator, An. cruzii, An. farauti or a combination of two or more thereof.

In an embodiment, candidate compounds can be screened in-silico using, for example, various computer implemented methods. Thus, in another embodiment, the present disclosure also relates to a computer implemented method for selecting a candidate compound for modifying mosquito behaviour, the method comprising identifying compounds having a similar structure to a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio- propanol, (E)-l-methylthio-l-propene, (Z)-l-methylthio-l-propene, or a derivative thereof, and selecting the identified compound as a candidate compound. Such methods may further comprise screening selected candidate compounds for the ability to modify mosquito behaviour.

Any example herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Labelled Y-tube olfactometer.

Figure 2. 1-methylthio-l-propene (MTPE) (as a mix of E and Z enantiomers) activation.

Figure 3. Allyl methyl sulphide (AMS) activation.

Figure 4. 3-mefhylthio-propanol (MTPL) activation.

Figure 5. Mosquito attraction comparison of (A) Allyl methyl sulphide (AMS); (B) 1- methylthio-l-propene (as a mix of E and Z enantiomers) (MTPE); (C) 3-mefhylthio- propanol (MTPL) with l-octen-3-ol / C0₂.

Figure 6. Structure and ion fragmentation patterns of thioethers useful for the disclosure.

Figure 7. (A) Mean EAG response of A. stephensi (compounds tested at 1/10 concentrations; 1-methylthio-l-propene tested at 1/100 concentration), error bars represent standard error of the mean, 6 mosquitoes tested; (B) Mean EPG response of A. stephensi (compounds tested at 1/10 concentrations; 1-methylthio-l-propene tested at 1/100 concentration), error bars represent standard error of the mean, 7 mosquitoes tested. Figure 8. (A) A. stephensi EAG dose response for 1-methylthio-l-propene (diamonds) and l-octen-3-ol (squares), error bars represent standard error of the mean, 7 mosquitoes were tested; (B) A. stephensi EPG dose response for 1-methylthio-l- propene, error bars represent standard error of the mean, 7 mosquitoes tested.

Figure 9. Expression of olfactory and gustatory receptors for Anopheles stephensi mosquitoes. RT-PCR expected band sizes Gr22: cDNA=151bp, gDNA=247bp, Or8: cDNA=180bp, gDNA=242bp, Or28, cDNA=218bp, gDNA = 280bp. A: Antennae, M: Maxillary palps, P: Proboscis.

Figure 10. (A) Mean EAG response of A. farauti (compounds tested at 1/10 concentrations; 1-methylthio-l-propene tested at 1/100 concentration), error bars represent standard error of the mean, 5 mosquitoes were tested; (B) Mean EPG response of A. farauti (compounds tested at 1/10 concentrations; 1-methylthio-l- propene tested at 1/100 concentration), error bars represent standard error of the mean, 6 mosquitoes were tested.

Figure 11. Mean EAG response of C. annulirostis, error bars represent standard error of the mean, 5 mosquitoes were tested.

Figure 12. Mean EAG response of A. aegypti, error bars represent standard error of the mean, 5 mosquitoes were tested.

Figure 13. Structure and ion fragmentation patterns of thioether derivatives useful for the disclosure.

Figure 14. Example log-concentration response curve for screened compound/s against the Anopheles gambiae Or28 or Anopheles gambiae Gr22 (or functionally equivalent) receptors. Agonist is a thioether, antagonist changes the response of the agonist when tested together.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: l - Amino acid sequence of Anopheles gambiae Gr22.

SEQ ID NO:2 - Amino acid sequence of Anopheles gambiae Or28.

SEQ ID NO:3 - Amino acid sequence of Anopheles gambiae Gr23.

SEQ ID NO:4 - Amino acid sequence of Anopheles gambiae 0x24.

SEQ ID NO:5 - Amino acid sequence of ^'Anopheles gambiae Orl .

SEQ ID NO:6 - Amino acid sequence of Aedes aegypti Or49.

SEQ ID NO:7 - Amino acid sequence of Culex quinquefasciatus Or38.

SEQ ID NO:8 - Amino acid sequence of Anopheles gambiae Or8.

SEQ ID NO:9 - Amino acid sequence of Aedes aegypti Or8.

SEQ ID NO: 10 - Amino acid sequence of Culex quinquefasciatus Orl 13. SEQ ID NO: 11 - Amino acid sequence of Culex quinquefasciatus Orl l8.

SEQ ID NO: 12 - Amino acid sequence of Aedes aegypti Grl .

SEQ ID NO: 13 - Amino acid sequence of Culex quinquefasciatus Grl .

SEQ ID NO: 14 - Amino acid sequence of Aedes aegypti Gr2.

SEQ ID NO: 15 - Amino acid sequence of Culex quinquefasciatus Gr2.

SEQ ID NO: 16 - Amino acid sequence of Anopheles gambiae Gr24.

SEQ ID NO: 17 - Amino acid sequence of Aedes aegypti Gr3.

SEQ ID NO: 18 - Amino acid sequence of Culex quinquefasciatus Gr3.

SEQ ID NO: 19 - Amino acid sequence of Anopheles stephensi Gr22.

SEQ ID NO:20 - Amino acid sequence of Anopheles stephensi Or28.

SEQ ID NO:21 - Amino acid sequence of Anopheles stephensi Or8.

SEQ ID NO:22 - Amino acid sequence of Anopheles farauti Gr22.

SEQ ID NO:23 - Amino acid sequence of Anopheles farauti Or28.

SEQ ID NO:24 - Amino acid sequence of Anopheles farauti Or8.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., insect physiology and biology, organic chemistry, biochemistry, insect formulations, insect trap design and mosquito behaviour).

As understood in the art the use of "E" and "Z" in reference to 1 -methyl thio-1- propene (MTPE) is the notation used to describe the geometric isomerism, or stereochemistry, of the double bond in 1-methylthio-l-propene. Thus, in an embodiment, reference to MTPE in the present disclosure encompasses the E enantiomer, Z enantiomer or a mixture thereof.

As will be understood, an "aromatic" group means a cyclic group having 4m+2 π electrons, where m is an integer equal to or greater than 1. As used herein, "aromatic" is used interchangeably with "aryl" to refer to an aromatic group, regardless of the valency of aromatic group. A heteroaromatic group is an aromatic group or ring containing one or more heteroatoms, such as N, O, S, Se, Si or P. As used herein,

"heteroaromatic" is used interchangeably with "heteroaryl".

"Aryl" whether used alone, or in compound words such as arylalkyl, aryloxy or arylthio, represents an optionally substituted aromatic carbocyclic moiety.

"Heterocyclyl" or "heterocyclic" whether used alone, or in compound words such as heterocyclyloxy represents an optionally substituted carbocyclic group, in which one or more of the carbon ring atoms has been replaced by element(s) other than carbon, for example nitrogen, oxygen, sulfur or silicon. The term heterocyclyl encompasses heteroaryl.

"Heteroaryl" whether used alone, or in compound words such as heteroaryloxy represents an optionally substituted aromatic organic moiety, in which one or more of the ring members is/are element(s) other than carbon, for example nitrogen, oxygen, sulfur or silicon; the heteroatom(s) interrupting a carbocyclic ring structure and having a sufficient number of delocalized π electrons to provide aromatic character, provided that the rings do not contain adjacent oxygen and/or sulfur atoms.

The term "optionally substituted" means that a functional group is either substituted or unsubstituted, at any available position.

The term "halo" or "halogen" whether employed alone or in compound words such as haloalkyl, haloalkoxy or haloalkylsulfonyl, represents fluorine, chlorine, bromine or iodine. Further, when used in compound words such as haloalkyl, haloalkoxy or haloalkylsulfonyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different. Examples of haloalkyl include, without limitation, -CH₂CH₂F, -CF₂CF₃ and - CH₂CHFC1.

"Alkyl" whether used alone, or in compound words such as alkoxy, alkylthio, alkylamino, dialkylamino or haloalkyl, represents a monovalent straight or branched chain hydrocarbons group. Thus alkyl moieties include, for example, methyl, ethyl, n- propyl, iso-propyl and/or butyl, pentyl and hexyl.

"Alkenyl" whether used alone, or in compound words such as alkenyloxy or haloalkenyl, represents monovalent straight or branched chain hydrocarbons groups containing at least one carbon-carbon double bond, including, ethylene, 1-propenyl, 2- propenyl, and/or butenyl, pentenyl and hexenyl.

"Alkynyl" whether used alone, or in compound words such as alkynyloxy, represents monovalent straight or branched chain hydrocarbons groups containing at least one carbon-carbon triple bond, including, ethynyl, 1-propynyl, 2-propynyl, and/or butynyl, pentynyl and hexynyl.

"Cycloalkyl" represents a carbocyclic ring system, e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

"Alkylene" represents a bivalent straight or branched chain saturated hydrocarbon group.

"Alkenylene" represents a bivalent straight or branched chain unsaturated hydrocarbon with at least one carbon-carbon double bond. "Alkynylene" represents a bivalent straight or branched chain unsaturated hydrocarbon with at least one carbon-carbon triple bond.

"Carboxyl" represents a -C(=0)OH moiety.

"Alkylaminocarbonyl" represents a -C(=0)NHR or -C(=0)NRR' group in which R and R' is an alkyl group as defined supra.

"Cyano" represents a -CN moiety.

"Hydroxyl" represents a -OH moiety.

"Alkoxy" represents an -O-alkyl group in which the alkyl group is as defined supra. Examples include methoxy, ethoxy, n-propoxy, iso-propoxy, and the different butoxy, pentoxy, hexyloxy and higher isomers.

"Alkenyloxy" represents an -O-alkenyl group in which the alkenyl group is as defined supra. An example is allyloxy.

"Amino" represents an -NH₂ moiety.

"Alkylamino" represents an -NHR or -NRR' group in which R and R' is an alkyl group as defined supra. Examples include, without limitation, methylamino, ethylamino, n-propylamino, isopropylamino, and the different butylamino, pentylamino and hexylamino.

As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a thio compound" optionally includes a plurality of thio compounds.

As used herein, the term "about", unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Screening Methods

The present disclosure relates to a method of screening for a compound able to modify mosquito behaviour. In an embodiment, such compounds will modify the ability of a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio-propane, 3-methylthio-propanol, (E)-l-methylthio-l-propene, (Z)-l- methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof, to bind and/or activate a mosquito neuron receptor.

In an embodiment, a mosquito neuron receptor can be contacted with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio-propanol, (E)- 1 -methylthio-l-propene, (Z)-l -methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof. The receptor can then be assessed to determine whether the candidate compound modifies the effect of the thio compound to bind and/or activate the receptor. Exemplary mosquito neuron receptors are discussed below.

It is considered that terms such as "contacting", "exposing" or "applying" are terms that can, in context, be used interchangeably in the present disclosure. The term contacting, requires that the compounds (i.e. candidate compound and thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3- methylthio-propanol, (E)-l -methylthio-l-propene, (Z)-l -methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof be brought into contact with a mosquito neuron receptor. Exemplary mixtures of thio compounds selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio- propanol, (E)-l -methylthio-l-propene, (Z)-l -methylthio-l-propene include 1- methylthio-l-propene and allyl methyl sulphide; 1 -methylthio-l-propene and 3- methylthio-l-propanol; 1 -methylthio-l-propene and 1-methylthio-propane; allyl methyl sulphide and 3-methylthio-l-propanol; allyl methyl sulphide and 1-methylthio-propane; 1 -methylthio-l-propene, allyl methyl sulphide and 3-methylthio-l-propanol; 1- methylthio-l-propene, allyl methyl sulphide and 1-methylthio-propane; 1 -methylthio-l- propene, 3-methylthio-l-propanol and 1-methylthio-propane; allyl methyl sulphide, 3- methylthio-l-propanol and 1-methylthio-propane; 1 -methylthio-l-propene and Z enantiomer of 3-methylthio-l-propanol; allyl methyl sulphide and Z enantiomer of 3- methylthio-l-propanol; 1-methylthio-propane and Z enantiomer of 3-methylthio-l- propanol; 1 -methylthio-l-propene, allyl methyl sulphide and Z enantiomer of 3- methylthio-l-propanol; 1 -methylthio-l-propene, 1-methylthio-propane and Z enantiomer of 3-methylthio-l-propanol; allyl methyl sulphide, 1-methylthio-propane and Z enantiomer of 3-methylthio-l-propanol; 1 -methylthio-l-propene and E enantiomer of 3-methylthio-l-propanol; allyl methyl sulphide and E enantiomer of 3- methylthio-l-propanol; 1-methylthio-propane and E enantiomer of 3-methylthio-l- propanol; 1 -methylthio-l-propene, allyl methyl sulphide and E enantiomer of 3- methylthio-l-propanol; 1 -methylthio-l-propene, 1-methylthio-propane and E enantiomer of 3-methylthio-l-propanol; allyl methyl sulphide, 1-methylthio-propane and E enantiomer of 3-methylthio-l-propanol; 1-methylthio-l-propene and a mixture of E and Z enantiomers of 3-methylthio-l-propanol; allyl methyl sulphide and a mixture of E and Z enantiomers of 3-methylthio-l-propanol; 1-methylthio-propane and a mixture of E and Z enantiomers of 3-methylthio-l-propanol; 1-methylthio-l-propene, allyl methyl sulphide and a mixture of E and Z enantiomers of 3-methylthio-l- propanol; 1-methylthio-l-propene, 1-methylthio-propane and a mixture of E and Z enantiomers of 3-methylthio-l-propanol; allyl methyl sulphide, 1-methylthio-propane and a mixture of E and Z enantiomers of 3-methylthio-l-propanol. Exemplary derivatives of thio compounds selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio-propanol, (E)- 1-methylthio-l-propene, (Z)- 1-methylthio-l-propene are shown in (Figure 13). Thus, exemplary derivatives include ethyl propyl sulphide, propyl sulphide, 1-propylthio-pentane, 1 -propyl thio- butane, 2-methyl-3-methylthio-l-propene, 3-ethylthio-l-propene, (Z)-l-methylthio-l- butene, l-methylthio-2-butene, 1-1-propenylthio-propane or a mixture of two or more thereof.

Various candidate compounds suitable for screening using the methods of the present disclosure are discussed below.

As the skilled person would appreciate, there are a wide variety of different screening procedures which could be adapted to screen candidate compounds. For example, candidate compounds may be screened using in vitro and in vivo heterologous assay systems comprising various neuron receptors.

Thus, in an embodiment, the mosquito neuron receptor or variant thereof is present in a cell. Neuron receptors can be expressed in various cell types such as Xenopus oocytes (Wetzel et al., 2001), HEK293 (Thomas and Smart, 2005), Sf9 (Schneider and Seifert, 2010), S2 (Smart et al, 2008), HeLa (Sato et al., 2008), Bm5 (Tsitoura et al., 2010) or high five (Tsitoura et al., 2010) or yeast cells. Such cells will typically comprise a genetically engineered reporter system to detect receptor binding or activation (see, for example, Fukutani et al., 2012; and Dowell and Brown, 2009; WO 2014/169336). One of skill in the art would be aware of various methods of providing an appropriate in vitro assay system expressing a desired neuron receptor. For example, cells can be transfected with an expression system carrying a desired neuron receptor and a suitable reporter. Cells expressing the neuron receptor can then be contacted with candidate compounds to identify those compounds that modify the ability of a selected thio compound to agonise, antagonize or otherwise interact with the receptor. In an embodiment, the screening method comprises contacting compounds with a library of neuron receptors (for example taste or odorant, or a combination thereof) expressed in cells, and identifying specific receptors which preferentially bind candidate compounds.

Exemplary in vivo heterologous screening systems include Drosophila "empty neuron: systems. In these systems a neuron receptor is expressed in place of an endogenous receptor in the Drosophila antennae, using for example, the Gal4-UAS system, and receptor responses to compounds are measured using single sensillum electrophysiological recordings.

In another example, compounds are screened using a cell free system (see e.g.

US 4,668,624; US 5,807,717; Roberts et al. 1973). Such systems contain or are supplemented with all factors required for the translation of mRNA, for example ribosomes, tRNAs, aminoacyl synthetases, elongation factors and initiation factors. Neuron receptors can be synthesized inside a variety of cell free mediums such as cell- sized lipid bilayer capsules such as giant vesicles or in wheat- germ cell-free expression systems.

Previously described olfactory- and contact-mediated bioassays (Trexler et al., 1998; Dekker et al., 2002; WO2003/103395) or variations thereof can also be used to screen candidate compounds.

In another embodiment, high throughput screening methods are used which involve providing a library containing a large number of candidate compounds. Such libraries are then screened in one or more assays to identify those library members (e.g. particular chemical species or subclasses) that display a desired level of binding or activation.

High throughput screening systems are commercially available and typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detectors appropriate for the assay. These configurable systems provide rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems (e.g. Invitrogen, PerkinElmer, Bayer Pharma etc.) provide detailed protocols for use.

For the screening of a library of volatile candidate compounds, purified receptor or cells can be exposed to air or other gas mixtures comprising the compound(s). Alternatively, cells can be exposed to a solution or suspension of the volatile compound in cell culture media. For example, the compound can be dissolved in cell culture media if the compound is water soluble or water-immiscible. Otherwise, a suitable substrate may be soaked in the compound and placed over cells in culture. In another example, combinatorial libraries of candidate compounds immobilized on a solid support (e.g., a "chip") are synthesized using for example, photolithography (see for example US 5,143,854; WO 1990/15070; WO 1992/10092). The immobilized compounds are contacted with a labelled receptor and the support is scanned to determine the location of the label, to thereby identify candidate compounds binding to the receptor.

Other exemplary screening systems can incorporate Surface Plasmon Resonance (SPR) or Biomolecular Interaction Analysis (BIA; e.g., Biacore) to detect biospecific interactions in real time, without labelling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface. The changes in the refractivity generate a detectable signal, which are measured as an indication of realtime reactions between biological molecules. In other examples, gel electrophoresis, ELISA or immunoblot may be incorporated into the methods of the present disclosure to detect interaction of compounds with a mosquito neuron receptor.

In another example, X-ray crystallography can be used to screen candidate compounds and identify receptor interaction (e.g. US 6,297,021; WO 2004/057340). For example, a crystal of a neuron receptor can be obtained and contacted with one or more candidate compounds. Contact between receptor and candidate compound can be initiated by soaking a crystal in a solution of one or more candidate compounds or co- crystallizing a neuron receptor in the presence of one or more candidate compounds. An X-ray crystal diffraction pattern is then obtained to determine whether a complex is formed between the receptor and the candidate compound. In this example, structural information from identified receptor/candidate compound complexes can be used to design new compounds that bind tighter, bind more specifically or have better biological activity than previously tested candidate compounds.

Other exemplary screening methods include, but are not limited to high- resolution NMR, phage display, affinity chromatography, isothermal titration calorimetry (ITC), immunoprecipitation and GST pull downs coupled with mass spectroscopy.

In performing the methods of the present disclosure a plurality of candidate compounds can be contacted with a mosquito neuron receptor. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 5,000, at least 10,000, at least 20,000, at least 40,000, at least 50,000, at least 100,000, at least 200,000 or more candidate compounds can be contacted with mosquito neuron receptors.

Receptor Binding and Activation

Various methods of detecting whether a candidate compound modifies the ability of a selected thio compound to bind and/or activate a mosquito neuron receptor are known in the art. For example, kinetic studies can be performed to estimate association (K_on) and dissociation (k_off) rates. In another example, saturation experiments can be performed to determine affinity constant (k) or dissociation constant (k_d) for a candidate compound. In another example, competition/modulation experiments can be performed to calculate the equilibrium inhibitor constant (Kj) of a candidate compound. For these examples, a preparation containing the neuron receptor is provided in aliquots. Suitable labelled candidate compounds are incubated with a thio compound selected from the group consisting of allyl methyl sulphide, 1- methylthio-propane, 3-methylthio-propanol, (E)-l -methylthio-l-propene, (Z)-l- methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof at varying concentrations in aliquots of the neuron receptor for a defined time at a defined temperature in a defined buffer. The bound and/or free concentration of compounds is then measured and the data analysed mathematically to extract and compare quantitative estimates of rate constants, affinity constants and cooperativities. Exemplary assays are reviewed in Hulme and Trevethick (2010).

Detecting whether a candidate compound modifies the ability of a selected thio compound to activate a neuron receptor can also be detected via various means. In an example, a suitable reporter of receptor activity such as calcium levels can be monitored. In this example, a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio-propane, 3-methylthio-propanol, (E)-l -methylthio- l-propene, (Z)-l -methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof (control), candidate compound (test) and ionomycin (to determine maximal fluorescence) can be provided with a suitable calcium indicator such as Fluo4 (Invitrogen). In this example, the fluorescent calcium indicator can be detected using a suitable imaging system (e.g. Metafluor®). A change in fluorescence (e.g. AF) indicates interaction between the candidate compound and the receptor. Alternative reporter systems suitable for assessing receptor activation are discussed above. In another example, receptor activation can be determined using electrophysiology. Various electrophysiology methods suitable for measuring receptor activation are known in the art. Appropriate methods will depend upon the system in which the receptor is being studied. For example, single channel patch clamping, whole cell clamping or the use of voltage and ion-sensitive dyes and resins may be suitable for in vitro analysis. In other examples, iontophoresis and local drug application or stimulation recording may be suitable for in vivo analysis.

Results from binding/activation assays can be represented and compared visually, for example by generating a response curve for screened compound/s against a mosquito neuron receptor (Figure 14).

Mosquito Activity

In another embodiment, the methods of the present disclosure encompass contacting mosquitoes directly with candidate compounds. In this embodiment, mosquito behaviour can be assessed directly after contact with a candidate compound and, if required monitored over a period of time. For example, mosquitoes can be contacted with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio-propane, 3-methylfhio- propanol, (E)-l-methylthio-l-propene, (Z)-l-methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof. Mosquitoes can then be assessed to determine whether the candidate compound modifies the effect of the thio compound on the behaviour of the mosquito. In an example, the method comprises selecting a compound which reduces the ability of the selected thio compound to activate the mosquito.

Various methods of contacting mosquitoes with candidate compounds are known in the art. Suitable methods will depend on the behaviour being assessed. In an example, two cups (test and control) are placed randomly in diagonal corners of a suitable cage. Each cup releases or is filled with either candidate compound (test) or a thio compound selected from the group consisting of allyl methyl sulphide, 1- methylthio-propane, 3-methylthio-propanol, (E)-l -methyl thio- 1-propene, (Z)-l- methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof (control) in formulation.

For olfactory bioassays, a mesh screen covered with insect glue can be placed above test and control cups. Positive or negative responses to test formulations can be measured by the numbers of mosquitoes trapped on the sticky screen during an exposure period (e.g. 24 hr). For egg-laying bioassays, cups are filled with solution and gravid females are released in each replicate cage. After an exposure period (e.g. 24 hr), eggs laid on the surface of the solution in test and control cups are counted.

In another example, mosquitoes can be visually assessed following exposure to test and control formulations with mosquito activation being measured by counting the number of mosquitoes which take flight following exposure. Mosquito attraction/repulsion can also be visually assessed to determine whether a candidate compound initiates an attractive or repulsive directional response in mosquitoes.

In other examples, assays can also be performed in a semi-field tunnel or in an outdoor field setting (see, for example, Lorenz et al., 2013; Ritchie and Devine, 2013).

The ability of candidate compounds to mask a subject can be assessed by exposing a test subject to mosquitoes in the presence of test and control formulations for a period of time and then recording the number of bites received by the subject.

In an example, identified compounds which modify mosquito behaviour can be assessed to determine how selective they are for the chosen target. This process is known as cross-screening and can be used to identify compounds that only interfere with the chosen neuron receptor, but not other, related receptors.

Computer Implemented Screening

Compounds having a similar structure to a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio- propanol, (E)-l -methyl thio- 1-propene, (Z)-l-methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof may be more likely to modify mosquito behaviour. This is supported by the previously described "similarity property principle" which reasons that structurally similar molecules (e.g., activating compounds) are more likely to have similar properties (Hendrickson 1991; Martin et al, 2002).

Candidate compounds having a "similar structure" to a selected thio compound can be identified using multiple computational approaches (e.g. Cerius2, Accelrys Software Inc; Dragon, Talete; Maximum-Common-Substructure (MCS), Cao et al., 2008b; atom-pair (AP), Carhart et al, 1985; Cao et al., 2008a). Exemplary features which can be assessed include, molecular weight, functional group counts, carbon chain length, three-dimensional relationships within molecules, shortest path distances between all atom pairs in a molecule, identification of the largest two-dimensional substructure that exists between two compounds. Structural information on thio compounds selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio-propanol, (E)-l-methylthio- 1 -propene, (Z)-l -methyl thio- 1 -propene and derivatives thereof are known in the art and are provided in Figures 6 and 13. This information can be used as a training data set for comparison with structural information available for various candidate compounds. Thus, in another embodiment, the methods of the present disclosure encompass computer implemented screening of candidate compounds to predict compounds that agonise, antagonize or otherwise interact with a mosquito neuron receptor.

In an example, the present disclosure relates to a computer implemented method of screening for a compound which modifies mosquito behaviour, the method comprising identifying candidate compounds having a similar structure to a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio- propane, 3-methylthio-propanol, (E)-l-methylthio-l -propene, (Z)-l-methylthio-l- propene, a derivative thereof, or a mixture of two or more thereof, and selecting the identified compound as a candidate compound.

In an example, identified candidate compounds are further screened based on their smell, presence in natural sources, human safety profile, and production cost.

Identified candidate compounds can be screened using the above exemplified methods to determine if the compound modifies the ability of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3- methylthio-propanol, (E)-l-methylthio-l -propene, (Z)-l-methylthio-l -propene, a derivative thereof, or a mixture of two or more thereof, to bind and/or activate a mosquito neuron receptor.

In another example, identified candidate compounds can be screened using the above exemplified methods to determine if the compound modifies the ability of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio- propane, 3-methylthio-propanol, (E)-l -methyl thio- 1 -propene, (Z)-l-methylthio-l- propene, a derivative thereof, or a mixture of two or more thereof, to modify mosquito behaviour. Compounds shown to modify mosquito behaviour can be added to the training data set.

Methods utilising a training data set of compounds known to modify mosquito behaviour to screen for similar candidate compounds have been described previously (e.g. Tauxe et al., 2013; Boyle et al, 2013). In these examples, libraries of candidate compounds are screened and ranked based on their similarity to the training data set. Algorithms exist in statistical analysis (supervised learning; principal component analysis; machine based learning) and in artificial intelligence (unsupervised learning; Kohonen neural networks) for these types of analysis. Data analysis programs or packages are available commercially which include these functions (e.g. SPSS, SPSS, Inc., Chicago, Illinois; SAS, SAS Institute, Inc., Cary, NC; Neuroshell 2, Word Systems Group, Inc.; Stuttgart Neural Network Simulation, The University of Stuttgart, Stuttgart, Germany; Support Vector Machine (SVM), Cortes and Vapnik 1995).

The computer implemented method may be implemented using a system (e.g. a computer system) comprising one or a plurality of processors which may operate together (referred to for convenience as "processor") connected to a memory. The memory may be located inside the computer system or located elsewhere, such as in the form of a storage area network (SAN). The memory may be a non-transitory computer readable medium, such as a hard drive, a solid state disk or CD-ROM. Software, that is executable instructions or program code, such as program code grouped into code modules, may be stored on the memory, and may, when executed by the processor, cause the computer system to perform functions.

Exemplary functions include determining that a task is to be performed, for example, to assist a user to access a reference database of structural information on compounds; compare and statistically analyse structural information; assess structural similarity; screen candidate compounds; fit a statistical model to the structural information; validate a statistical model using training data.

Mosquito Behaviour

The methods of the present disclosure can be used to identify compounds able to modify mosquito behaviour in various ways. The term "modify" is used in the context of the present disclosure to refer to a change or alteration in mosquito behaviour. For example, compounds identified using the methods of the present disclosure may activate mosquitoes. The terms "activate", "activating", "activator" and variations thereof as used in the context of the present disclosure relate to a compound that generates a response, such as being one or more of excited, confused, irritated, flight, attracted or repelled, by a mosquito. In an example, the methods of the present disclosure can be used to identify a compound that reduces the likelihood a subject, such as a human subject with a Plasmodium infection, will be bitten by a mosquito.

Thus, the methods of the present disclosure can be used to identify compounds that can modify mosquito behaviour in at least three different ways, namely "attractants" that pull mosquitoes towards a particular position such as into a trap placed away from humans, "repellents" that push mosquitoes away from a particular position and "maskers" that block attraction to humans. The terms "repellent" and "attractant" are used in the context of the present disclosure to refer to a compound that generates a directional response in mosquitoes. In an example, the formulations of the present disclosure can act as a mosquito "attractant". In another example, the methods of the present disclosure can be used to identify compounds that can act as a mosquito "repellent".

Mosquito attraction or repulsion is not initiated without mosquito activation. Thus, the terms "attractant" and "repellent" as used in the context of the present disclosure also encompass "activator". However, mosquito "activation" can occur without "attraction" or "repulsion". Thus, in an example, an "activator" does not encompass an "attractant" or "repellent".

In another example, the methods of the present disclosure can be used to identify compounds that can act as a mosquito "masker". In other examples, the methods of the present disclosure can be used to identify compounds that can act as arrestants that cause mosquitoes to stay at a site longer and/or as stimulants that cause mosquitoes to oviposition, i.e., lay eggs, or to lay more eggs at a single site.

Thus, in an embodiment, the methods of the present disclosure further comprise testing the ability of a candidate compound to modify the behaviour of a mosquito. Behaviour modifying compounds can then be used as discussed below. In an example, the methods of the present disclosure can further comprise selecting a compound which:

i) activates a mosquito,

ii) activates and attracts a mosquito,

iii) activates and repels a mosquito,

iv) does not activate, or reduces activation of, a mosquito in the presence of human subject(s), or

v) does not activate, or reduces activation of, a mosquito in the presence of human subject(s) with a Plasmodium infection.

Neuron Receptors

Candidate compounds can be screened using various mosquito "neuron receptors" or variants thereof. The term "neuron receptor" is used in the context of the present disclosure to refer to receptors that trigger an electrical signal by regulating the activity of ion channels upon activation. Thus, the term "neuron receptors" encompasses neurons of mosquito maxillary palps. For example, candidate compounds can be screened using CpA, CpB, or CpC neuron receptors or variants thereof. In an example, candidate compounds can be screened using CpA or CpC neuron receptors or variants thereof. In another example, candidate compounds can be screened using CpA neuron receptors or variants thereof. In another example, candidate compounds can be screened using CpC neuron receptors or variants thereof.

In an example, candidate compounds can be screened using odorant receptors (Or), gustatory receptors (Gr), ionotropic receptors and variants thereof. In an example, candidate compounds are screened using odorant receptors, gustatory receptors or variants thereof. In an example, candidate compounds are screened using odorant receptors or variants thereof. Exemplary odorant receptors include Or8 (e.g. SEQ ID NOs: 8, 9, 21, 24), Or28 (e.g. SEQ ID NOs: 2, 20, 23) and the canonical receptor orco (Or7) (e.g. SEQ ID NO: 5). In another example, candidate compounds are screened using gustatory receptors or variants thereof. Exemplary gustatory receptors include C0₂ receptors or variants thereof. Other exemplary gustatory receptors include Gr22 (e.g. SEQ ID NO: 1, 22), Gr23 (e.g. SEQ ID NO: 3), Gr24 (e.g. SEQ ID NO: 4) or variants thereof. In another example, candidate compounds are screened using ionotropic receptors and variants thereof.

One of skill in the art would appreciate that equivalents of the above referenced receptors exist across different insect genera and species. In an embodiment, the methods of the present disclosure encompass screening candidate compounds in such equivalent receptors. For example, candidate compounds can be contacted with Anopheles gambiae Or28 or an equivalent thereof such as Aedes aegypti Or49 or Culex quinquefasciatus Or38 which are not homologues but are expressed in the CpC neuron. In another example, candidate compounds can be contacted with Anopheles gambiae Or8 or an equivalent thereof such as Aedes aegypti Or8 or Culex quinquefasciatus Orl l3 and Culex quinquefasciatus Orl l8 which are homologues and are expressed in the CpB neuron. In another example, candidate compounds can be contacted with Anopheles gambiae Gr22 or an equivalent thereof such as Aedes aegypti Grl or Culex quinquefasciatus Grl which are homologues and are expressed in the CpA neuron. In another example, candidate compounds can be contacted with Anopheles gambiae Gr23 or an equivalent thereof such as Aedes aegypti Gr2 or Culex quinquefasciatus Gr2 which are homologues and are expressed in the CpA neuron. In another example, candidate compounds can be contacted with Anopheles gambiae Gr24 or an equivalent thereof such as Aedes aegypti Gr3 or Culex quinquefasciatus Gr3 which are homologues and are expressed in the CpA neuron.

In an example, the neuron receptor comprises two or more different subunits. In an example, the neuron receptor comprises Or7 or a variant thereof and a further subunit. For example, one of the subunits can be mosquito Or7 or a variant thereof and a further subunit can be mosquito Or28 or a variant thereof. In another example, one of the subunits can be mosquito Or7 or a variant thereof, and a further subunit can be mosquito Or8, or a variant of one or both thereof. In another example, one of the subunits can be mosquito Gr22 or a variant thereof, and a further subunit(s) can be mosquito Gr23 and/or Gr24, or a variant of one or both thereof. Non-naturally occurring combinations of subunits may also be used to screen candidate compounds. In an example, receptors are provided with two or more subunits that respond to thio compounds. For example, one of the subunits can be mosquito Or28 or a variant thereof, and a further subunit can be mosquito Gr22, or a variant thereof.

The screening of candidate compounds is not restricted to any particular genus or species of mosquitoes. However, in various examples it may be desirable to screen candidate compounds using receptors from one or more of the mosquito genera or species discussed below. For example, the neuron receptor may be from an Anopheles spp., Aedes spp., Culex spp., Culiseta spp. or Haemagogus spp. For example, the neuron receptor may be from an Anopheles spp.. Exemplary Anopheles spp. include An. arabiensis, An. funestus, An. gambiae, An. moucheti, An. nili, An. stephensi, An. bellator, An. Cruzii or An. farauti. Exemplary Culex spp. include Culex quinquefasciatus.

Information on the neuron receptors described above (e.g. amino acid sequence) can be readily obtained from publically available databases such as National Centre for Biotechnology Information (NCBI) and Universal Protein Resource (UniProt). Alternatively, the amino acid sequence can be derived by direct sequencing of the native protein (e.g. with an automated amino acid sequencer). For example, the amino acid sequences of An. gambiae Gr22 (SEQ ID NO: l) can be obtained from UniProt under reference >gill 18500886lgblABK97612.ll gustatory receptor 22 [Anopheles gambiae] and An. gambiae Or28 (SEQ ID NO:2) can be obtained from UniProt under reference >gil31206485 Iref IXP_312203. II AGAP002722-PA [Anopheles gambiae str. PEST].

One of skill in the art would appreciate that various modifications can be made to the receptors discussed above that retain the receptors biological activity (e.g. trigger an electrical signal by regulating the activity of ion channels upon activation; bind thio compounds). Thus, reference to "neuron receptors" in the present disclosure also includes biologically active variants, mutants, modifications, analogous and/or derivatives of the neuron receptors described herein.

Thus, with regard to a defined neuron receptor, it will be appreciated that % identity figures higher than those provided below will encompass preferred embodiments. For example, it is preferred that the neuron receptor comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to the known amino acid sequence of the neuron receptor or the relevant nominated SEQ ID NO. For example, the neuron receptor can comprise an amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO:24, or a sequence at least 90% identical to an amino acid sequence as set forth in any one of SEQ ID NO:l to SEQ ID NO:24. In another example, the neuron receptor comprises an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NO:l to SEQ ID NO:24. In another example, the neuron receptor comprises an amino acid sequence at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO:24.

In the context of the present disclosure, the % identity of a variant to an amino acid sequence encoding the native neuron receptor is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the query sequence is at least 200 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 200 amino acids. Even more preferably, the query sequence is at least 300 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 300 amino acids. More preferably, the two sequences are aligned over their entire length.

Amino acid sequence mutants of neuron receptor can be prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the neuron receptor, or by in vitro synthesis of the desired neuron receptor. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired characteristics.

Mutant (altered) neuron receptors can be prepared using any suitable technique known in the art. For example, a polynucleotide encoding the neuron receptor can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub- cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, polynucleotides are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine whether biological activity of the neuron receptor has been retained.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

Table 1. Exemplary Substitutions.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.

If desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the neuron receptor. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4- diaminobutyric acid, -amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β- alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Coc- methyl amino acids, Noc-methyl amino acids, and amino acid analogues in general.

Neuron receptors may also be differentially modified during or after synthesis.

For example, neuron receptor may be detectably labelled. Appropriate isotope, fluorescent and photoaffinity markers as well as antigens such as V5 epitope and polyhistidine sequence (PIB/V5-His vector; Invitrogen) are suitable exemplary detectable labels.

Other exemplary modifications include biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the neuron receptor.

Neuron receptors can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated neuron receptor is produced by culturing a cell capable of expressing the neuron receptor under conditions effective to produce the neuron receptor, and recovering the neuron receptor. Various suitable cell types such as E. coli or yeast cells are known in the art. Other exemplary cell types suitable for expressing neuron receptors are discussed above. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit neuron receptor production. An effective medium refers to any medium in which a cell is cultured to produce the desired neuron receptor. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microti ter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Candidate Compounds

The term "candidate compound" is used in the context of the present disclosure to refer to an agent to be screened for modifying mosquito behaviour. Candidate compounds may include, for example, small molecules such as small organic compounds (e.g. , organic molecules having a molecular weight between about 50 and about 2,500 Da), peptides or mimetics thereof, ligands including peptide and non- peptide ligands, polypeptides, nucleic acid molecules such as aptamers, peptide nucleic acid molecules, and components, combinations, and derivatives thereof. In an example, candidate compounds are labelled prior to screening. For example, compounds can be labelled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radio-emission or by scintillation counting. Alternatively, candidate compounds may be labelled with a suitable fluorescent or photo affinity marker. In another example, candidate compounds can be enzymatically labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

In an example, the candidate compound is a "volatile organic compound". The term "volatile organic compound", which may also be abbreviated to "VOC", refers to compounds that have a high vapor pressure at ambient temperature (e.g. about 20°C or about 25°C). The high vapor pressure of VOCs results from a low boiling point, which causes large numbers of molecules to evaporate or sublimate from a liquid or solid form of the compound into a gaseous state (i.e. volatisation).

Thus, in an example, candidate compounds can have vapor pressure ranging from as low as about lOPa to about 300kPa at 20°C. In an example, candidate compounds have vapor pressure ranging from about 50Pa to about 200kPa, about 70Pa to about 175kPa, about lOOPa to about 150kPa, about 200Pa to about lOOkPa, about 300Pa to about 90kPa, about 400Pa to about 80kPa, about 500Pa to about 70kPa, about 600Pa to about 60kPa, about 700Pa to about 50kPa, about 800Pa to about 40kPa, about 900Pa to about 30kPa, about IkPa to about 20kPa, about 5kPa to about 150kPa, about lOkPa at 20 °C. For example, candidate thio compounds can have vapor pressure of about 9kPA such as for example, 9.1kPA at 25°C.

In another example, candidate compounds can have a boiling point of less than

100°C, less than 90°C, less than 80°C, less than 70°C, less than 60°C, less than 50°C or less.

In another example, candidate compounds are present in the breath of a human or animal. In an example, a candidate compound is present in the breath of a human. Exemplary compounds found in breath are shown in Table 2.

Table 2. Exem lar Thio Com ounds Found in Breath.

In an example, a candidate compound is present in the breath of a human or animal bitten by a mosquito.

In an example, a candidate compound is present in the breath of a human or animal infected with a disease. For example, the human or animal may be infected with malaria. In an example, a candidate compound is present in the breath of a human or animal infected with a malaria parasite from the genus Plasmodium. For example, a human may be infected with any one of the Plasmodium species referenced below. For example, the Plasmodium species may be Plasmodium falciparum.

The present inventors have found that thio compounds are useful for activating mosquitoes. Thus, in an example, the candidate compound is a thio compound or a combination or derivative thereof. In an example, the thio compound is a thioether. In an example, the candidate compound is a thioether represented by the following Formula 1 :

Formula 1

wherein Rl and R2 are substituents that may be the same or different, and are each independently selected from the group consisting of an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, and an optionally substituted heterocyclic group.

In an example of the compound of Formula 1, Rl and R2 are independently selected from an optionally substituted Ci_-6alkyl, an optionally substituted C₂-₆alkenyl, an optionally substituted C_2-6alkynyl, an optionally substituted Ci ^cycloalkyl, and an optionally substituted heterocyclic group.

In another example of the compound of Formula 1, Rl and R2 are independently selected from an optionally substituted Ci.₆alkyl and an optionally substituted C_2-6alkenyl.

In another example of the compound of Formula 1, Rl and R2 are independently selected from an optionally substituted Ci_-6alkyl and an optionally substituted C_2-6alkenyl, wherein when said Ci_₆alkyl or C₂.₆alkenyl is substituted, it is independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; -CN; -OC]-₆alkyl; -NH₂; NH(Ci-₆alkyl); N(Cj-₆alkyl)₂; -CONH₂; phenyl optionally substituted with up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, Ci-₆alkyl,- OCi-₆alkyl, -NH₂, NH(C ₆alkyl), N(Ci-₆alkyl)₂; and 5- to 6-membered heterocyclyl optionally substituted with up to three substituents each independently selected from halo, hydroxyl, -CN, Ci-₆alkyl, -OCi-₆alkyl, -NH₂, NH(Ci-₆alkyl), N(C ₆alkyl)₂.

In another example of the compound of Formula 1, Rl and R2 are independently selected from optionally substituted C].₆alkyl and optionally substituted C_2-6alkenyl, wherein when said or C_2-6alkenyl is substituted, it is independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; -CN; -OCi-₆alkyl.

In another example of the compound of Formula 1, Rl and R2 are both Cj. ₆alkyl, each of said Ci_-6alkyl being independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; - CN; -OC_r6alkyl; -NH₂; NH(C_r6alkyl); N(C_r6alkyl)₂; -CONH₂; phenyl optionally substituted up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, Ci-₆alkyl,-OCi-₆alkyl, -NH₂, NH(C ₆alkyl), N(C,- ₆alkyl)₂; and 5- to 6-membered heterocyclyl optionally substituted with up to three substituents each independently selected from halo, hydroxyl, -CN, Ci-₆alkyl,-OCi- ₆alkyl, -NH₂, NH(C ₆alkyl), N(C_r6alkyl)₂.

In another example of the compound of Formula 1, Rl and R2 are both Q. ₆alkyl, each of said C^ lkyl being independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; - CN; -OC-ealkyl.

In another example of the compound of Formula 1, Rl and R2 are both C₂. ₆alkenyl, each of said C_2-6alkenyl being independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; - CN; -OCrealkyl; -NH₂; NH(C ₆alkyl); N(C,-₆alkyl)₂; -CONH₂; phenyl optionally substituted up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, Cr₆alkyl,-OC)-₆alkyl, -NH₂, NH(Ci-₆alkyl), N(Ci- ₆alkyl)₂; and 5- to 6-membered heterocyclyl optionally substituted with up to three substituents each independently selected from halo, hydroxyl, -CN, Ci-₆alkyl,-OCi- ₆alkyl, -NH₂, NH(Cr₆alkyl), N(C_r6alkyl)₂.

In another example of the compound of Formula 1, Rl and R2 are both C₂_ ₆alkenyl, each of said C_2-6alkenyl being independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; - CN; -OCi-₆alkyl.

In another example of the compound of Formula 1, Rl and R2 are each independently unsubstituted Ci_₆alkyl. In another example, Rl and R2 are each independently Cr₄ alkyl. In that example, Rl and R2 may each independently be, for example, methyl, ethyl or propyl. In another example, Rl and R2 are each independently unsubstituted C_2-6alkenyl. In another example, Rl and R2 are each independently C₂-₄ alkenyl. In that example, Rl and R2 may each independently be, for example, allyl or 1-propenyl. In another example of the compound of Formula 1, Rl is an unsubstituted Ci_ ₆alkyl, for example, methyl, ethyl or propyl; and R2 is a Ci_₆alkyl substituted by hydroxyl, for example, a hydroxypropyl moiety.

In another example of the compound of Formula 1, Rl is substituted Ci.₆alkyl and R2 is substituted C₂-₆alkenyl, said Ci.₆alkyl and C_2-6alkenyl each being independently substituted with up to three substituents each independently selected from the group consisting of halo; hydroxyl; -CN; -OCi-₆alkyl; -NH₂; NH(Cr6alkyl); N(C]-₆alkyl)₂; -CONH₂; phenyl optionally substituted up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, Ci-₆alkyl, - OCi-₆alkyl, -NH₂, NH(Ci-₆alkyl), N(Ci-₆alkyl)₂; and 5- to 6-membered heterocyclyl optionally substituted with up to three substituents each independently selected from halo, hydroxyl, -CN, C ₆alkyl, -OC_r6alkyl, -NH₂, NH(C ₆alkyl), N(Ci-₆alkyl)₂.

In another example of the compound of Formula 1, Rl and R2 are independently selected from an unsubstituted Ci_₆alkyl, an unsubstituted C₂.₆alkenyl and a Ci_-6alkyl substituted with a hydroxyl group.

In another example of the compound of Formula 1, Rl is selected from unsubstituted Ci^alkyl and R2 is selected from unsubstituted C_2-6alkenyl. In that example, Rl may be, for example, methyl, ethyl or propyl, butyl or pentyl and R2 may be, for example, allyl or 1-propenyl.

In another example, a candidate thio compound is a thioether represented by the following Formula 2:

Formula 2

wherein n is an integer from 1 to 10; wherein L is a linker group; and wherein Rl and R2 have the same definitions as for the compounds of Formula 1, as defined above. For the avoidance of doubt, the examples of Rl and R2 groups provided above for the compounds of Formula 1 are equally applicable to the compounds of Formula 2.

In one example of a compound of Formula 2, L is a linker group which is an unsubstituted Ci-₆alkylene, C₂-₆alkenylene, or C₂-₆alkynylene.

In another example, a candidate thio compound is a compound represented by the following Formula 3:

R3. R4

S

Formula 3 wherein R3 is selected from the group consisting of hydrogen, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, and an optionally substituted heterocyclic group; and wherein R4 is selected from the group consisting of an optionally substituted aryl group, and an optionally substituted heterocyclic group.

In an example of the compound of Formula 3, R3 is selected from the group consisting of hydrogen; Ci.₆alkyl optionally substituted with up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, -OCi- ₆alkyl; C₂-₆alkenyl optionally substituted with up to three substituents each independently selected from the group consisting of halo, hydroxyl, -CN, -OCi-₆alkyl; and R4 is selected from the group consisting of a phenyl optionally substituted with halo, hydroxyl, -CN, Ci-₆alkyl, -OC]-₆alkyl; and 5- to 6-membered heterocyclyl optionally substituted with up to three substituents each independently selected from halo, hydroxyl, -CN, C ₆alkyl, -OCi-₆alkyl.

In another example of the compound of Formula 3, R3 is selected from the group consisting of hydrogen; Ci_₆alkyl optionally substituted with up to three substituents each independently selected from the group consisting of from halo, hydroxyl, -CN, -OC ₆alkyl; C₂-₆alkenyl optionally substituted with up to three substituents each independently selected from the group consisting of from halo,

hydroxyl, -CN, C]-₆alkyl, -OCi-₆alkyl; and R4 is

5 and

R6 are independently selected from the group consisting of hydrogen; C].₆alkyl optionally substituted with up to three substituents each independently selected from the group consisting of from halo, hydroxyl, -CN, -OC]-₆alkyl; C_2-6alkenyl optionally substituted with up to three substituents each independently selected from the group consisting of from halo, hydroxyl, -CN, -OCi-₆alkyl. e of the compound of Formula 3, R3 is hydrogen, and

wherein R5 and R6 are independently unsubstituted Ci.₆alkyl.

In one example, the compound of Formula 3 i

In an embodiment, a candidate thio compound is not a thiozole.

Exemplary combinations of candidate thio compounds (e.g. thioethers) include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, least 25, at least 30, at least 35, at least 40 thio compounds.

In an embodiment, candidate compounds can modify mosquito behaviour across a range of concentrations.

The appropriate concentration of the thio compound will vary depending on the system in which the compound is to be screened and the nature of the formulation comprising the candidate compound. For example, release in an enclosed space may require a lower concentration of thio compound. In contrast, release into an open area may require a higher concentration of thio compound.

Candidate compounds can be converted into formulations, such as solutions, micellar solutions, emulsions, microemulsions, suspensions, solids, powders, foams, pastes, granules, gases, sprays, aerosols, gels, waxes and active-compound-impregnated natural and synthetic materials before screening using the methods of the present disclosure.

In an embodiment, candidate compounds can be screened using methods of the present disclosure in conjunction with other active compounds. For example, the candidate compounds may be screened in conjunction with other activators, attractants, repellents or combinations thereof. Various exemplary actives are provided below.

Exemplary activators, attractants and repellents include appropriate concentrations of kairomones such as alkenols (e.g., octenol; l-octen-3-ol), alkynols (octynol), gases such as carbon dioxide, nitrogen dioxide, as well as substances such as carboxylic acids, lactic acid, butyric acids, caproic acids, propionic acids, valeric acids, ammonia, acetone and combinations thereof such as carbon dioxide and l-octen-3-ol.

Candidate compounds can be screened at a range of concentrations (either singularly or in combination) with a concentration of thio compound known to activate the receptor with changes in the activation of the receptor being measured. Exemplary activating concentrations may comprise about 0.00001% to 10%, about 0.00001 to 5%, about 0.00001 to 2%, about 0.00001 to 1%, about 0.00001 to 0.1% or about 0.00001 to 0.01% (v/v) compound. Mosquitoes

Mosquitoes are small, midge-like flies which comprise the family Culicidae. Females of most species are ectoparasites, whose tube-like mouthparts (called a proboscis) pierce a hosts skin to consume blood.

Examples of mosquito genera which can be screened using the methods of the present disclosure include, but are not necessarily limited to Aedeomyia, Aedes, Anopheles, Armigeres, Ayurakitia, Borachinda, Coquillettidia, Culex, Culiseta, Deinocerites, Eretmapodites, Ficalbia, Galindomyia, Haemagogus, Heizmannia, Hodgesia, Isostomyia, Johnbelkinia, Kimia, Limatus, Lutzia, Malaya, Mansonia, Maorigoeldia, Mimomyia, Onirion, Opifex, Orthopodomyia, Psorophora, Runchomyia, Sabethes, Shannoniana, Topomyia, Toxorhynchites, Trichoprosopon, Tripteroides, Udaya, Uranotaenia, Verrallina, Wyeomyia.

In an example, the mosquito genera are known to bite humans. In another example, the mosquito genera are a known vector of a disease agent to animals or humans. For example, mosquito genera can be selected from the group consisting of Anopheles, Aedes, Culex, Culiseta, Mansonia or Haemagogus. In an example, the mosquito genus is Culex. In this example, the Culex species can be C. annulirostis. In an example, the mosquito genus is Aedes. In this example, the Aedes species can be Ae. aegypti. In another example, the Aedes species can be Ae. albopictus. In an example, the mosquito genera are a known vector of a disease agent to humans.

In an example, the mosquito genera are a known vector of human malaria. In another example, the mosquito genera are a known vector of parasites from the genus Plasmodium. In another example, the mosquito genera are a known vector of Plasmodium selected from the group consisting of Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri or Plasmodium malariae. In an example, the mosquito genera are a known vector of Plasmodium falciparum. For example, the mosquito genera can be Anopheles. In an example, the Anopheles species is An. arabiensis, An. funestus, An. gambiae, An. moucheti, An. nili, An. stephensi, An. bellator, An. cruzii, An. farauti. For example, the Anopheles species is An. gambiae. For example, the Anopheles species is An. stephensi.

In other examples, the Anopheles species is Anopheles (Cellia) aconitus;

Anopheles (Nyssorhynchus) albimanus; Anopheles (Nyssorhynchus) albitarsis species complex; Anopheles (Cellia) annularis; Anopheles (Nyssorhynchus) aquasalis; Anopheles (Cellia) arabiensis; Anopheles (Anopheles) atroparvus; Anopheles (Cellia) balabacensis; Anopheles (Anopheles) barbirostris species complex; Anopheles (Cellia) culicifacies species complex; Anopheles (Nyssorhynchus) darling; Anopheles (Cellia) dims species complex; Anopheles (Cellia) farauti species complex; Anopheles (Cellia) flavirostris; Anopheles (Cellia) fluviatilis species complex; Anopheles (Anopheles) freeborni; Anopheles (Cellia) funestus; Anopheles (Cellia) gambiae; Anopheles (Cellia) koliensis; Anopheles (Anopheles) labranchiae; Anopheles (Anopheles) lesteri (formerly An. anthropophagus in China); Anopheles (Cellia) leucosphyrus and Anopheles (Cellia) latens; Anopheles (Cellia) maculatus Group; Anopheles (Nyssorhynchus) marajoara; Anopheles (Cellia) melas; Anopheles (Cellia) merus; Anopheles (Anopheles) messeae; Anopheles (Cellia) minimus species complex; Anopheles (Cellia) moucheti; Anopheles (Cellia) nili species complex; Anopheles (Nyssorhynchus) nuneztovari species complex; Anopheles (Anopheles) pseudopunctipennis species complex; Anopheles (Cellia) punctulatus species complex; Anopheles (Anopheles) quadrimaculatus; Anopheles (Anopheles) sacharovi; Anopheles (Cellia) sergentii species complex; Anopheles (Anopheles) sinensis species complex; Anopheles (Cellia) stephensi; Anopheles (Cellia) subpictus species complex; Anopheles (Cellia) sundaicus species complex; Anopheles (Cellia) superpictus.

In an embodiment, the mosquito is female.

One of skill in the art can easily identify mosquitoes falling in the above exemplified categories using publically available databases and their associated search engine(s) (e.g. http://www.map.ox.ac.uk/map/; Bionomics Search).

Dispensers

In an embodiment, compounds identified using the methods of the present disclosure can be released from various dispensers. Dispensers can be provided in various forms: rubber septa, hollow fibres, flakes, tape, laminated plastics, membranes over reservoirs, and polyethylene vials with acetate beads. Other, exemplary dispensers include cylinders, vaporizers, oils, candles, wicked apparatus, fans and treated articles such as clothes and mats.

In an embodiment, identified compounds can be incorporated into various insect traps and lures. Lures can vary in duration of effectiveness and distance of attraction due to differences in compound load and release rates. A controlled-release lure will allow the compound to be released in a concentration small enough to entice a mosquito into the trap, but strong enough to reach out and pull them in over a useful distance.

Exemplary trap types include container traps, tapes, combustibles and electric traps. Various traps and lures of these types are known in the art (see for example, US 5205064; US5799436; US6925752; US7987631). Suitable exemplary traps and lures are commercially available such as for example, Mega-Catch™, Mosquito Magnet®, Koolatron™ and Dragonfly (BioSensory Inc.). Other exemplary suppliers of suitable traps include Biogents AG, EnviroSafe Technologies International Limited, Woodstream Corporation, Bayer AG, Reckitt Benckiser.

In an example, formulations comprising identified compounds do not include C0₂. These formulations can be incorporated into existing insect dispensers that may not have otherwise been suitable for mosquitoes. For example, such formulations may be incorporated into existing flying insect dispensers such as codling moth or fruit fly dispensers (see for example, US8424239; US5683687).

Suitable traps and lures may also comprise lighting arrays flashing both visible and invisible spectrums at oscillating frequencies, blue light, additional fragrance strips or lures such as an octenol strips, C0₂ systems including for example, burn tanks of propane for C0₂, heating systems to assist in attracting mosquitoes approaching the trap, baits comprising an insecticide, catch systems such as vacuum or fan directed capture in a collection net or a liquid catch container.

Methods and Uses

Compounds identified using the methods of the present disclosure can be used in methods of modifying mosquito behaviour by activating mosquitoes. In another example, the compounds are used in a method of attracting mosquitoes. In another example, the compounds are used in a method of repelling mosquitoes. In another example, the compounds are used in a method of masking a subject from mosquitoes.

The present disclosure also relates to the use of identified compounds for modifying the behaviour of mosquitoes. For example, use of identified compounds for activating mosquitoes. In another example, use of identified compounds for attracting mosquitoes. In another example, use of identified compounds for repelling mosquitoes. In another example, use of identified compounds for masking a subject from mosquitoes.

The above exemplified methods and uses comprise releasing a compound to affect mosquito behaviour. The term "release" or "releasing" is used in the context of the present disclosure to refer to release of a compound into the atmosphere (e.g. the three dimensional space surrounding a subject or another particular site of release such as a mosquito trap). This may occur through a spray or similar release of a compound from a formulation, or through a volatile compound being released, either passively or actively, from a formulation. Compounds can be released via various methods such as evaporation, diffusion, atomisation or dispersion.

The location of release will differ depending on the nature of the identified compound. For example, attracting compounds can be released into the atmosphere to attract mosquitoes away from a subject. In this example, attracting compounds can be released into the atmosphere surrounding the perimeter of a subjects domicile or campsite.

In another example, repelling compounds can be released into the atmosphere to repel mosquitoes away from a subject. In another example, masking compounds can be released into the atmosphere to mask the subject from mosquitoes. In these examples, compounds can be released into the atmosphere surrounding or close to the subject such as inside the subjects domicile or campsite.

In an embodiment, performing these methods involves releasing the compound in an appropriate concentration to impart the desired effect on mosquito behaviour. For example, an appropriate activating, attracting, repelling, masking concentration will be released. Exemplary concentrations suitable for modifying mosquito behaviour are discussed above. Methods of determining the appropriate concentration of compound are also discussed above. Thus, the selection of the appropriate concentration for release is well within the skill set of one skilled in the art. EXAMPLES

EXAMPLE 1 - Materials and Methods

Overview

A y-tube olfactometer was used to establish whether the volatile components of human breath elicited behavioural responses in the malaria vector Anopheles stephensi.

Attraction to these components was compared to a standard "positive control" known to elicit a directional response in An. stephensi mosquitoes placed in an olfactometer. The standard used as a positive control is a mix of compressed air, 5% C0₂ and an octenol source. Unpaired t-test was used to test for the differences in means for all data sets presented. Mosquitoes

The MR4 strain of An. stephensi (New Delhi, India) was maintained at 27°C, 70% RH, 12: 12 L:D and 30 min dawn/dusk periods). 4 to 7 day old female mosquitoes, that had never received a blood meal, were used in experiments. Mosquitoes were maintained on 10% sugar solution ad libitum but starved for 12 hr prior to testing.

Test and Control Stimuli

a) Test: Allyl methyl sulphide (AMS)

b) Test: The mixture of the E and Z stereoisomers of 1-methylthio-l-propene (MTPE)

c) Test: 3-methylthio-l-propanol (MTPL; methylthio derivative; not found in breath)

d) Positive Control: l-octen-3-ol / 5% C0₂. Olfactometer

The Y tube olfactometer is made entirely of glass and consists of a release chamber, the Y tube itself, and a trap chamber attached to each arm (Figure 1). Each trap chamber is divided in two by a fine glass filter. On the far side of those filters, the trap chambers can connect to charcoal filters or to gas bags or gas cylinders. Air is pulled through the olfactometer by connecting the release chamber to a vacuum tap which vents the contents of the tube. Standard air flow (0.4 L / minute) is maintained using a 0.1-1.2 LPM flow meter (John Morris Pty, LTD). The air volume of the Y-tube is approximately 1 litre and is therefore replaced every 2.5 minutes.

One arm is attached to a gas bag of clean air (from a compressed air cylinder) plus 5% C0₂.

The other arm remains connected to a carbon filter and the air inlet to give a plume of compressed air plus test stimuli (i.e. standard air concentration of C0₂ only).

Test Sample Preparation

1 μΐ 10^" 2 to 10^" 5 % test stimuli in mineral oil (Sigma Aldrich) was spotted on lcm² Whatman filter paper. Test stimuli was delivered by placing treated filter paper between the air inlet and the trap chamber to give a plume of compressed air plus test stimuli (i.e. standard air concentration of C0₂ only). Control Sample Preparation

1 μΐ lCT⁴ % l-octen-3-ol in mineral oil (Sigma Aldrich) was spotted on 1cm² Whatman filter paper.

5% C0₂ by volume was added to a 10L FlexFilm gasbag (SKC Inc.) of compressed air.

When a C0₂ / l-octen-3-ol mix was required, 5% C0₂ was delivered by connecting the gas bag to the requisite Y tube arm and placing the l-octen-3-ol treated filter paper between the gasbag inlet and the glass filter of the trap chamber.

Purified air was introduced via charcoal filters attached to the far side of the trap chambers.

Mosquito Releases

All experiments were run under PC2 quarantine conditions, at 27°C, 70% RH, under infrared light. Observers were always in the same position during the tests, at the trap chamber end of the olfactometer, between the 2 arms of the Y tube.

Using an aspirator, 25-30 starved female An. stephensi were introduced into the release chamber (Figure 1).

The experiment was terminated when mosquitoes become habituated to the stimuli and did not respond further (usually 4-6 min). After each test, mosquitoes were carefully aspirated from the apparatus by disconnecting constituent parts. The apparatus was flushed with clean air (charcoal filters only) for 5 minutes before the next batch of stimuli was introduced.

A total of 10 replicates (different batches of mosquitoes) were run for each stimuli assessment. Treatment and control arms were alternated between replicates. After completion of 10 replicates, the apparatus was rinsed in acetone and allowed to dry overnight.

Measurement of Activation

Many arthropods respond to semiochemicals at specific dose ranges. At least under laboratory settings, chemicals that elicit a positive directional response at some concentrations may exhibit a more general, undirected excitation, or even repellency at other concentrations (Knols et al, 1997; Smallegange et al., 2005; Vale and Hall, 1985).

Initial experiments were therefore less concerned with directional responses and more with establishing the range of doses that appeared to cause some form of excitation. Those initial experiments were conducted in comparison to a neutral control (a negative would be repellent) of clean air drawn through a charcoal filter. The test stimuli were tested against mosquitoes at serial dilutions in the range 10^~2 to 10^~5 %.

Once mosquitoes had stopped responding, the numbers in the olfactometer arms, base leg and release chamber were counted. Mosquitoes were assumed to have been "activated" if they moved into the Y tube arms.

Measurement of Attraction

Once the range of doses that "activated" mosquitoes had been established, the capacity of those concentrations to initiate a directional response was tested. In this instance, experiments were conducted in comparison to a positive control of C0₂ / 1- octen-3-ol [10^"4 % v/v]. C0₂ / l-octen-3-ol were used as the positive control as this mixture was consistently more attractive to mosquitoes than C0₂ alone. A positive control was used to assess whether test stimuli were at least as attractive as a standard directional stimulus.

Test stimuli were tested against mosquitoes at the doses that had been shown to activate them and their capacity to attract was assessed by comparing the numbers of mosquitoes in the control and treatment arms of the olfactometer.

EXAMPLE 2 - Mosquito Activation and Attraction

Y-tube olfactometry experiments were performed on Anopheles stephensii.

Mosquito activation and attraction was measured following exposure to compounds allyl methyl sulphide (AMS), 3 -methyl thio-propanol (MTPL) and 1 -methyl thio-1- propene (as a mix of E and Z enantiomers) (MTPE).

The compounds elicit activation responses (i.e. mosquitoes become excited or irritated) and directional responses (i.e. mosquitoes display a chemotaxis towards the stimulus) at low concentrations.

Activation

Test compounds were compared against a neutral control of clean air drawn through a charcoal filter. MTPE initiates its greatest activation response at a concentration of 0.0001% v/v. Activation was reduced at lower (0.00001%) and higher (0.001 - 0.01%) concentrations (v/v) (Figure 2). AMS initiates its greatest activation response at a concentration of 0.00001% v/v. AMS caused activation at 0.00001% v/v (Figure 3). Methylthio derivative MTPL also initiated an activation response across a wide range of concentrations (0.00001 - 0.01%) (Figure 4).

Attraction

Test compounds were compared against a known attractant, a mixture of 1- octen-3-ol / C0₂ to determine whether they were also mosquito attractants. AMS was twice as attractive as l-octen-3-ol / C0₂ at 0.00001% (Figure 5A); MTPE was more attractive than l-octen-3-ol / C0₂ at 0.0001% (Figure 5B) and MTPL was 1.5 more attractive than l-octen-3-ol / C0₂ at 0.01% (Figure 5C).

Summary

A summary of the data from the activation and attraction experiments provided in Table 3.

Table 3. Summar of Mos uito Activation and Attraction

These data indicate that the compounds (Figure 6) elicit strong, activation and attraction responses in mosquitoes suggesting that they are useful activators such as attractants. Further, the data indicates that the test compounds elicit activation and attraction responses in mosquitoes without the addition of any other attractant, synergist or potentiator such as C0₂. EXAMPLE 3 - Electrophvsiology Studies

Electroanntennogram (EAG) and electropalpogram (EPG) were performed on A. stephensi, A. farauti, C. annulirostis and A. aegypti female mosquitoes to determine the molecular basis for activation and attraction behavior.

Five to eight days old Anopheles stephensi, two to fourteen days old Culex annulirostris and Aedes aegypti as well as four days old Anopheles faraulti non- blooded females, fed on 10% sucrose ad-libitum and then starved for >12 hours, were used. Mosquito head was excised and placed on the reference electrode coated with electrode gel (Parker Laboratories, Orange, NJ). The distal end of the antennae or palp (< 0.5 mm), cut to ensure a good electrical contact, were carefully placed on the recording electrode (Syntech, Germany). Signals were fed to a lOx amplifier and processed with ID AC 4 and PC -based interface and EAG software package (Syntech, Germany).

l-octen-3-ol, an odour attractive to mosquitos first identified from cows (Hall et al, 1984) and human sweat (Cork and Park, 1996), elicits significant EAG response from female An. funestus and An. gambiae antennae (Cork and Park, 1996; Costantini et al., 2001). Thus l-octen-3-ol was used in the EAG analysis as a positive control with thio compounds, (E and Z)-l -methylthio-l-propene, 3-methyl-thio-l-propanol, ally methyl sulphide, and carbon dioxide (5%) and mineral oil (negative control). Pure chemicals were diluted in mineral oil to a 10 times v/v stock solution, from which decadal dilutions were made. A 10 μΐ aliquot of each solution was applied to a filter paper strip (1x3.5 cm; Whatman No. 4, Fisher Scientific) and the solvent was evaporated under a fume hood before inserting the paper strip into 5 ml disposable plastic syringe (Terumo syringes). A 500 ms pulse (5 ml/s) was delivered by stimulus controller CS-55 (Syntech, Germany) to deliver chemical stimulants to a humidified continuous air flow (10 ml/s) over the electrophysiolgical preparation. The chemicals were tested randomly and applied with 0.5-1 min intervals between stimulations.

Initial screening was performed using a 10 times dilution (1/10, v/v) source dose solution. Screening of 1 -methylthio-l-propene was also performed usin a 100 times dilution (1/100 v/v). Then 1 -methylthio-l-propene and l-octen-3-ol were selected for further dose-response study. A minimum of five mosquitoes were tested for each compound to calculate the average EAG amplitude ± standard error (SE).

EAG in A. stephensi showed that antennae significantly respond to all tested thio compounds and also to l-octen-3-ol which was tested as a control (Figure 7A). 1- methylthio-l-propene elicited the strongest response in antennae, surprisingly stronger than the response to l-octen-3-ol. EPG results show that 1 -methylthio-l-propene, allyl methyl sulphide and C0₂ elicit significant responses on the maxillary palps, while methylthio-l-propanol and l-octen-3-ol do not (Figure 7B). Again, 1-methylthio-l- propene elicited the strongest response even in comparison with C0₂. The EAG and EPG responses observed in A. stephensi for 1-methylthio-l-proene were dose dependent (Figure 8 A and B).

A. stephensi maximilary receptors Gr22, Or8 and Or28 receptors were also analysed at the molecular level using gene expression analysis. RT-PCR results showed that Gr22, Or8 and OR28 are selectively expressed in the maxillary palps but not in the antennae and proboscis (Figure 9).

The pattern of response in A. farauti was similar to A. stephensi with EAG

(Figure 10A) and EPG (Figure 10B) showing antennae and maxillary palps respond to all tested thio compounds. Again, 1-methylthio-l -propene elicited the strongest response in both tissues.

l-octen-3-ol and 3-methylthio-l-propanol elicited the stongest responses in C. annulirostis antennae (Figure 11) while 3-methylthio-l-propanol elicited the strongest response in A aegypti (Figure 12).

EXAMPLE 4 - Compound Identification - Sf9 cells

Sf9 cells are used to screen for compounds that alter the interaction of a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio- propane, 3-methylthio-propanol, (E)- 1-methylthio-l -propene, (Z)- 1-methylthio-l - propene, a derivative thereof, or a mixture of two or more thereof with the Anopheles gambiae Or28 and/or Anopheles gambiae Gr22 receptors, the ligand receptors expressed in the maxillary palp neurons that are activated by the thio compounds.

Candidate compounds are assessed at a range of concentrations (either singularly or in combination) with a concentration of thio compound known to activate the receptor. Changes in the activation of the receptor are measured.

Sf9 cells are plated into 12-well plates and left to settle for 20 min before being transfected by 500 ng of PIB/V5-His vector containing the receptor and 3 pL of Fugene HD transfection reagent (Promega, USA) in 100 pL per well of Sf-900 medium (Invitrogen, USA). Forty-eight hours after transfection, cells are prepared for calcium imaging and data analysis (Schneider et al., 2010; Kiely et al., 2007; Zhang et al., 2011). Briefly, Fluo4 (Invitrogen) is used as the calcium indicator. Fluorescence images are recorded using a Leitz digital still camera. Images are recorded every 10 s for 50 s following the addition of: saline (negative control), the test ligands and ionomycin (to determine maximal fluorescence). Images are analysed using the Metafluor® imaging system and AF is calculated as the ratio of change in fluorescence from basal levels (saline) upon the addition of ligand relative to change in fluorescence from basal levels following the addition of ionomycin (Figure 14). Cells responding to saline are removed from subsequent analysis.

EXAMPLE 5 - Compound Identification - Cell Free System

Cell free systems where mosquito neuron receptors are synthesized inside cell- sized lipid bilayer capsules called giant vesicles (Hamada et al., 2014) or in wheat-germ cell-free expression systems (Carraher et al., 2013) are produced. Candidate compounds are screened to identify compounds that agonise, antagonize or otherwise interact with the receptor.

EXAMPLE 6 - In vivo Compound Identification

In vivo heterologous systems such as the Drosophila "empty neuron" systems (Dobritsa et al., 2003; Kurtovic et al, 2007) are used to screen for interacting compounds. A mosquito neuron receptor is expressed in place of an endogenous receptor in the Drosophila antennae, using the Gal4-UAS system. Receptor responses to test compounds are measured using single sensillum electrophysiological recordings. Test compounds are screened to identify compounds that agonise, antagonize or otherwise interact with the receptor.

EXAMPLE 7 - Assessing Identified Compounds

Efficacy of identified compounds to activate mosquitoes can be assessed by releasing them into the atmosphere over a period of time. In brief, test and control compounds are provided in formulation for release. The test formulation releases a candidate compound and the control formulation releases a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio-propane, 3- methylthio-propanol, (E)-l -methyl thio- 1-propene, (Z)-l-methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof. Mosquito activation can be assessed visually. An increase in the number of mosquitoes activated by the test formulation relative to the control formulation indicates that the candidate compound will be an effective mosquito activator.

Efficacy of compounds to attract mosquitoes to commercially available traps can also be assessed over a period of time. A test and control trap is provided. The test trap is provided with a candidate compound and the control trap is provided with a lure containing a thio compound selected from the group consisting of allyl methyl sulphide, 1 -methyl thio-propane, 3-methylthio-propanol, (E)-l-methylthio-l-propene, (Z)-l-methylthio-l-propene, a derivative thereof, or a mixture of two or more thereof. Mosquito attraction can be assessed based on the number of mosquitoes caught in each trap. An increase in the number of mosquitoes caught in the test trap relative to the control indicates that the candidate compound will be an effective addition to commercially available traps.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU 2015905320 filed 22 December 2015, the entire contents of which are incorporated herein by reference.

All publications discussed above are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

REFERENCES

Boyle et al. (2013) eLife 2:e01120.

Carraher et al. (2013) Protein Expr Purif. 90: 160-169.

Cao et al. (2008a) Bioinformatics 24: 1366-74.

Cao et al. (2008b). Bioinformatics 24: 1733^.

Carhart et al. (1985). J Chem Inf Comput Sci 25:64-73.

Cork and Park (1996) Med Vet Entomol 10: 269-276.

Cortes and Vapnik (1995) Machine Learning 20:273-297.

Costantini et al. (2001) Med Vet Entomol 15:259-266.

Dekker et al. (2002) Med Vet Entomol. 16:91-98.

Dobritsa et al. (2003) Neuron 37:827-841.

Dowell and Brown (2009) Methods in Molecular Biology 552:213-229.

Fukutani (2012) Biotechnology and Bioengineering 109:205-212.

Hall et al. (1984) Insect Science and Its Application 5:5.

Hamada et al. (2014) Chem Commun (Camb) 50:2958-2961.

Harayama, (1998) Trends Biotechnol, 16:76-82.

Hendrickson (1991) Science 252: 1189.

Hulme and Trevethick (2010) British Journal of Pharmacology 161: 1219-1237.

Kiely et al. (2007) J Neurosci Methods 159:189-194.

Knols et al. (1997) Annals of Tropical Medicine and Parasitology 91 :S 117-S 118.

Kurtovic et al. (2007) Nature 446:542-546.

Lorenz et al. (2013) Parasites and Vectors 6: 1-12.

Martin et al. (2002) J Med Chem 45:4350-8.

Needleman and Wunsch (1970) J Mol Biol, 48:443-453.

Ray (2015) Current opinion in Neurobiology 34: 158-164.

Ritchie and Devine (2013) Parasites and Vectors, 6: 1-9.

Roberts et al. (1973) Proc.Natl.Acad.Sci. USA, 70:2330.

Sato et al. (2008) Nature 452: 1002-1006.

Schneider and Seifert (2010) Pharmacol Ther. 128:387-418.

Smallegange et al. (2005) Chemical Senses 30: 145-152.

Smart et al. (2008) Insect Biochem Mol Biol 38:770-780.

Tauxe et al. (2013) Cell 155: 1365-1379.

Thomas and Smart (2005) J Pharmacol Toxicol Methods 51 : 187-200.

Trexler et al. (1998) J Med Entomol 35:967-976.

Tsitoura et al. (2010) PLoS One 5:el5428.

Vale and Hall (1985) Bulletin of Entomological Research 75:219-231. Wetzel et al. (2001) Proc Natl Acad Sci USA 98:9377-9380.

Zeoli et al. (1982) Insect Suppression with Controlled Release Pheromone Systems, vol. 1, CRC Press Inc., Boca Raton, FL.: 131-144.

Zhang et al. (2011) PLoS One 6:e24111.

Claims

1. A method of screening for a compound which modifies mosquito behaviour, the method comprising:

(a) contacting a mosquito neuron receptor, or a variant thereof, with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio-propanol, (E)-l-methylthio- 1-propene, (Z)-l -methyl thio- 1-propene, a derivative thereof, or a mixture of two or more thereof,

(b) determining if the candidate compound modifies the ability of the thio compound to bind and/or activate the receptor.

2. The method of claim 1 , wherein the candidate compound is a thio compound.

3. The method of claim 2, wherein the thio compound is a thioether.

4. The method of claim 3, wherein the thioether is a compound of Formula 1 :

R-L R2

S

Formula 1

wherein Rl and R2 are independently selected from an optionally substituted Q. ₆alkyl and an optionally substituted C₂-₆alkenyl.

5. The method of claim 4, wherein Rl and R2 are independently selected from an unsubstituted C^aHcyl, an unsubstituted C₂-₆alkenyl and a Ci^alkyl substituted with a hydroxyl group.

6. The method according to any one of claims 1 to 5, wherein the receptor is a mosquito CpA or CpC neuron receptor, or a variant thereof.

7. The method according to any one of claims 1 to 6, wherein the neuron receptor is a mosquito odorant (Or) or a gustatory (Gr) receptor, or a variant thereof.

8. The method of claim 6 or claim 7, wherein the receptor is selected from the group consisting of mosquito Or28, Gr22, Gr23, Gr24, or a variant thereof.

9. The method of claim 8, wherein the receptor comprises two or more different subunits and:

i) one of the subunits is mosquito Or28 or a variant thereof, and a further subunit 5 is mosquito Or7 or a variant thereof, or

ii) one of the subunits is mosquito Gr22 or a variant thereof, and a further subunit(s) is mosquito Gr23 and/or Gr24, or a variant of one or both thereof.

10. The method according to any one of claims 1 to 9, wherein the receptor is from 10 an Anopheles spp., Aedes spp., Culex spp., Culiseta spp. or Haemagogus spp..

11. The method according to any one of claims 1 to 10, wherein the receptor, or variant thereof, is present in a cell.

15 12. The method according to any one of claims 1 to 11, wherein binding of the candidate compound to the receptor is determined via gel electrophoresis, ELISA, immunoblot, or surface plasmon resonance.

13. The method according to any one of claims 1 to 12, wherein receptor activation 20 is determined by electrophysiology.

14. The method according to any one of claims 1 to 13 which further comprises testing the ability of the candidate compound to modify the behaviour of a mosquito.

25 15. The method of claim 14 which further comprises selecting a compound which i) activates a mosquito,

ii) activates and attracts a mosquito,

iii) activates and repels a mosquito,

iv) does not activate, or reduces activation of, a mosquito in the presence of 30 human subject(s), or

v) does not activate, or reduces activation of, a mosquito in the presence of human subject(s) with a Plasmodium infection.

16. A method of screening for a compound which modifies mosquito behaviour, the 35 method comprising: (a) contacting a mosquito with a candidate compound in the presence of a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio- propane, 3-methylthio-propanol, (E)-l-methylthio-l-propene, (Z)-l-methylthio-l- propene, a derivative thereof, or a mixture of two or more thereof,

(b) determining if the candidate compound modifies the effect of the thio compound on the behaviour of the mosquito.

17. The method of claim 16 which further comprises selecting a compound which i) activates the mosquito,

ii) activates and attracts the mosquito,

iii) activates and repels the mosquito,

iv) does not activate, or reduces activation of, the mosquito in the presence of human subject(s), or

v) does not activate, or reduces activation of, the mosquito in the presence of human subject(s) with a Plasmodium infection.

18. The method of claims 16 or claim 17, wherein the mosquito is an Anopheles spp., Aedes spp., Culex spp., Culiseta spp., Haemagogus spp. or a combination of two or more thereof.

19. A computer implemented method for selecting a candidate compound for modifying mosquito behaviour, the method comprising identifying compounds having a similar structure to a thio compound selected from the group consisting of allyl methyl sulphide, 1-methylthio-propane, 3-methylthio-propanol, (E)-l-methylthio-l- propene, (Z)-l-methylthio-l-propene, or a derivative thereof, and selecting the identified compound as a candidate compound.

20. The method of claim 19, further comprising screening the selected candidate compound for the ability to modify mosquito behaviour.