EP4615446A1 - Reduction or prevention of a malaria parasite or dengue virus transmission with 1 -methyl-9h-pyrido[3,4-b]indole - Google Patents

Abstract

The present application relates to compositions comprising 1-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in the reduction or prevention of transmission of malaria and dengue, as well as to the corresponding methods. The application further relates to l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the growth of a parasite into an ookinete, an oocyst, or a sporozoite; and to a mosquito nectar feed comprising l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and one or more of a sugar source.

Description

REDUCTION OR PREVENTION OF A MALARIA PARASITE OR DENGUE VIRUS TRANSMISSION WITH 1 -METHYL-9H- PYRIDO[3,4-B]INDOLE

FIELD OF THE INVENTION

The present application relates to compounds and pharmaceutically acceptable salts thereof, compositions thereof, and their use in the reduction or prevention of transmission of malaria by the Anopheles mosquito vector and dengue by the Aedes mosquito vector.

BACKGROUND TO THE INVENTION

Infectious diseases are responsible for a wide variety of diseases of medical and veterinary importance. Many of these diseases are transmitted by insect vectors. Vector-borne diseases are infections transmitted by the bite of infected arthropod species, such as mosquitoes, ticks, triatomine bugs, sandflies, blackflies, as well as ectoparasites such as ticks and fleas.

Mosquitoes are vectors for a variety of infectious diseases. In particular, three medically relevant genus of mosquitoes which transmit diseases are Anopheles, Culex and Aedes. The genus Culex and Aedes belong to the sub-family Culicinae, while the Anopheles belongs to the sub-family Anophelinae. The Anopheles genus is a vector for malaria. The Aedes genus is a vector for dengue virus.

Malaria is a disease caused by protozoan parasites of the genus Plasmodium that infect and destroy red blood cells, leading to fever, severe anaemia, cerebral malaria, and if untreated, death. There are five species of Plasmodium parasite: falciparum, vivax, ovale, maiariae, and knowlesi. Plasmodium falciparum is the most virulent. In 2019, there were an estimated 229 million people infected with malaria in 87 malaria endemic countries, and malarial disease was responsible for an estimated 409,000 deaths (WHO malaria report: https://www.who.int/teams/qlobal-malaria-proqramme/reports/world-malaria-report-2021).

Dengue virus is a single positive-stranded RNA virus of the Flaviviridae family that causes Dengue fever. It is transmitted by Aedes mosquitoes. It is endemic in the tropics and subtropics, worldwide, where an estimated 100,000,000 cases occur annually. At least four serotypes of the virus have been identified, and the virus causes approximately 400 million infections annually. Infections with dengue virus can be asymptomatic or cause a spectrum of clinical disease ranging from mild fever to the more life-threatening dengue hemorrhagic fever and dengue shock syndrome which is frequently fatal.

These diseases are of significant medical importance. A number of drugs are available to treat and/or prevent some parasites or vector-borne diseases. However, not all parasites or vector-borne diseases can be treated efficiently. For example, there is currently no chemotherapeutic drug or vaccine available against the Dengue virus. Furthermore, in the case of antimalarial drugs, treatment with the drugs currently available is becoming less effective due to increased resistance in some Plasmodium strains. There is therefore the need to effectively control parasites and vectors of diseases to prevent transmission. In this regard, mosquitoes can be targeted by a wide range of insecticides and insect repellents. Mosquitoes can be targeted with insecticides when they are in a larval state or once they have developed into adults. However, mosquitoes have developed widespread resistance to currently used insecticides.

One approach to addressing this issue is to develop agents that are capable of reducing or preventing the transmission of vector borne diseases without negatively impacting the insect vector thereby circumventing the generation of resistance. In this regard, PCT/EP2020/069569 (published as WO 2021/009050) discloses bacteria of the Delftia genus, and its use in reducing malaria transmission in mosquitoes.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a composition for use in a method of reducing or preventing transmission of malaria or dengue, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing the mosquito vector into contact with the composition.

According to a second aspect of the invention, there is provided composition for use in a method of reducing or preventing transmission of the malaria parasite or dengue virus, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing the mosquito vector into contact with the composition.

In a third aspect of the invention, there is provided a method of reducing or preventing transmission of dengue or malaria, or malaria parasitic or dengue viral infection comprising a step of bringing at least one Anopheline vector or Culicinae vector of these vector borne diseases into contact with l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention, there is provided the use of l-Methyl-9H-pyrido[3,4- b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing malaria or dengue, or a malaria parasitic or dengue viral infection.

In a further aspect of the invention, there is provided l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the formation of malaria parasite into an ookinete, an oocyst, or a sporozoite or dengue viral infection. In a further aspect of the invention, there is provided a mosquito nectar feed comprising l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and glucose as a sugar source.

The present invention may be advantageous in a number of respects. In particular, the present inventors have found that the compound l-Methyl-9H-pyrido[3,4-b]indole (also known as Harmane, or 1-methyl-p-carboline) is produced by bacteria of the Delftia genus that is responsible for hindering transmission of malaria in the Anopheles mosquito. When introduced into an environment with Anopheles mosquito vector, the compositions of the invention prevent parasite development in the mosquito and therefor interrupts disease transmission. For example, l-Methyl-9H-pyrido[3,4-b]indole inhibits formation of ookinetes which are early stages of malaria parasite development in the mosquito gut. l-Methyl-9H-pyrido[3,4-b]indole can be used to combat the spread of malaria parasites. The compound may be used similarly in inhibition of dengue viral infections.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by reference to the accompanying drawings, which are non-limiting.

Fig. 1 shows a Liquid Chromatography-High Resolution accurate Mass Spectrometer trace for the active component of Delftia tsuruhatensis.

Fig. 2 shows a Heteronuclear single quantum coherence spectroscopy of the active component of Delftia tsuruhatensis.

Fig. 3 shows a ^TH NMR spectrum of the active component of Delftia tsuruhatensis.

Fig. 4 shows the effect of feeding harmane to An. gambiae mosquitoes in blocking Plasmodium development

Fig. 5 shows the effect of contacting harmane with An. gambiae mosquitoes in blocking Plasmodium development

Fig. 6 shows the duration of harmane inhibitory action in mosquitoes.

Fig. 7 shows harmane inhibition of ookinete formation in vitro.

Fig. 8 shows the effect of treatment of Delftia supernatant with proteinase K on the inhibition of P. falciparum development in An. gambiae mosquitoes.

Fig. 9 shows the effect of different concentrations of Delftia supernatant on Plasmodium parasite development in An. gambiae mosquitoes.

Fig. 10 shows the duration of Delftia supernatant inhibition of P. falciparum oocyst formation in An. gambiae mosquitoes.

Fig. 11 shows the inhibition of Plasmodium development in An. gambiae mosquitoes by supernatants of different Delftia strains. Fig. 12 shows results of a screen of Deiftia supernatant fractions for blocking activity of P. falciparum oocyst development in An. gambiae mosquitoes.

Fig. 13 shows the effect of harmane on BHK21 cell growth.

Fig. 14 shows the effect of harmane on dengue virus growth using cell culture.

Fig. 15 shows the effect of harmane on dengue virus multiplication in Aedes aegypti mosquitoes.

Fig. 16 shows the effect of harmane on mosquito mortality.

Fig. 17 shows the fitness cost of Deiftia o Ae. aegypti mosquitoes.

Fig. 18 shows the effect of Deiftia bacteria on dengue virus multiplication in Ae. aegypti mosquitoes.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In one aspect, the present invention provides a composition for use in a method of reducing or preventing transmission of malaria or dengue, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing at least one vector into contact with the composition. l-Methyl-9H-pyrido[3,4-b]indole is also known as harmane, and is represented by the following structure:

This compound has been found to be the active compound secreted by Deiftia bacteria, and is capable of suppressing malaria parasite and/or dengue virus transmission in a variety of vectors. In particular, it is shown that l-methyl-9H-pyrido[3,4-b]indole is capable of selectively suppressing inhibiting ookinetes and oocysts of Plasmodium in mosquito midgut. Thus, compositions of the present invention may reduce or prevent malaria transmission and/or malaria parasite transmission in a mosquito. In other cases, compositions of the present invention may reduce or prevent dengue transmission and/or dengue virus transmission in a mosquito. It will further be understood that the compounds of the invention, such as a compound of Formula (I) may exist in different tautomeric forms. Tautomers refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment that the compound is in. The compound may also be protonated or deprotonated depending on the pH of its surrounding environment. The compound may also be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include but are not limited to those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in P H Stahl and C G Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Edition, John Wiley & Sons, March 2011.

Where the compound functionality allows, suitable pharmaceutically acceptable salts of a compound of Formula (I) can be formed, which include acid or base addition salts. Acid addition salts may be formed by reaction with the appropriate acid, optionally in a suitable solvent such as an organic solvent, to give the salt which can be isolated by crystallisation and filtration. Base addition salts may be formed by reaction with the appropriate base, optionally in a suitable solvent such as an organic solvent, to give the salt which can be isolated by crystallisation and filtration.

Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecylsulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1 , 2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydrabamine (/V,/V-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1 ,5- disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-a mi nobenzenesulfonate, p-aminosalicyclate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate.

Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminium, 2-amino-2-(hydroxymethyl)-l ,3-propanediol (TRIS, tromethamine), arginine, benethamine (AAbenzylphenethylamine), benzathine (A(/V-dibenzylethylenediamine), bis-( 2- hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl- 2-pyrrolildine-r-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (AAmethylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, f-butylamine, and zinc.

The compound will be administered in the appropriate "effective amount". This effective amount will depend upon a number of factors including, for example, the size and weight of the subject, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of distribution, and will ultimately be at the discretion of the skilled person.

In a second aspect, the present invention provides a composition for use in a method of reducing or preventing transmission of a malaria parasite or dengue virus, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing at least one vector into contact with the composition.

In one embodiment, the parasitic disease is Malaria.

In one embodiment, the composition is for use in reducing or preventing: (i) malaria, and/or (ii) malaria parasite transmission. In an embodiment the composition is suitable for preventing malaria transmission in a mosquito. In another embodiment the composition is suitable for prevention malaria parasite transmission. In another embodiment the composition is suitable for prevention malaria parasite transmission in a mosquito.

As defined herein "reducing or preventing malaria or malaria parasite transmission" is defined as precluding malaria e.g. by inhibiting mosquito stages of the malaria parasite (ookinete and thereby oocyst formation). It has been found that the compound l-Methyl-9H-pyrido[3,4- bjindole is the active compound secreted by Delftia bacteria, specifically Delftia tsuruhatensis, and is capable of suppressing malaria transmission in mosquitoes by blocking malaria parasites.

Specifically, it has been shown here that, when introduced into a mosquito containing environment, harmane can prevent malaria parasite transmission by inhibiting ookinetes and oocysts of Plasmodium 'm mosquito midgut. In some embodiments, the mode of introduction to the mosquito can be through contact with sugar baits, nectar baits, blood baits, and/or other feeding baits where the l-Methyl-9H-pyrido[3,4-b]indole can be transmitted to and/or into the mosquito through cuticular uptake and/or ingestion. Thus, compositions of the present invention may reduce or prevent malaria transmission and/or malaria parasite transmission in a mosquito. The mosquito may be any mosquito capable of transmitting malaria, e.g. mosquitoes of the Anopheles genus. It is envisaged that the compositions and methods of the present invention extend to any Anopheles species of mosquito. In an embodiment, the mosquito is Anopheles gambiae, Anopheles stephensi, Anopheles culicifacies, or Anopheles coluzzi. In an embodiment of the invention the mosquito is Anopheles gambiae or Anopheles stephensi. In an embodiment the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

The malaria parasite may be any malaria parasite. In some embodiments the malaria parasite is a Plasmodium parasite. In some embodiments the malaria parasite is Plasmodium falciparum, Plasmodium berghei, Plasmodium vivax, or a combination thereof. In some embodiment the parasite is Plasmodium falciparum. In other embodiments, the parasite is Plasmodium berghei. In still other embodiments, the parasite is Plasmodium vivax.

In an embodiment, the composition is for use in reducing or preventing: (i) Dengue, and/or (ii) Dengue virus transmission. In an embodiment, the composition is for use in reducing or preventing: (i) Dengue, and/or (ii) Dengue virus transmission in a mosquito. In one embodiment, the composition is for use in reducing or preventing Dengue. In another embodiment, the composition is for use in reducing or preventing Dengue virus transmission (e.g., in a mosquito). The mosquito may be any mosquito. In one embodiment, the mosquito is a mosquito of the ecfesgenus. In one embodiment, the mosquito is Aedes aibopictus, or Aedes aegypti.

As defined herein "reducing or preventing dengue virus transmission" is defined as precluding dengue e.g. by inhibiting mosquito stages of the dengue virus load. It has been found that the compound l-Methyl-9H-pyrido[3,4-b]indole is the active compound secreted by Deiftia bacteria, specifically Deiftia tsuruhatensis, and is capable of suppressing dengue transmission in mosquitoes by blocking dengue virus.

In another aspect, the present invention provides a method of reducing or preventing transmission of malaria or dengue comprising a step of bringing at least one vector of the disease or parasite into contact with l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

The step of bringing the vector parasite or virus into contact with the compound may occur in any suitable way. For instance, a person does not physically have to contact a vector, parasite or virus with l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The compound could be left in a place where it will come into contact with the vector. The compound may be in the form of a composition as described above or below.

In certain embodiments of the invention, the contacting may be achieved by treating an area with a composition of the present invention, for example, by using a spray formulation, such as an aerosol or a pump spray. In certain embodiments of the invention, an area can be treated, for example, via aerial delivery, by truck-mounted equipment, or the like. In some embodiments, the composite on is sprayed by e.g., backpack spraying, aerial spraying, spraying/dusting etc. The vector of parasite or virus may be any vector of parasite or virus capable of transmitting disease. In one embodiment, the vector is a mosquito.

For malaria, the mosquito may be any mosquito capable of transmitting malaria, e.g. mosquitoes of the Anopheles genus. It is envisaged that the compositions and methods of the present invention extend to any species of mosquito. In one embodiment, the mosquito is a mosquito of the Anopheles genus. In one embodiment, the mosquito is Anopheles gambiae, Anopheles stephensi, Anopheles culicifacies, or Anopheles coluzzi. In one embodiment, the mosquito is Anopheles stephensi or Anopheles gambiae. In an embodiment the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae. The malaria parasite may be any malaria parasite. In an embodiment the malaria parasite is a Plasmodium parasite. In an embodiment the parasite is Plasmodium falciparum. In another embodiment, the parasite is Plasmodium berghei. In still other embodiments, the parasite is Plasmodium vivax. In an embodiment, the parasite is selected from Plasmodium malariae, Plasmodium ovale curtisi, P. ovale wallikeri and Plasmodium knowlesisel.

For Dengue virus the method involves contacting a mosquito with 1-Methyl-9H- pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The mosquito may be any mosquito capable of transmitting Dengue virus. It is envisaged that the compositions and methods of the present invention extend to any species of mosquito. In one embodiment, the mosquito is of the ecfesgenus. In one embodiment, the mosquito is Aedes aibopictus, or Aedes aegypti.

In another aspect, the present invention provides the use of l-Methyl-9H-pyrido[3,4- b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing a malaria or dengue, or a malaria parasitic or dengue viral infection.

In another aspect, the present invention provides l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the growth of a malaria parasite into an ookinete, an oocyst, a sporozoite or replication of the dengue virus in its mosquito vector.

COMPOSITIONS

The compositions of the invention may be in any suitable form and may include any suitable carrier. The composition may be a feed composition, i.e. the composition may be in a form which can be presented to a vector or parasite for consumption through oral administration. In some embodiments, the feed composition is an attractive sugar bait, a sugar source, or a nectar feed. In one embodiment, the feed composition is a sugar source. The sugar source may be an attractive sugar bait or be comprised within an attractive sugar bait. Attractive sugar baits typically comprise a sugar and a toxic ingredient. It is envisaged that an attractive sugar bait according to the invention will comprise the composition of the invention instead of the toxic ingredient, i.e. the attractive sugar bait may comprise sugar and a composition of the invention. In some embodiments, the attractant is a sugar source comprising a mixture of fruit juices and/or syrups. In an embodiment, the composition is in the form of a bait. The bait is designed to lure the mosquito vector to come into contact with the composition. In one embodiment, upon coming into contact therewith, the composition is then internalized by the vector (e.g., mosquito), by ingestion and/or through cuticular uptake for example. An attractant can also be used. The attractant can be a a sugar or a mixture of sugars or fruit juices or fruit pulps, pheromone, such as a male or female pheromone. The attractant acts to lure the vector or parasite (e.g., mosquito) to the bait. The bait can be in any suitable form, such as a solid, paste, pellet or powdered form.

The baits can be provided in a suitable "housing" or "trap". Such housings and traps are commercially available and existing traps can be adapted to include the compositions of the invention. The housing or trap can, for example, be box-shaped and can be provided in preformed condition or can be formed of foldable cardboard for example. Suitable materials for a housing or trap include plastics and cardboard, particularly corrugated cardboard. The inside surfaces of the traps can be lined with a sticky substance in order to restrict movement of the vector or parasite (e.g., mosquito) once inside the trap. The housing or trap can contain a suitable trough inside which can hold the bait in place. A trap is distinguished from a housing because the mosquito cannot readily leave a trap following entry, whereas a housing acts as a "feeding station" which provides the mosquito vector with a preferred environment in which they can feed and feel safe from predators.

In another embodiment, the present invention provides a mosquito nectar feed comprising l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and one or more of a sugar source.

COMBINATIONS

It is envisaged that the present invention is deployed along with other malaria or dengue eradication efforts. For example, the compositions, methods and compound for use of the present invention may be used alongside known anti-vector or anti-parasite agents (such as anti-malarial agents). In one embodiment, the compositions or compound for use of the present invention may be used in combination with one, two or three additional anti-parasite agent or anti-malarial agents. Integrated Vector Management (IVM) suggests making full use of the tools available.

The at least one other anti-malarial agent may also be selected from ferroquine, KAF156, cipargamin, DSM265, artemisone, artemisinone, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, OZ609, OZ277 and SAR97276. In the treatment of P. falciparum infections, the at least one, two or three additional anti-malarial agents may be selected from the following list, wherein at least one of the anti-malarial agents is an artemisinin-based agent: artemether and lumefantrine, artesunate and amodiaquine, artesunate and mefloquine, dihydroartemisinin and piperaquine, or artesunate and sulfadoxine- pyrimethamine (SP). The above combination treatments are known as artemisinin-based combination therapies (ACTs). The choice of ACT is usually based on the results of therapeutic efficacy studies against local strains of P. falciparum malaria. In the treatment of P. vivax infections, an ACT may be used, as described above. Alternatively, the at least one other anti- malarial agent may be chloroquine, particularly in areas without chloroquine resistant P. vivax. In areas where resistant P. vivax has been identified, infections may be treated with an ACT, as described above. The combinations of therapeutic agents may conveniently be presented for use in the form of a pharmaceutical composition or formulation and may be administered together or separately and, when administered separately, this may occur separately or sequentially in any order (by the same or by different routes of administration).

The compositions or bacteria for use of the invention may be used in conjunction with use insecticide treated nets (ITNs), including long-acting insecticidal nets(LLINs) and/ IRS (Indoor residual sprays). ATSB (attractive toxic sugar baits) lure mosquitos to feed on sugar with toxic mosquito— killing compounds. For efficiency, ATSB's could also include the harmane compound discussed above, instead of the toxic compound.

EXAMPLES

The invention will now be illustrated by way of the following non-limiting examples. While particular embodiments of the invention are described below a skilled artisan will appreciate that various changes and modifications can be made. References to preparations carried out in a similar manner to, or by the general method of, other preparations, may encompass variations in routine parameters such as time, temperature, work-up conditions, and minor changes in reagent amounts, etc.

Materials

Microorganism strains

- Detia tsuruhatensis GSK TCI

- Deiftia tsuruhatensis Carb* , and Mutl**

* Carb-USA: Deiftia tsuruhatensis originally from ATCC, obtained from Dr. Ramesh Goel, University of Utah, USA.

** Mutl-USA; Deiftia tsuruhatensis, obtained from Dr. Goel (2).

- Deiftia acidovorans. Ingrid* * Ingrid: Deffia acidovorans, obtained from Dr. Ingrid Faye, Stockholm University, Sweden.

- Pseudomonas putida GSK TC2

- Pantoea agglomerans

Mosquitoes

- An. gambiae

-An. stephensii

Parasite:

P. falciparum SA

P. bergheiM A 234

Media :

M9 minimal medium:

Filter-sterilize and store at 4°C.

Autoclave and store at room temperature.

Medium for BHK21: DMEM+10% Heat inactivate FBS+1% L- glutamine+1% Pen-strep+5pg plasmocin.

Overlay medium: DMEM+2 % Heat inactivate FBS+1% L- glutamine+1% Pen-strep+5pg plasmocin + 0.8% methycellulose

Drug: harmane (CHEM-IMPEX INT'L INC, Cat#21682)

DMSO (Amresco Life Science, Cat#0231-500ML)

Example 1: Bioassay guided purification of the active natural product from the supernatant of Delftia tsuruhatensis

The fermentation supernatant (10L) was loaded onto a C-18 reversed-phase silica gel column (160 x 30 mm; Sepra™ C-18-E (50 pm, 65 A)) for flash fractionation. Unretained material in the column (follow-through was collected for activity testing) and the column was eluted subjected to isocratic elution (H2O/CH3CN 95:5) followed by a gradient of acetonitrile (CH3CN) in water from 5% to 100% in 40 min, and an isocratic step at 100% of CH3CN for 20 min at 10 mL/min. 18 mL fractions were collected. UV detection at 210 and 280 nm was used.

Additionally, lOOmL of unfermented medium were loaded onto a 60 x 15 mm C-18 cartridge and eluted with 100 mL of 100% of CH3CN, that were dried down (12.5 mg) and were dissolved in MeOH 1 mL.

500 piL of of the supernatant, aliquots of 500 piL of each fraction, 500 piL of the follow- through, obtained while loading the 10L supernatant in the C-18 column, and 100 piL of the blank medium extract were transferred into an AB-Gene 0765 800 piL 96-well storage plate and dried down in a HT-8 Genevac vacuum centrifuge for shipment and activity evaluation.

LC-HRMS dereplication methodology

Active fractions were analyzed using an Agilent 1200 Rapid Resolution HPLC interfaced to a Bruker maXis mass spectrometer. The volume of sample injected was 2 ptL. A Zorbax SB- C8 column (2.1 x 30 mm, 3.5 pirn particle size) was used for the separation. Two solvents were used as mobile phase: solvent A FbCXCHsCN 90:10, solvent B water: CH3CN 10:90, both with 13 mM ammonium formate and 0.01% TFA. The gradient composition was:

The mass spectrometer was operated in positive ESI mode. The instrumental parameters were: 4kV capillary voltage, drying gas flow of 11 L/min at 200°C, nebulizer pressure at 2.8 bars. TFA-Na cluster ions were used for mass calibration of the instrument prior to samples injection. Each sample run was recalibrated by infusion with the same TFA-NA calibrant before the chromatographic front.

NMR dereplication methodology

For the NMR analyses, samples were dissolved in CD3OD. After dissolution, each sample was transferred to a 1.7 mm tube. Acquisitions (ID ^XH spectra and 2D HSQC spectra) were carried out on a Bruker AVANCE III 500 MHz spectrometer equipped with a 1.7 mm TCI microcryoprobe. All spectra were registered at 24 °C.

Results

A molecular formula of C12H10N2 was determined for the active component from LC- HRMS as shown in Fig. 1.

The HSQC and^ NMR spectra for the active component matched that of harmane as shown in Figs. 2 and 3, respectively.

BIOLOGICAL DATA

Mosquitoes rearing and parasite culture

Anopheles gambiae strain Keele strain) and Anopheles stephensi (Nijmegen strain) were reared as described and known in the art. For fitness evaluation, the mosquitoes were fed on Swiss Webster mice.

Female An. gambiae were infected with P. falciparum gametocyte cultures via membrane feeding and P. falciparum NF54 gametocytes were produced. Briefly, parasites were maintained in 0+ human erythrocytes using RPMI 1640 medium supplemented with 25 mM HEPES, 50 mg/L hypoxanthine, 25 mM NaHCO3, and 10% (v/v) heat-inactivated type O+ human serum (Interstate Blood Bank, Inc.) at 37 °C and with a gas mixture of 5% O2, 5% CO2, and balanced N2. For feeding, 14-17-day-old mature gametocytes were pelleted by centrifugation (5 min, 2,500 g), resuspended with O+ human RBCs to 0.15%-0.2% gametocytemia and diluted to 40% hematocrit with human serum. All manipulations were done maintaining the cultures, tubes, and feeders at 37 °C.

Supernatant preparation

Deiftia an Pantoea agg/omerans {control) were grown in LB liquid medium overnight (200 rpm, 28 °C). Bacteria were washed, resuspended in M9 medium (10⁹/ml) and incubated (200 rpm, 28 °C) for 8 h, after which they were centrifuged, and the supernatant passed through a 0.22 pm filter to generate Deiftia supernatant (D-8h) and P. aggiomerans supernatant (P-8h). An aliquot of the Deiftia supernatant was passed through a 3 kDa centrifugal filter (Amicon Ultra-3K, REF: UFC500396) (D-8h<3KD). Another supernatant aliquot was boiled for 10 min (D- 8h boiled). An aliquot of the Deiftia cell suspension in M9 medium (10⁹/ml) was heated at 70 °C for 15 min to generate dead bacteria (Dead-B).

Deiftia supernatant treatment with proteinase K M9 medium (control) and D-8h were incubated with 1.25 pg/ml proteinase K in 30 mM Tris HCI, pH 8.0 at 50 °C for 1 h (M9+K and D-8h+K). Mosquitoes were fed with P. falciparum gametocytes (150 pl infected red blood cell+150 pl normal human serum) with added 50 pl M9, M9+K, D-8h or D-8h+K. Number of oocysts per midgut was determined 7 d feeding. Parasite numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

Delftia supernatant blocking of P. falciparum oocyst development

0 day, 1 day, 2 days and 4 days after 2-day old mosquito fed with or without Delftia supernatant. Day 4, the mosquitoes were fed by P. falciparum gametocytes. The number of oocysts in the midgut were determined 7 d post-blood meal. Data was pooled from two independent experiments. Parasite numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

Delftia supernatant blocking of oocyst formation

P. falciparum gametocytes (150 pl infected red blood cells + 150 pl normal human serum to a final 0.02% gametocytemia) were mixed with different Delftia supernatant concentrations in M9 medium and fed to An. gambiae mosquitoes. The number of oocysts was determined 7 d post feeding. Parasite numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

Delftia supernatant blocking of ookinete formation

An. gambiae mosquitoes were fed on a P. faiciparumA e e blood meal (150pl Red blood cell + 150pl normal human serum) with 50 pl M9 medium, D-8h, D-8h boiled, Dead-B and P-8h. The number of ookinetes in the midgut were determined 22 h post feeding. Each midgut was put into 20 pl PBS, homogenized by pipetting and transferred to 8-well slides, 1 midgut/well. After drying at room temperature, the samples were fixed in 80% methanol for 15 s and allowed to dry at room temperature. Samples were blocked with 5% BSA 1 h at room temperature and then incubated for 1 h at room temperature with Pfs25 antibody (Mab4b7) (38) in blocking buffer (1:250-1:500 dilution). After 3 washes with PBS for 5 min, samples were incubated for 1 h at room temperature with Alexa fluor 488 goat anti-mouse IgG (Life Technologies, Cat#: Al 1001) in blocking buffer (1:250-1:500 dilution). After washing 3 times with PBS for 5 min, slides were allowed to dry and covered with cover slips. Ookinetes were counted using fluorescent microscopy. Parasite numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism). Harmane inhibitory activity against asexual blood stages of P. falciparum

This assay is based on the parasite incorporation of labelled hypoxanthine that is proportional to Plasmodium falciparum growth. Briefly, cultures of 3D7A and Dd2 parasitized red blood cells (RBCs) (0.5% parasitemia and 2% hematocrit in RPMI-1640 supplemented with 25 ml Albumax and 5 pM hypoxanthine for final volume of 500 ml) were exposed to 2-fold serial dilutions. A total of 10 concentrations were used starting with lOpM. Plates were incubated at 37°C using [5% CO2, 5% 02, 90% N2]. After 24 h incubation, ³H-hypoxanthine (0.025 pCi/pl in RMPI-1640) was added and incubated for an additional 24 h, following which plates were kept frozen at -80°C. Parasites were harvested on a glass fiber filter using a TOMTEC Cell Harvester 96. Filters were dried and melt-on scintillator sheets were used to determine the incorporation of ³H-hypoxanthine by measuring radioactivity in a MicroBeta counter.

Exflagellation assay

A total of 1 ml of P. falciparum NF54 gametocyte culture was centrifuged for 1 min at 700 X G and after discarding the supernatant, the pellet was resuspended in an equal volume of normal human serum and incubated for 10-15 min at room temperature. After placing 10 pl of this suspension on a glass slide and covering with a coverslip, the average number of exflagellation centers per field was determined using a light microscope at 400X magnification. At least 10 to 20 random fields were counted. Exflagellation numbers among control and experimental groups were compared using the Student's test.

Harmane inhibition of Plasmodium development in An. gambiae mosquitoes

Feeding assay: P. falciparum NF54 gametocytes (150pl Red blood cell+150pl normal human serum) were fed to 2-day-old mosquitoes after addition of 50pl of M9 medium or D-8h with without 1 nM, 10 nM, 25 nM, 50 nM and 150 nM harmane. The number of oocysts was determined 7 d post feeding. Oocyst numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

Contact assay: Harmane was dried on 9 cm glass Petri dishes to a final coverage of lnmol/m², 10 nmol/m², 100 nmol/m², 1 pmol/m² and 10 pmol/m². Atovaquone (ATQ) was dried on 9 cm glass Petri dishes to a final coverage of 100 nmol/m², 1 pmol/m², 10 pmol/m² and 100 pmol/m². A total of 50 pl D-8h (positive control), and M9 medium and or P-8h Pantoea supernatant as a negative controls) were also dried on 9 cm glass Petri dishes. Two-day-old mosquitoes were sugar-starved overnight (only water offered), knocked down with CO2 for 2-3 minutes and transferred to the coated Petri dishes for 1 h at 27 °C. Mosquitoes were knocked down and transferred to mosquito cups and 3 h later, were fed on a P. faiciparum^ ecte blood meal. Oocyst numbers were determined 7 d post feeding. Oocyst numbers among control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

Harmane inhibitory activity of P. falciparum asexual growth

This assay is based on parasite incorporation of labelled hypoxanthine that is proportional to Plasmodium falciparum growth. Briefly, 3D7A- and Dd2-parasitized red blood cell (RBC) cultures (0.5% parasitemia and 2% hematocrit in RPMI-1640 supplemented with 25 ml Albumax and 5 pM hypoxanthine in 500 ml) were exposed to 2-fold serial dilutions of the compound. A total of 10 concentrations were used starting with 10 pM. Plates were incubated at 37°C in 5% CO2, 5% 02, 90% N2. After 24 h incubation, ³H-hypoxanthine (0.025 pCi/pl in RMPI-1640) was added and further incubated for an additional 24 h following which plates were frozen at -80 °C. Then, parasites are harvested on a glass fiber filter using a TOMTEC Cell Harvester 96. Filters were dried and melt-on scintillator sheets were used to determine the incorporation of ³H-hypoxanthine by measuring radioactivity in a Microbeta counter.

Harmane inhibitory activity of P. falciparum male and female gamete formation

Dual Gamete Formation Assay or DGFA. Mature Stage 5 gametocytes were exposed to 2-fold serial dilutions of the compound. A total of 10 concentrations were used starting with lOpM and ending at O.OlpM. Compound was incubated for 48 hours followed by activation in ookinete media using the DGFA protocol by Ruecker (39).

In vitro ookinete production assay

Mice were treated with phenylhydrazine three days prior to infection by intra peritoneal inoculation of P. berghei ANKA parasites, obtained from a donor mouse between the second and sixth passage from cryopreserved stock. Parasitaemia was checked on day three postinfection or more, by microscopic examination of thin blood films and the presence of exflagellating gametocytes was determined (to make sure they have >15 exflagellations/20 x field). Gametocytaemic blood was collected into a heparinized syringe via cardiac puncture. This was achieved by passing the blood through a pre-equilibrated 5 ml sterile column containing 1 ml glass wool and 3 ml of Whatmann CF11 cellulose powder (Beckton & Dickenson). Wash the blood with RPMI and add blood (1 ml) to fresh complete ookinetes medium (19 ml). Transfer the blood solution to 24-well plate (0.5ml/well), mix with DMSO (control) or harmane and shake the plate for 24 h at 19 °C. Cytotoxicity assays

Harvest 80% confluent BHK21 cells and dilute the cell suspension 50 times. Transfer cell suspension to 24-well plate and add harmane to a final concentration of 0 (control), 1 nM, 10 nM, lOOnM, luM and lOuM in individual wells. Cells were cultured at 37 °C and 5% CO2 for 2 d, and count cell numbers in each well. Inhibition = 100 X [cell number(control)-cell number (harmane treated well)]/ cell number(control)

Dengue infection blocking assay

BHK21 cells were cultured in 24-well plates, then used for dengue infection when the cells were 80% confluent. The cells were diluted 10 times and transferred to 24 well plates. Harmane was diluted to a final concentration of 1 nM, 10 nM, 100 nM, 1 uM and 10 uM in complete DMEM medium and added to the cells, which were then incubated at room temperature on a rocking platform for 15 min, then incubated for lh in an incubator at 37 °C with 5% CO2. Approximately 1 h after the addition of the test compound, dengue virus was added to each well, and incubated with slow rocking for 15 min, and then incubated for 45 min in an incubator at 37°C with 5% CO2. Add 1 ml of overlay medium to each well, and cells were incubated at 37 °C with 5% CO2 for 5 d. The plates were then fixed and visualized with methanol/acetone mixture (1:1 volume) and 1% crystal violet mixture for 30 min at RT, and plaque-forming units were counted. Results from drug-treated cells were compared to results from DMSO controls. The experiments were performed in triplicate.

Discussion of the figures

Referring to Fig. 4: Harmane fed to An. gambiae mosquitoes inhibits Plasmodium development.

(A) P. falciparum gametocytes were fed to mosquitoes with or without the indicated concentrations of harmane. M9: M9 medium control; D-8h: supernatant of Delftia cultured in M9 medium for 8 h. The horizontal red bars indicate median values. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Data pooled from three independent experiments

(B) Dose-response curve fit for oral feeding of harmane {nonlinear regression, n = 13, df = 12, sum of squares = 1054, R² = 0.9082. The IC50 for HA feeding was calculated by interpolation and indicated below the graph. Mean inhibition relative to control oocyst intensity is indicated. Error bars are 95% confidence intervals (Cl)}. Fifty percent inhibition observed at 45.9 nM. Relates to data in panel A. Statistical analysis by Mann-Whitney U test. ***: P value <0.001 ; **: P value <0.001 ; *: P value <0.05; NS, not significant. Referring to Fig. 5: Contact inhibition of Plasmodium development in An. gambiae mosquitoes.

(A) The Delftia supernatant contains a compound that penetrates the mosquito cuticle and inhibits P. falciparum development. Female mosquitoes were exposed for 60 min to D-8h, atovaquone (ATQ) or P-8h (Pantoea supernatant) dried on a glass plate before infection with P. falciparum gametocytes. D-8h feeding: supernatant mixed with infectious blood and fed to mosquitoes, as a positive control. Data pooled from two independent experiments.

(B) and (F) Harmane (HA) or (C) and (G) atovaquone (ATQ) were dried on glass plates at the final concentrations indicated. Mosquitoes were exposed to these plates for 60 min and were then fed P. falciparum gametocytes. M9: 3 ml of M9 medium dried on the plate. D-8h: 3 ml Delftia supernatant dried on the plate. Data pooled from two independent experiments. Statistical analysis by Mann-Whitney U test. ***: P value <0.001 ; **: P value <0.01 ; NS, not significant. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts.

(D) Dose-response curve fit for harmane (HA) exposure {nonlinear regression, n = 17, df = 16, sum of squares = 6698, R² = 0.6122. The IC50 for HA exposure was calculated by interpolation and indicated below the graph. Mean inhibition relative to control oocyst intensity is indicated. Error bars are 95% confidence intervals (Cl)}. Relates to panel B. Fifty percent HA inhibition observed at 102.2 nmol/m².

(E) Dose-response curve fit for atovaquone (ATQ) exposure {nonlinear regression, n = 13, df = 12, sum of squares = 4120, R2 = 0.8085. The IC50 for ATQ exposure was calculated by interpolation and indicated below the graph. Mean inhibition relative to control oocyst intensity is indicated. Error bars are 95% confidence intervals (Cl)}. Fifty percent ATQ inhibition observed at 3,634 nmol/m². Relates to panel C.

Referring to Fig. 6: Duration of harmane inhibitory action in mosquitoes.

(A) Schematic outline of the experiments. Harmane was administered to female mosquitoes either via feeding (B) or via contact (C) at 0, 1, 2 and 4 d prior to feeding P. fa/ciparun rn ected blood. Oocyst number per midgut was determined 7 d later. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Data pooled from two independent experiments. Statistical analysis by Mann-Whitney U test. ***: P value <0.001; NS, not significant.

Referring to Fig. 7: Harmane inhibition of ookinete formation in vitro.

(A) Schematic outline of the experiment. (B) P. berghei gametocyte-containing blood was incubated after addition of DMSO (control) or 1 pM harmane at 0 or 4 h post culture setup and ookinete numbers were measured 24 h later. Data pooled from two independent experiments. Statistical analysis by Student's test. **: R value <0.01.

Referring to Fig. 8: Treatment of Delftia supernatant with proteinase K does not alter inhibition of P. falciparum development in An. gambiae mosquitoes.

M9 medium and D-8h (supernatant of Delftia cultured in M9 medium for 8 h) were treated with 1 .25 pg/ml proteinase K at 50 °C for 1 h, indicated by M9+K and D-8h+K. Mosquitoes were fed P. falciparum gametocytes with M9, M9+K, D-8h or D-8h+K and oocyst formation was monitored. Horizontal lines are median values. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Statistical analysis by Mann-Whitney U test. ***: P value <0.001 ; NS, not significant. Data from one experiment.

Referring to Fig. 9: Effect of different concentrations of Delftia supernatant on Plasmodium parasite development in An. gambiae mosquitoes. A total of 50 pl of M9 medium or of straight (100%) or diluted (1% or 10%) Delftia supernatant was added to 300 pl of blood carrying P. falciparum parasites (150 pl infected red blood cells + 150 pl normal human serum to a final 0.02% gametocytemia) and fed to mosquitoes. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Data from one experiment. Statistical analysis by Mann-Whitney U test (GraphPad, Prism). ***: P value <0.0001.

Referring to Fig. 10: Duration of Delftia supernatant inhibition of P. falciparum oocyst formation in An. gambiae mosquitoes.

Zero, one, two and four days after feeding mosquitoes with Delftia 8-hour culture supernatant in M9 medium, mosquitoes were fed P. falciparum gametocytes and oocyst formation was measured. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Statistical analysis by Mann-Whitney U test. ***: P value <0.001; NS, not significant. Data pooled from two independent experiments.

Referring to Fig. 11: Inhibition of Plasmodium development in An. gambiae mosquitoes by supernatants of different Delftia strains. P. falciparum gametocytes were combined with supernatants of different strains that were cultured for 8 h in M9 medium and fed to mosquitoes. Pantoea\ P. aggiomerans control; Dtsul: D. tsuruhatensis strain originated at GSK used in this work; Dtsu2: D. tsuruhatensis strain from ATCC; Dtsu3: D. tsuruhatensis strain from Gilcrease et al. (57); Dacido, De/ftia acidovorans strain obtained from Dr. Ingrid Faye, Stockholm University, Sweden. Number: number of mosquitoes analyzed; Prevalence: proportion of mosquitoes carrying one or more oocysts. Data pooled from two experiments. Statistical analysis by Mann-Whitney U test. ***: /’value <0.0001; NS: not significant.

Referring to Fig. 12: Screen of Delftia supernatant fractions for blocking activity of P. falciparum oocyst development in An. gambiae mosquitoes.

(A) A total of 10 L of Delftia supernatant was fractionated on a C18 column and 1/36 of each fraction (equivalent to 278 mL of supernatant) was dried in a multi-well plate. “PL_ROW” and “PL_COLMN” indicate position of the fraction in the plate. The seven pooled fractions for testing are indicated by color shading and named to the right. Crude: 500 pL dried Delftia supernatant; FL: 500 pL dried flow-through of the C18 column; M9 FL: as a control, M9 medium alone was passed through the column and 100 ml flow-through was collected and dried.

(B) Fractions resuspended in DMSO and tested for inhibition of P. falciparum oocyst formation in An. gambiae mosquitoes. C: DMSO control; D-8h: fresh Delftia 8-hour culture supernatant in M9 medium positive control.

(C) Assays of individual P4 and P5 fractions.

(D) Assays of individual P6 and P7 fractions. Data from one experiment. Statistical analysis by Mann-Whitney U test. ***: R value <0.001 ; **: R value <0.01 ; *: R value <0.05; NS, not significant.

Referring to Figs. 13 and 14, whereas harmane does not affect BHK21 cell growth in culture (except for the highest concentration tested: 10 pM), it significantly inhibits dengue virus infection at 1 pM.

Referring to Fig. 15, 3-to-5-day-old mosquitoes were fed a blood meal containing approximately IO^6-7 PFU/ml Dengue virus.

(A) The blood meal contained the indicated concentrations of harmane.

(B) Prior to taking the bloodmeal, mosquitoes were exposed for 60 min to DMSO (control) or the indicated concentrations of harmane (HA) dried on a glass plate.

After 7 days, the mosquitoes were dissected, and midguts homogenized and analysed. The number of plaque forming units (PFUs) were determined as described under "Dengue infection blocking assay and this is shown in Figure 15. Figure 15A shows that harmane concentration of 100 nM and lower does not significantly inhibit dengue virus multiplication in mosquitoes, whereas 1 pM and 10 pM strongly inhibited. Figure 15B shows that contacting mosquitos with 10'¹ mmol/m² (100 pmol/m²) harmane prior to infection can inhibit dengue virus infection. (Data pooled from 3 independent experiments.) Fig. 16 shows survival rates for 3-to-5-day-old Aedes mosquitoes which were fed a blood meal containing approximately 10⁶'⁷ PFU/ml Dengue virus and different concentrations of harmane, as follows: 0, 10 nM, 100 nM, lpM and 10 pM. Mosquito survival after 7 days was not affected, even with 10 uM harmane. (Data pooled from 3 independent experiments.)

Figure 17 shows the fitness cost of Delftia o mosquitoes.

Referring to Fig. 17A and 17B, two-day-old Aedes aegypti female mosquitoes were fed with Delftia or Pantoea bacteria. The "control" group was not fed bacteria.

(A) the blood feeding rate and (B) the blood meal size were determined 2 days later by measuring hemoglobin content of individual guts after the blood meal. Error bars represent SD of the mean. Data pooled from three independent experiments (30 mosquitoes per sample). Statistical analysis used the t-test.

Referring to Fig. 17C and 17D, two-day-old female mosquitoes were fed with Delftia or Pantoea bacteria. The "control" group was not fed bacteria. Five days later, mosquitos were provided a blood meal.

(C) fertility (number of laid eggs) and (D) fecundity (proportion of the laid eggs that hatched) were measured. Horizontal lines are median values. No significant differences were detected using the Mann-Whitney U test. Combined from three biological replicates (96 mosquitoes total). NS: not significant; *: R value <0.05.

Referring to Fig. 17E and 17F, two-day-old Aedes female mosquitoes were fed with Delftia or Pantoea bacteria. The "control" group was not fed bacteria.

(E) Male and (F) Female mosquito survival was measured. No significant differences were detected in the male group. A slight difference was observed between the survival rates of Deiftia-t&A and control mosquitoes in the female group. Survival rates were calculated by Kaplan-Meier survival curves, and multiple comparisons by Log-rank test. Combined from three biological replicates (around 330 mosquitoes total). NS: not significant; *: P value <0.05.

The data in Fig. 17 shows that Delftia does not impose a fitness cost to mosquitoes.

Referring to Figure 18: Delftia inhibition of dengue infection in Aedes mosquitos.

(A) and (B) Aedes mosquitoes were fed Delftia bacteria and subsequently infected with dengue virus 2 days later. (C) Aedes mosquitoes were fed Deffia bacteria and subsequently infected with dengue virus 16 days later.

Figures 18A and 18B show that Aedes mosquitoes which were fed Deffia bacteria were found to have fewer PFUs in the midgut and salivary gland. Figure 18C shows that this effect is long lasting. This demonstrates that Deffia can inhibit the dengue virus infection at midgut and salivary gland stages, and the effects last at least 16 days. The number of plaque forming units (PFUs) were determined as described under "Dengue infection blocking assay. Deffia bacteria inhibit Dengue virus multiplication in Aedes aegypti 78.4%. (Data pooled from 3 independent experiments.)

JOURNAL PUBLICATION

Further biological data was published after the priority date of the present application in Huang, Science, 381, 533-540 (2023).

Claims

CLAIMS:

1. A composition for use in a method of reducing or preventing transmission of malaria or dengue virus, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing at least one Anopheline or Culicinae mosquito vector into contact with the composition.

2. A composition for use in a method of reducing or preventing transmission of a malaria parasite or dengue virus, wherein the composition comprises l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of bringing at least one mosquito vector into contact with the composition.

3. The composition for use according to claim 1, wherein the composition further comprises at least one carrier, delivery vehicle, adjuvant, solvent, stabilizer, or preservative.

4. The composition for use according to any one of claims 1 to 3, wherein the composition comprises an attractant.

5. The composition for use according to claim 4, wherein the attractant is a sugar source or pheromone.

6. The composition for use according to claim 1, wherein the vector borne disease is selected from dengue and malaria.

7. The composition for use according to claim 2, wherein the parasitic disease is Malaria.

8. The composition for use according to any previous claim, wherein the composition is for use in reducing or preventing: (i) malaria, and/or (ii) malaria parasite transmission.

9. The composition for use according to claim 8, wherein the malaria parasite is Plasmodium falciparum.

10. The composition for use according to claim 9, wherein the composition is for use in reducing or preventing: (i) malaria, and/or (ii) malaria parasite transmission in a mosquito.

11. The composition for use according to claim 10, wherein the mosquito is a mosquito of the Anopheles genus.

12. The composition for use according to claim 11, wherein the mosquito is Anopheles Gambiae, Anopheles stephensi, Anopheles culicifacies, Anopheles gambiae, or Anopheles coluzzi.

13. The composition for use according to any one of claims 1 to 7, wherein the composition is for use in reducing or preventing: (i) dengue, and/or (ii) dengue virus transmission.

14. The composition for use according to any one of claims 13, wherein the composition is for use in reducing or preventing: (i) dengue, and/or (ii) dengue virus transmission in a mosquito.

15. The composition for use according to claim 13 or claim 14, wherein the mosquito is a mosquito of the Aedes genus.

16. The composition for use according to claim 15, wherein the mosquito is Aedes albopictus, or Aedes aegypti.

17. A method of reducing or preventing transmission of dengue or malaria comprising a step of bringing at least one mosquito vector of these diseases into contact with 1-Methyl-9H- pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

18. Use of l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing malaria or dengue.

19. l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the growth of a parasite into an ookinete, an oocyst, or a sporozoite.

20. A mosquito nectar feed comprising l-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and one or more of a sugar source.