Patent Application: US-81298907-A

Abstract:
the invention provides a method of enhancing the effectiveness of pesticides , as well as pesticidal formulations . furthermore , it also provides the means for the de novo rational design of pesticides . the present invention also relates to a method of screening agents for potential use in insecticides , particularly against mosquitoes .

Description:
the present invention will now be further described by way of example and with reference to the figures which show : fig1 shows a schematic view of the p450 mono - oxygenase complex ; p450 catalyses the insertion of a single oxygen molecule into an organic substrate ( s ) to produce a mono - oxygenation product ( s — oh ) and water . two electrons are supplied by nadph and shuttled consecutively to the heme centre of p450 via the isoalloxazine rings of fad and fawn . the complex is tethered to the endoplasmic reticulum . the reaction scheme is shown at the bottom . fig2 shows immunolocalisation of cpr . cpr is labelled in green in all images . a , b , c — midgut , a inset and c counterstained red with nuclear pore antiserum , b counterstained red with integrin antiserum . cpr is abundantly localised in the perinuclear region d , merged brightfield fluorescence image of abdomen wall showing intense staining of oenocytes . e , oenocytes — nuclei counterstained blue with to - pro3 . cpr appears dispersed throughout the cell . cells contain large vesicle structures f female antennae — counterstained blue with dapi . cpr is localised to a subset of cells . g malpighian tubules — counterstained red with nuclear pore antiserum . cpr is localised specifically to the large principal ( type i ) cells of the tubules . fig3 shows silencing of cpr expression , a , immunoblot of total protein extracts from dissected body parts taken from mosquitoes injected with dscpr ( cpr ) and dsgfp ( gfp ). filters were probed with cpr antisera ( α - cpr ), then stripped and probed with tubulin antiserum ( α - tub ). the percentage of cpr remaining after knockdown was estimated by densitometric scanning of western filters using imagej software and corrected for loading using tubulin . the figures indicated are mean and sd from three independent experiments . image shows random selected whole mount abdomens stains taken from control ( con ) or dscpr injected mosquitoes . oenocyte staining is drastically reduced . b , effect of cpr rnai knockout on susceptibility to permethrin . each independent experiment shows mean percentage numbers of mosquitoes dead , 24 hrs after permethrin treatment . numbers refer to experiment number and * indicates the use of a different region of cpr to make dsrna . fig4 demonstrates the different binding behaviours of cpr extracted from the mosquito a . gambiae and the closely related dipteran species , drosophila melanogaster ( fruit fly ) with respect to 2 ′- 5 ′- adp . in this example , whole flies have been solubilized with a detergent / buffer solution , loaded onto 2 ′- 5 ′- adp sepharose mini - columns , washed and column bound proteins eluted with 50 mm 2 ′- amp . ( 2 ′- 5 ′- adp sepharose interacts strongly with nadp +- dependent dehydrogenases and other enzymes which have affinity for nadp + ( amersham - pharmacia biotech 1999 handbook on affinity chromatography : principles and methods , edition ab ). thus a mixture of different nadp + binding proteins will be eluted . to identify cpr , samples of whole fly extracts from the pre - 2 ′- 5 ′- adp adsorption , post - 2 ′- 5 ′- adp adsorption and post 2 ′- 5 ′- adp elution stages were transferred onto nitrocellulose by immunoblotting ( western blotting ) and cpr identified using rabbit antisera to a . gambiae cpr . in the an . gambiae lanes ( fig4 ), a prominant cpr band (˜ 75 kda ) is evident in the total protein and unbound ( flow - through ) lanes , but not in the eluted fraction . by contrast , in the d . melanogaster lanes , cpr bands are evident in total protein , the unbound fraction and elution fraction . thus , the mosquito cpr is clearly different to the fruit fly with respect to the binding of 2 ′- 5 ′- adp . the test described in fig4 can be used as a diagnostic tool to distinguish non - 2 ′- 5 ′- adp binding versus 2 ′- 5 ′- adp binding cprs . this is important in the context of the development of inhibitors against cprs as it allows one to examine different species , strains or individuals to determine if they have non - 2 ′- 5 ′- adp binding cpr . such enzymes present good pesticide targets since they differ to the 2 ′- 5 ′- adp binding human cpr counterpart 19 . fig5 shows inhibition of human and mosquito cpr by 2 ′ 5 ′ adp and diphenyl iodonium ( dpi ). micromolar ic 50 values for inhibition of cytochrome c reduction by human ( squares ) and mosquito ( circles ) are indicated in each graph on bottom left . human cpr ( ic 5o = 28 μm ) is ˜ 10 fold more sensitive to 2 ′ 5 ′- adp inhibition ( ic 50 = 262 μm ), while mosquito cpr ( ic 50 = 28 μm ) is ˜ 10 fold more sensitive to dpi than human ( ic 50 = 361 μm ). measurement of cytochrome c reduction was carried out at 25 ° c . with 50 μm cytochrome c and 0 . 75 pmol purified a . gambiae cpr or human cpr as described 19 , using different concentrations of 2 ′ 5 ′- adp or dpi . a . gambiae and human cpr reactions were initiated by the addition of 30 μm or 15 μm nadph respectively , corresponding to their apparent k m values . errors are deviation from the fit of the curve ( grafit 5 . 06 ). fig6 shows a sds - polyacrylamide gel of purified a . gambiae cpr . lane 1 shows the kilodalton molecular weight standards , with sizes indicated on left . lane 2 shows a partially purified histidine tagged agcpr , which has been eluted off a nickel affinity column with 300 mm imidazole . lane 3 shows purified agcpr that has been cleaved with thrombin to remove the n - terminal histidine tag . fig7 shows inhibition of human and mosquito p450 activities by 2 ′- 5 ′- adp . micromolar ic 50 values calculated for the inhibition of mosquito cyp6z2 br dealkylation ( squares ) and human cyp3a4 bq oxidation ( circles ) are shown . human p450 activity is approximately 20 fold more sensitive ( ic 50 = 10 ± 1 microm ) than a . gambiae p450 ( ic 50 = 234 ± 28 microm ). error bars show standard deviation for two independent experiments . measurement of p450 activity was carried out using e . coli membranes coexpressing a . gambiae cyp6z2 and agcpr or human cyp3a4 and human cpr . 7 - benzyloxyresorufin ( br ) and 7 - benzyloxyquinoline ( bq ) assays were performed in 200 μl reactions consisting of 50 mm potassium phosphate buffer , ph 7 . 4 ; 5 pmol cyp6z2 or 20 pmol cyp3a4 ; 5 μm br or 100 μm bq , 125 microm nadph and 0 - 1 mm 2 ′- 5 ′- adp . rates were recorded for 5 min at 37 ° c . using a fluorescence plate reader ( labsystems fluoroskan ascent - fl ) set to measure λ ex 530 λ em585 ( br assay ) or λ ex 405 λ em530 ( bq assay ). percentage activities were calculated and ic 50 curves were plotted using grafit version 5 . dscpr and dscpr * constructs were created by insertion of a 700 and 500 base pair xhoi restriction enzyme fragments of the cpr cdna clone , respectively , into pll10 ; a plasmid which carries two t7 polymerase primer sites in opposite orientation surrounding a multiple cloning site 5 . dsrna was generated as described by osta 20 , following the standard protocol of the ambion megascript kit using a template consisting of 500 ng of kpni digested plldscpr and an equal quantity of an ecori digestion of the same plasmid . rnas were quantified spectrophotometrically , diluted to 3 μg / ml and analysed by ethithium bromide gel electrophoresis . rnas synthesised from premixed , reverse complementary t7 polymerase templates spontaneously form dsrna in vitro , which migrates slightly slower than the equivalent dsdna and requires no additional annealing steps prior to use . batches of 100 one to two day old female mosquitoes were divided into two groups and injected with 69 μl of either dscpr or dsgfp rnas using a hand held drummond nanoinjector ii . four days later , sub - groups of 19 - 20 mosquitoes were exposed to permethrin for 20 minutes using the standard who exposure kits ( http :// www . who . int / whopes / resistance / en / who_cds_cpe_pvc — 2001 . 2 . pdf ) and impregnated papers ( 0 . 75 % permethrin ). 24 hours later , dead mosquitoes were counted . three replicate biological experiments were performed . midgut and abdomen immuno - staining was performed essentially as described 21 . cpr was localized by sequential incubation with affinity purified cpr antisera ( 1 / 200 ) and appropriate fluorescent tag . co - staining was performed with to - pro 3 ( 1 / 5000 - molecular probes , dapi , or nuclear pore mab414 ( babco ) and mouse anti - integrin antibodies with appropriate secondary antibodies as described in the figure legends , localization of cpr in heads and their appendages was performed essentially as described 22 . cloning of a . gambiae ( ag ) cpr cdna is described in nikou et al 2003 . two nucleotide changes were found in the coding sequence relative to the published sequence 23 ; t689c and c1375t , producing val230ala and his459tyr changes respectively . the membrane anchor sequence was deleted by removal of amino acids 2 - 63 by pcr , using pfu polymerase ( stratagene ) and the following oligonucleotides ; forward primer : cgc g gat cc g atg acg atg acg atg gtg gag acc and reverse primer : ttc gga tcc tta gct cca cac gtc cgc cga . ( the bamhi sites are underlined and the start and stop codons are indicated in bold ). the pcr product was digested with bamh i and subcloned into the expression vector pet - 15b ( novagen ). this expression vector has an in - frame 6 × histidine tag and thrombin cleavage sequence , which enables metal affinity purification and tag removal . this facilitates purification of the mosquito cpr which does not readily bind to 2 ′- 5 ′- adp sepharose , the usual affinity matrix for this enzyme class . constructs were confirmed by dna sequencing . agcpr was expressed in e . coli strain bl21 ( de3 ) plyss and nickel - affinity purified as described previously for human cpr domains 24 . the human cpr was purified as described by dohr 19 . protein purity was & gt ; 95 % as assessed by sds - page gel electrophoresis . rabbit polyclonal antibodies to agcpr were made by moravian - biotechnology ltd , brno , czech republic . both antisera were affinity - purified against ˜ 100 μg purified recombinant cpr bound to nitrocellulose using immunopure gentle ag / ab binding and elution buffers ( pierce , rockford , ill ., u . s . a .) according to manufacturers instructions . cytochrome c assays and enzyme kinetic measurements were carried out 25 ° c . as described 19 , using 0 . 75 pmol purified agcpr or hcpr . apparent kinetic parameters ( non - linear fitting : michaelis - menten equation ) and ic 50 values were calculated using grafit version 5 . 06 . 0 . 1 g of frozen adult a . gambiae or d . melanogaster flies were added to a pre - chilled mortar and ground to a powder in the presence of liquid nitrogen . the powder was transferred to a 50 ml falcon tube and 2 . 5 mls of cold solubilisation buffer ( 20 mm tris - cl ph 8 ; 10 % glycerol ; 100 mm kcl and 20 mm chaps ) added . the suspensions were mixed on a rotary wheel at 4 ° c . for 2 hrs to solubilise the membrane bound proteins , then centrifuged ( 5 min ; 12 , 000 rpm ; 4 ° c .) to clarify . 200 μl of 2 ′ 5 ′ adp sepharose was loaded into a mini - column ( a 1 ml gillson pipette tip , plugged with glass - wool ) and equilibriated with 5 ml of solubilisation buffer . the clarified whole fly protein extracts were applied to the column and the flow - through collected . the column was washed with 5 ml of solubilisation buffer to remove non - binding proteins . to elute the bound proteins , 3 × 200 μl of 50 mm 2 ′ amp in 0 . 5 × solubilisation buffer was added to the mini - columns , and the 3 × 200 μl elution fractions collected in eppendorf tubes . to detect cpr , 25 μl each of solubilised pre - column extract ( total fly protein ); flow through ( unbound protein ) and eluted proteins were run on a nupage 4 - 12 % bis - tris gel ( invitrogen ; uk ) and transferred onto protran ® nitrocellulose membrane ( schleicher & amp ; schuell ; germany ) according to the manufacturer &# 39 ; s instructions . the membrane was blocked overnight at 4 ° c . in tbst & amp ; 5 % milk powder , then incubated for 1 hr with a 1 : 10000 dilution of anti - ag cpr primary antibody . after washing ( 3 × 10 min in tbst ) the membrane was incubated for 1 hr with 1 : 3000 dilution of hrp anti - rabbit igg ( sapu ; uk ). the membranes were washed as before and the proteins were visualised by chemiluminescence using the ecl ( amersham biosciences ) kit according to the manufacturers instructions . functional expression of cyp6z2 - cyp6z2 cdnas was isolated as described 23 . for e . coli expression , cyp6z2 cdna was fused to a bacterial ompa leader sequence as previously described ( pritchard , m . p ., et al . ( 1998 ) pharmacogenetics 8 , 3342 ; pritchard et al ( 1 . 997 ) arch biochem biophys 345 , 342 - 354 ) by pcr with ompa forward primer : g gaa ttc cat atg aaa aag aca gct at ( nde i site underlined , initiation codon in bold ); ompa / cyp6z2 fusion primer ( reverse orientation ): gag cac gag aaa gat cac ggc cgc aac aag agc aag agt ata aac agc cat cgg agc ggc ctg ctg cgc tac ggt agc gaa ; cyp6z2 reverse primer : cgg gaa ttc tca ctt tct atg gtc tat cct cat ( ecor i site underlined , stop codon in bold ). the pcr fragment was digested with nde i / ecor i and ligated into pb13 ( modified pcw vector ). for functional expression cyp6z2 was co - expressed with full - length a . gambiae cpr cloned into a compatible pacyc vector . a . gambiae cpr cdna was fused by pcr to a pelb leader sequence ( forward primer : c g gg atc c at atg aaa tac ctg ctg ccg acc gct gct gct gct ctg ctg ctc ctc gct gcc cag ccg gcg atg gcc atg gac gcc cag aca gaa acg gaa gtg ( bamh i site underlined and start codon in bold ) and reverse primer : ccg gaa ttc tta gct cca cac gtc cgc cga gta tcg ttt ( ecor i site underlined and stop codon in bold ). the pcr product was digested with bamh i / ecor i and cloned into pb13 . a cassette containing the ptac - ptac promotor from pb13 and pelb agcpr was excised using an ecor v / bgl ii digest , and cloned into ecor v / bamh i digested pacyc 184 ( new england biolabs ), disrupting the tetracycline resistance gene ( pacyc - agcpr ). competent e . coli jm109 cells were co - transformed with ompa 6z2 & amp ; pacyc - agcpr , and expression , membrane isolation and determination of p450 and cpr content was carried out as previously described ( pritchard , m . p ., et al . ( 1998 ) pharmacogenetics 8 , 33 - 42 ). for p450 assays , e . coli membranes co - expressing human cyp3a4 and cpr were produced as described . 7 - benzyloxyresorufin ( br ) and 7 - benzyloxyquinoline ( bq ) assays were performed in 96 well white plates in a total volume of 200 μl consisting of 50 mm potassium phosphate buffer , ph 7 . 4 ; 5 pmol cyp6z2 or 20 pmol cyp3a4 ; 5 μm br or 100 μm bq , with 0 - 1 mm 2 ′- 5 ′- adp . plates were preincubated for 5 min at 37 ° c . before addition of 5 μl of 5 mm nadph . rates were recorded for 5 min at 37 ° c . using a fluorescence plate reader ( labsystems fluoroskan ascent - fl ) set to measure λ ex 530 λ em585 ( br assay ) or λ ex 405 λ em530 ( bq assay ). percentage activities were calculated and ic 50 curves were plotted using grafit version 5 . the tissue distribution of the p450 complex has been poorly described in mosquitoes . using an affinity purified antibody against cpr , a peptide of the expected size was detected in a . gambiae extracts derived from dissected head ( with appendages ), gut ( with malphigian tubules ), and the abdomen wall . cpr was only weakly detected in the thorax , which is largely comprised of muscle , fat body and salivary glands ( not shown ). the same antibody revealed that the cellular distribution of cpr can be divided into three main tissues ; antenna , midgut epithelia and oenocytes . in the head , staining was prominent in a subset of cells within the antennae ( fig2 f ), which is consistent with reported high levels of cpr in this organ in drosophila melanogaster 25 . it has been proposed to be involved in the metabolism of chemical odorants , clearing the olfactory organ from accumulating chemicals . cpr was also observed in midgut epithelial cells ( fig2 a - c ), and was highly expressed in the foregut epithelium . p450 dependant metabolism within these tissues is probably important to neutralise toxins acquired during feeding on plant material 2 and possibly in the blood meal 26 . the principal ( type 1 ) cells of the malphigian tubules were also specifically labelled ( fig2 g ). these cells are involved in maintaining ion homeostasis and are known sites of p450 dependant ecdysone metabolism in drosophila and other insects 27 , 28 . very intensely stained large cells (& gt ; 25 μm ) on the abdomen wall of adults were observed ( fig2 d , e ). these cells were found in distinct subcuticular clumps that form rows on each abdominal segment ( fig2 d ), predominantly on the ventral half of the abdomen wall . they clearly overlap the brown cuticle pigmentation in the overlay of brightfield and fluorescence images ( fig2 d ) and may thus be involved in its formation . based on anatomical description and comparison with other insects , particularly drosophila 29 , these cells are identified as oenocytes . oenocytes are a major focus of drosophila developmental research as their identity is controlled by a single homeotic gene ( abdominal a ) 30 , however , their functional role is still unresolved . based on gene products expressed , these cells have been ascribed numerous endocrine and secretory functions including the regulation of ecdysteroids and production of pheromones 31 , glycogen storage 32 , and hydrocarbon / lipid synthesis 33 . it is probable that cpr is associated with several of these metabolic activities , many of which have been linked to p450 activities . in drosophila , oenocytes are thought to be the major site of heme biosynthesis 34 , suggesting a role in cytochrome p450 production . in adult a . gambiae the oenocytes are restricted to the ventral body wall , which contrasts with drosophila in which these cells are evenly distributed on both abdominal surfaces . this may reflect different physiological requirements in the two species . overall , the tissue distribution in mosquito suggests key physiological roles for cpr in metabolic processes and pheromone production / metabolism , consistent with known or expected functional involvement of insect p450s 2 . in addition , the abundant presence of an archetypal detoxification enzyme in the oenocytes suggests that this cell type has some functional equivalence to hepatocytes . to facilitate cpr silencing , dsrnas corresponding either to the cpr gene ( dscpr ) or to the control green fluorescent protein , gene ( dsgfp ) were injected into 1 - 2 day old adults . these were allowed to recover for 4 days . no significant difference in survival was noted between experimental and control samples following injection and recovery ( not shown ). western analysis of extracts taken from abdomen , gut and heads dissected from dsgfp and dscpr treated mosquitoes indicted that cpr depletion was most efficient in the abdomen (− 90 %), with a smaller reduction evident in midgut extracts (− 50 %) and negligible differences in the head extracts ( fig3 a ). in whole mount abdomens , we also noted a strong reduction in cpr staining in oenocytes ( fig3 b ). we have thus established that cpr expression can be effectively knocked down in the abdomen , particularly in the oenocytes , allowing us to examine their role in insecticide metabolism in vivo . the present inventors investigated the response to permethrin , a pyrethroid insecticide currently used in malaria control programmes 1 . dsrna - treated mosquitoes were challenged with a fixed permethrin dose at different exposure times and their survival monitored 24 hours later , according to the who guidelines e . g . http :// www . who . int / whopes / resistance / en / who_cds_cpe_pvc — 2001 . 2 . pdf . initial experiments defined an appropriate exposure in the laboratory strain we used . at a level at which the dsgfp controls showed approximately 40 % lethality , the dscpr treated mosquitoes showed a 2 fold increase in susceptibility to permethrin (˜ 80 %). this finding is reminiscent to the severely compromised ability of mice carrying a conditional deletion of hepatic cpr 4 to metabolise drugs such as pentobarbital or the analgesic acetaminophen 4 . our results indicate a key physiological role for cpr in protection against permethrin in the mosquito , presumably through p450 metabolism . they also suggest cpr as a viable target for the development of inhibitors to enhance and prolong the effectiveness of permethrin and potentially other insecticides metabolised by the p450 enzyme complex . mosquito cpr exhibits distinctive biochemical differences in relation to fruit fly and human cpr to further characterise a . gambiae cpr the present inventors compared the biochemical properties of the mosquito , fruit - fly and human enzymes . since the a . gambiae cpr contains an nadph binding domain 23 , it night be expected to be purified easily from whole fly extracts through affinity purification using 2 ′- 5 ′- adp sepharose ( 2 ′- 5 ′- adp sepharose interacts strongly with nadp +- dependent dehydrogenases and other enzymes which have affinity for nadp + ( amersham - pharmacia biotech 1999 handbook on affinity chromatography : principles and methods , edition ab ). however , comparison of the purification of crude 2 ′- 5 ′- adp binding proteins from crude extracts of a . gambiae and the closely related dipteran species d . melanogaster show that the mosquito cpr does not bind 2 ′- 5 ′- adp ( fig4 ) and is thus clearly different with respect to the molecular recognition of adenosine . soluble forms ( i . e . lacking the n - terminal membrane anchor ) 19 of the catalytic regions of human and a . gambiae cpr were expressed in e . coli 19 . the soluble histidine tagged form of a . gambiae cpr purified over nickel agarose ( fig6 ) also failed to bind to 2 ′- 5 ′- adp resin , indicating that the lack of binding was not artefactual . the relative enzymatic activities of human and a . gambiae cpr were measured through the reduction of cytochrome c ( a surrogate electron acceptor used for measuring diflavin reductase activity 3 ). these revealed functional similarities with human cpr as well as significant and unexpected biochemical differences . the binding affinities for cytochrome c were similar with k m cytc values for human and mosquito enzymes of 19 μm and 23 μm respectively . rates of cytochrome c reduction were also alike , characterized by k cat values of 3023 and 3099 nmoles cytochrome c reduced / nmole cpr / min − 1 . a two - fold decrease in affinity for nadph relative to the human protein was noted ( k m nadph 30 μm and 14 μm respectively ). overall , these experiments show comparable steady state rates of electron transfer from nadph to cytochrome c . an unexpected difference , however , was observed in the binding affinity for adenosine molecules ( fig4 ). nadph is comprised of nicotinamide and adenosine - ribose moieties ( i . e 2 ′, 5 ′- adp ), which are proposed to bind in a bipartite mode to separate binding pockets of cpr 19 . a . gambiae cpr failed to bind to 2 ′, 5 ′- adp sepharose , a standard affinity matrix used for purifying cprs and related nadph binding enzymes . we therefore compared the inhibitory effects of the 2 ′, 5 ′- adp fragment and found a ten - fold higher ic 50 for a . gambiae cpr ( ic 50 262 μm ) in comparison to human cpr ( ic 50 28 μm ) ( fig5 ). the structural reasons are still unclear , but may be associated with interactions involving 2 ′- phosphate , which is the major contributor to the high affinity binding of nadph to cpr 35 . importantly , we also found that a . gambiae cpr was an order of magnitude more sensitive than human cpr to diphenyliodonium chloride ( dpi ) ( fig5 ), a widely used inhibitor of flavin - containing enzymes 36 . these results highlight significant differences in binding interactions with small molecules that may be exploited to develop a mosquito cpr specific inhibitor . mosquito cpr exhibits distinctive biochemical differences in relation to human cpr in p450 coupling to confirm whether such differences in 2 ′- 5 ′- adp binding could be detected with physiological redox partners , we performed inhibition assays by co - expressing full length mosquito and human cprs in e . coli membranes with one of their respective p450 partners , cyp6z2 23 and cyp3a4 ( pritchard , m . p . et al ( 1997 ) arch biochem biophys 345 , 342 - 354 ). measurements were made of the oxidation of the fluorogenic substrates 7 - benzyloxyresorufin ( br ) and 7 - benzyloxyquinoline ( bq ), which are metabolised by cyp6z2 and cyp3a4 respectively ( fig7 ). consistent with inhibition of cytochrome c reduction , the ic 50 of 2 ′, 5 ′- adp for the mosquito cyp6z2 catalysed br o - dealkylation was 234 μm , which was approximately twenty - fold greater than the ic 50 ( 10 μm ) for human cyp3a4 bq oxidation . furthermore , reciprocal pairings of mosquito and human cprs and p450s were also examined ( i . e . cyp6z2 was co - expressed with human cpr and cyp3a4 with a . gambiae cpr ), but we were unable to detect significant p450 activity with either cross pairing . in view of the high sequence homology of human and mosquito cprs ( 40 % identical and 60 % similar residues ) 23 and the fact that cpr homologues are generally interchangeable ( feyereisen , r . ( 1999 ) annu rev entomol 44 , 507 - 533 ), the failure of a . gambiae cpr to couple with human cyp3a4 suggests possible differences in surface charge or hydrophobicity affecting cpr - p450 contact , or interactions with the membrane . in conclusion , this work identifies oenocytes as being one of the primary sites for cpr expression and by association p450 metabolism . this is strongly supported by the increase in susceptibility to permethrin following the depletion of cpr gene expression in oenocytes . our results firmly establish the critical role of p450 monoxygenase complex in insecticide metabolism . they also identify cpr as a viable target for the development of functional inhibitors for use as insecticides . there are two compelling reasons to investigate this further . firstly , cpr is encoded by a single gene that has a house - keeping role in furnishing electrons to all microsomal p450s . its inhibition is therefore likely to be lethal to early development stages . secondly , the low affinity of a . gambiae cpr for 2 ′, 5 ′- adp distinguishes it from all other cprs that have been purified 37 , including those from other insects . although this initial findings merits further investigation it encourages die efforts to design or screen for selective inhibitory molecules that will specifically target a . gambiae . 1 . ranson , h . et al . evolution of supergene families associated with insecticide resistance . science 298 , 179 - 81 ( 2002 ). 2 . feyereisen , r . insect p450 enzymes . annu rev entomol 44 , 507 - 33 ( 1999 ). 3 . paine , m . j . i ., scrutton , n . s ., munro , a . w ., roberts , g . c . k . & amp ; wolf , c . r . in cytochromes p 450 : structure , mechanism and biochemistry ( ed . ortiz de montellano , p . r .) 115 - 148 ( kluwer academic / plenum publishers , new york , 2004 ). 4 . henderson , c . j . et al . inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase . j . biol . chem . 278 , 13480 - 13486 ( 2003 ). 5 . blandin , s . et al . reverse genetics in the mosquito anopheles gambiae : targeted disruption of the defensin gene . embo rep 3 , 852 - 6 ( 2002 ). 6 . montgomery , m . k . rna interference : historical overview and significance . methods mol biol 265 , 3 - 21 ( 2004 ). 7 . mello , c . c . & amp ; conte , d ., jr . revealing the world of rna interference . nature 431 , 338 - 42 ( 2004 ). 8 . sanchez - vargas , i . et al . rna interference , arthropod - bone viruses , and mosquitoes . virus res 102 , 65 - 74 ( 2004 ). 9 . andreadis , t . g ., becnel , j . j . & amp ;; white , s . e . infectivity and pathogenicity of a novel baculovirus , cuninpv from culex nigripalpus ( diptera : culicidae ) for thirteen species and four genera of mosquitoes . j med entomol 40 , 512 - 7 ( 2003 ). 10 . becnel , j . et al . epizootiology and transmission of a newly discovered baculovirus from the mosquitoes culex nigripalpus and c . quinquefasciatus . j gen virol 82 , 275 - 82 ( 2001 ). 11 . shapiro , a . m ., becnel , j . j . & amp ; white , s . e . a nucleopolyhedrovirus from uranotaenia sapphirina ( diptera : culicidae ). j invertebr pathol 86 , 96 - 103 ( 2004 ). 12 . travanty , e . a . et al . using rna interference to develop dengue virus resistance in genetically modified aedes aegypti . insect biochem mol biol 34 , 607 - 13 ( 2004 ). 13 . kohl , a ., billecocq , a ., prehaud , c ., yadani , f . z . & amp ; bouloy , m . transient gene expression in mammalian and mosquito cells using a recombinant semliki forest virus expressing t7 rna polymerase . appl microbiol biotechnol 53 , 51 - 6 ( 1999 ). 14 . sambrook , j . & amp ; russell , d . w . molecular cloning : a laboratory manual ( ed . j ., s .) 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