Patent Application: US-201113113532-A

Abstract:
the present invention relates to compounds that modulate the shikimate pathway and / or a pathway branching from the shikimate pathway in members of the amoebida order . in particular these compounds may be useful in the treatment or prevention of diseases caused or contributed to by members of the amoebida order .

Description:
the present invention will now be described by way of example and with reference to the figures which show : fig1 . glyphosate inhibition of a . castellanii over a 96 hr period as determined by percent alamarblue reduction relative to untreated control cultures . fig2 . the cdna sequence of a . castellanii dhqs and partial epsp synthase . fig3 . the predicted amino acid sequence of a . castellanii dhqs and partial epsp synthase . fig4 . partial cdna sequence of a . castellanii chorismate synthase . fig5 . partial cdna for dahp synthase sequence from a . castellanii . fig6 . partial cdna for tryptophan synthase beta subunit from a . castellanii . fig7 . partial gdna for anthranilate synthase from a . castellanii . fig8 . partial gdna for dahp from a . polyphaga fig9 . the cdna sequence of a . polyphaga dhqs and partial epsp synthase . fig1 . partial gdna sequence of dhqs , epsp synthase and shikimate kinase from a . castellanii . acanthamoeba polyphaga ( strain no . 1501 / 18 ) were obtained from ccap ( cumbria , uk ). acanthamoeba castellanii ( neff strain ) was kindly obtained from keith vickerman ( glasgow , uk ). a . castellanii and a . polyphaga trophozoites were grown in ( a ) rich media containing 20 % mycological peptone ( fluka , uk ) and 0 . 9 % maltose ( sigma , uk ) and supplemented with 1 % penicillin , streptomycin and amphotericin b ( all sigma , uk ) or ( b ) modified 11 containing 20 % rich media . acanthamoeba spp . were incubated at room temperature in 75 cm 2 tissue culture flasks until 90 - 95 % confluence , then harvested or sub - cultured using accutase ( sigma , uk ). 80 % modified m11 ( l - arginine 0 . 825 g / l , l - methionine 0 . 3 g / l , l - leucine 0 . 9 g / l , l - isoleucine 0 . 6 g / l , l - valine 0 . 7 g / l , glycine 1 . 5 g / l , l - lysinehcl 1 . 25 g / l , l - threonine 0 . 5 g / l , glucose 18 g / l , sodium citrate 1 . 0 g / l biotin 0 . 25 mg / l , b 12 0 . 00125 mg / l , thiaminehcl 1 . 25 mg / l , znso 4 . 7h 2 o 1 mg / l , mncl 2 . 4h 2 o 2 . 3 mg / l , ( nh 4 ) 6 mo 7 o 24 . 4h 2 o 0 . 4 mg / l , cocl 2 0 . 017 mg / l , cuso 4 . 5h 2 o 0 . 0033 mg / l , h 3 bo 3 0 . 1 mg / l , edta 0 . 01 mg / l , mgso 4 . 7h 2 o 0 . 985 g / l , cacl 2 . 2h 2 o 0 . 0588 g / l , ( nh 4 ) 2so 4 feso 4 . 6h 2 o 0 . 0196 g / l , nahpo 4 . 2h 2 o 0 . 445 g / l , kh 2 po 4 0 . 34 g / l ), 20 % peptone media ( mycological peptone , 200 g / l , maltose 9 g / l ) the susceptibility of the a . castellanii to glyphosate as assessed by the alamarblue assay . the susceptibility of a . castellanii to glyphosate was determined by a modification of the method described by mcbride et al ., ( 2005 ). a . castellanii cells were grown in 75 cm 2 flasks until 90 - 95 % confluence and harvested using accutase ( sigma , uk ). a . castellanii and a . polyphaga cells were seeded in triplicate at 1 × 10 4 cells per well in 50 μl of medium in 96 well tissue culture plates ( greiner , uk ) and allowed three hours to adhere . 50 μl and 200 μl of medium containing glyphosate ( sigma poole , uk ), in the range 9 mm to 0 . 27 μm , was freshly prepared and added to the wells containing cells . test plates were incubated for 96 hours in normal culture conditions . six hours prior to the end of incubation 10 μl of alamarblue reagent was added to the test wells of the 96 well plate . test plates were incubated for 6 hrs at room temperature in the dark . alamarblue reduction was assessed as previously described . the percent inhibition of alamarblue reduction was calculated using the formula : ( εox ) λaλ 1 −( εox ) λ 1 aλ . of treated acanthamoeba /( εox ) λa ° λ 1 −( εox ) λa ° λ of untreated acanthamoeba × 100 [ where : ° λ 1 = absorbance of untreated control with alamarblue at 570 nm ); ° λ 2 = absorbance of untreated control with alamarblue at 600 nm )]. this number was subtracted from 100 to give percent alamarblue reduction relative to untreated control cultures . degenerate pcr for the shikimate pathway genes in a . castellanii degenerate primers were designed to amplify a portion of dhqs from acanthamoeba cdna ( acdhqsdegf , 5 ′- ggy ggy ggy gti aty ggy g - 3 ′ and acdhqsdegrl , 5 ′- tcv gcy tcc tti acc atr cc - 3 ′) 5 ′ race for the shikimate pathway genes from a . castellanii 5 ′ race was performed using a 5 ′, 3 ′ race kit ( roche molecular biochemicals , uk ). 5 ′ race was performed using the 5 ′/ 3 ′ race kit according to the manufacturer &# 39 ; s instructions ( roche molecular biochemicals ). first strand cdna was synthesized from 2 μg total rna using 25 pmol of a gene - specific primer , 10 u of amv reverse transcriptase and 10 mm of each dntp and in 1 × cdna synthesis buffer ( 50 mm tris - hcl , 8 mm mgcl 2 , 30 mmkcl , 1 mm dithiothreitol , ph8 . 0 ) in a final volume of 20 μl . the reaction was incubated for 60 minutes at 55 ° c . and for a further 10 minutes at 65 ° c . the cdna was subsequently purified by the high pure pcr product purification kit ( roche molecular biochemicals ) according to the manufacturer &# 39 ; s instructions and eluted in 50 μl 10 mm tris - hcl , ph8 . 3 . a da - tail was attached to the 5 ′ end of the first strand cdna by incubating 19 μl of the purified cdna sample with 2 . 5 μl 10 × reaction buffer ( 100 mm tris - hcl , 15 mm mgcl 2 , 500 mm kcl , ph8 . 3 ) and 2 mm datp for 3 minutes at 94 ° c . after being chilled on ice 10 u of terminale transferase was added and the reaction was incubated for 20 minutes at 37 ° c . and terminated at 70 ° c . for 10 minutes . nested pcr was performed to amplify the unknown region obtained from 5 μl of da - tailed cdna in a 25 μl reaction containing 0 . 5 μl oligodt - anchor primer ( 5 ′- gac cac gcg tat cga tgt cga ctt ttt ttt ttt ttt ttv - 3 ′ v = a , c or g ), 25 pmol of a second gene - specific primer designed upstream from the first , 10 mm of each dntp , 1 u of expand high fidelty taq polymerase ( roche molecular biochemicals ) and ix reaction buffer . the following temperature cycling conditions were used . denaturation at 94 ° c . for 2 minutes , followed by 10 cycles of denaturation at 94 ° c . for 15 seconds , annealing at 55 ° c . unless stated otherwise and elongation at 72 ° c . for 40 seconds . this was repeated for a further 25 cycles , but 20 seconds for each cycle were added onto the elongation step . a final elongation step was at 72 ° c . for 7 minutes . a second round of nested pcr was usually necessary to obtain the desired result , under the same conditions from 1 μl of a 1 : 20 dilution of purified pcr product from the first nested pcr . this employed a third gene - specific primer designed further upstream from the second together with the oligodt - anchor primer . the final product was run on a 1 . 5 % agarose gel , purified and cloned into pdrive for sequencing previously as described . custom race primers were designed from the sequence of dhqs obtained by degenerate pcr ( 5 ′ race1 , 5 ′- gga tgc cgt itt cgt tgg cgt c - 3 ′; 5 ′ race2 , 5 ′- ctg tcg ctc ctc aaa act c - 3 ′; 5 ′ race3 ; 5 ′- tag act gcc ttg ggg tgg tgg - 3 ′) genome walking for the shikimate pathway genes from a . castellanii . genome walking was used to obtain portions of the dhqs genomic sequence and to obtain further portions of genomic dna . long - distance genome walking pcr , adaptation of asymmetric pcr was used in order to isolate the unknown 5 ′ genomic sequence of gra3b adjacent to the known sequence . the method used was as described by min and power ( 1997 ). initially the known sequence of dhq synthase was extended by asymmetric pcr from 50 ng of gdna using 50 pmols of a gene - specific reverse primer gw1 in a 50 μl reaction containing , 2 . 5 u of expand high fidelity taq polymerase , 5 μl of 10 × expand high fidelity taq polymerase with mgcl 2 ( roche molecular biochemicals , germany ) and 0 . 2 mm of each dntp ( promega , uk ). conditions for pcr were 35 cycles of denaturation at 96 ° c . for 10 seconds , annealing at 64 ° c . for 10 seconds , and extension at 68 ° c . for 4 minutes . the single - stranded gdna ( ssgdna ) product was purified using the pcr purification kit ( qiagen , usa ) and eluted in 30 μl of 10 mm tris - hcl , ph8 . 3 and adjusted to a volume of 10 μl by centrifugal evaporation . the ssgdna was incubated at 95 ° c . for 5 minutes and immediately chilled on ice for 2 - 3 minutes . a dc - tail was added to the unknown 5 ′ end with the dna tailing kit ( roche molecular biochemicals ). 4 μl of 5 × tailing buffer ( 1m potassium cacodylate , 125 mmtris - hcl , 1 . 25 mg / ml bsa , ph6 . 6 ), 3 μl of 5 mmcocl 2 , 1 μl of 100 pmol dctp and 50 u of terminal deoxyribonucleotidyl transferase were added to 4 μl of the purified ssgdna and incubated at 37 ° c . for 20 minutes followed by incubation at 70 ° c . for 10 minutes . 5 μl of the tailing reaction was used in a nested pcr containing the reagents used in the initial extension step . primers used were the gene - specific reverse primer gw2 , designed upstream from gw1 and a oligodg - anchor primer ( 5 ′- cga gga att cgg ggg ggg ggg g - 3 ′), engineered to contain an ecori site near its 5 ′ end . cycling conditions were 40 cycles of denaturation at 96 ° c . for 30 seconds , annealing at 65 ° c . for 15 seconds and extension at 68 ° c . for 5 minutes . a final extension was at 65 ° c . for 5 minutes . the product was run on a 1 % agarose gel , purified and cloned into pdrive for subsequent sequence analysis . following sequencing of the product a second round of genome walking was performed by repeating this procedure , but substituting gw1 and gw2 primers with gw3 and gw4 primers . a third round of genome walking was performed with primers gw5 and gw6 based on the sequence obtained with primers gw3 and gw4 . the acanthamoeba genome project http :// www . tigr . org / was interrogated , using the tblastn algorithm and known protein sequences , for dna sequences with the potential to encode shikimate pathway enzymes or enzymes that are involved in the synthesis of tryptophan , phenylalanine or tyrosine . where sequences with potential to code for these enzymes were identified , pcr primers were obtained and the sequences amplified , cloned and verified . from these searches , sequences for chorismate synthase and dahp synthase were identified and primers designed ( dahp synthase : dahpfor , 5 ′- gag ttc ttg gac acc atc agc - 3 ′; dahprev , 5 ′- ctt cct ccg ttc tcg cac ggc cgc - 3 ′ and chorismate synthase : csfor , 5 ′- atg agc agc tit ggc aga gcg - 3 ′; csrev , 5 ′ cgt ctt ctg cgc ctg gct aat c - 3 ′). partial sequences for the 2 enzymes involved in tryptophan synthesis , tryptophan synthase beta subunit and anthranilate synthase were identified and primers designed . ( tryptophan synthase : tryptsfor , 5 ′ ggc acg ttc cat ccg ttc atc - 3 ′; tryptsrev ctg gat ggc gtg gta gac ggc and anthranilate synthase : anthransfor , 5 ′ atc cga gcg ctg gcg ggc aag - 3 ′; anthransrev , 5 ′- gga ggt cac aac aaa gtc cgg c - 3 ′). glyphosate was found to inhibit a . castellanii in a dose dependent manner with an 1050 between 0 . 00439 and 0 . 00219 mm ( fig1 ). degenerate pcr gave a portion of the dhqs cdna . this was extended in the 5 ′ direction by 5 ′ race , which gave a putative initiation codon . genome walking yields a portion of the dhqs gene that extends into a region that encodes a predicted epsp synthase providing evidence of a pentafunctional arom polypeptide in acanthamoeba . two successive rounds of genome walking gave specific products that when cloned and sequenced were found to have areas of predicted homology with previously identified dhq synthases and epsp synthases . that the genome walking products and the original degenerate pcr product were contiguous was confirmed by amplifying a portion of cdna from the initiation codon to within the epsp synthase using the primer : dhqsfor 5 ′- gac gcc aac gaa aac ggc atc c - 3 ′ and epsprev , 5 ′- tgg aga tcg act tgg agc ccg gg - 3 ′ ( fig2 ). the predicted polypeptide sequence this cdna codes for has considerable homology with the dhqs of other diverse species ( fig3 ). this provides evidence that acanthamoeba is likely to have a pentafunctional arom polypeptide . a further round of genome walking gave a specific product that when sequenced extends from the epsp synthase into a region that shares predicted homology to previously identified shikimate kinases , the third enzyme present in the classical pentafunctional arom structure ( fig1 ). portions of chorismate synthase , dahp synthase , tryptophan synthase beta subunit and anthranilate synthase are amplified from a . castellanii cdna . pcr gave portions of cdna that encode partial sequences of chorismate synthase , dahp synthase , tryptophan synthase beta subunit and anthranilate synthase from a . castellanii ( fig4 , 5 , 6 and 7 ). portions of dahp synthase , dhqs and epsp synthase are amplified from a . polyphaga gdna . pcr gave portions of gdna that encode a partial sequence of dahp synthase , and the dhqs domain and a partial epsp synthase domain from a . polyphaga ( fig8 and 9 ). auran , j . d ., m . b . starr , and f . a . jakobiec . 1987 . acanthamoeba keratitis . a review of the literature . cornea . 6 : 2 - 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