Patent Application: US-99898807-A

Abstract:
discovery and characterization of an apicomplexan fab i gene and encoded enzyme and discovery of the triclosan as a lead compound , provide means to rationally design novel inhibitory compositions useful for prevention and treatment of apicomplexan related diseases .

Description:
a plant - like fab i was identified in plasmodium falciparum . the nucleotide sequence and deduced amino acid sequence was prepared and correct sequences were confirmed . fab i is a single chain , discrete enzyme . all requisite residues for fab i enzyme activity were confirmed . the p . falciparum enayl acyl carrier protein reductase has a putative plastid targeting sequence and unique polar insertions . the fab i structure is modeled on e . coli and b . napus fab i structure alone and complexed to triclosan . key amino acids were identified for 2 ° structure . residues for binding triclosan were conserved providing explanation for inhibition by triclosan . triclosan inhibits p . falciparum , t . gondii ( nm ) in a pattern similar to the action of mefloquine . soluble protein can be overexpressed . information obtained from p . falciparum because fab i was purified include that the n terminal sequence is the same as b . napus fab i , enzyme activity is nadh dependent and inhibited by triclosan . fab i is involved in synthesis of 10 , 12 c fatty acids . in a p . berghei murine model , triclosan administered subcutaneously ( 3 or 38 mg / kg ) was nontoxic , cleared parasitemia and prevented death . synergy was demonstrated in vitro with cerulein , an inhibitor of fab f , b , h . t . gondii in vitro . growth inhibition was assessed over a 4 - day period as described previously by roberts et al ., 1998 ; zuther et al ., 1999 ; and mack et al ., 1984 , all incorporated by reference , using human foreskin fibroblasts ( hff ) infected with 105 tachyzoites of the rh strain of t . gondii . uptake of 3h uracil was determined . evaluation of slides of preparations containing hff , toxoplasma and inhibitors were made . p . falciparum . the in vitro assays ( oduala et al ., 1988 ; milhous et al ., 1985 ) were conducted using a modification of the semiautomated microdilution technique for assessing antifolate antagonists . instead of dialysed human plasma , 10 % albumaz i ( gibco brl ), a serum - free substitute , was used to supplement the rpmi 16 - 40 media . all test compounds were dissolved in dmso and diluted 400 - fold into complete media before serial dilution over 11 concentrations . incubation was at 37 ° c . in 5 % o2 , 5 % co2 and 90 % n2 for 48h , [ 3h ]- hypoxanthine incorporation was measured as described previously ( 13 , 14 ). w2 is susceptible to mefloquine , but resistant to pyrimethamine , sulpha - doxine , but similar to tm90c2a and tm90c2b , and tm91c235 is less susceptible to mefloquin . 1 the activity of triclosan , mefloquine , and chloro - quine were tested against a series of p . falciparum isolates and clones with differing susceptibilities to antimalarial drugs . d6 , a clone from the african sierra i / unc isolate , is chloroquine and pyrimethamine susceptible ; w2 is a clone of the indochina i isolate and is chloroquine and pyrimethamine resistant . tm90c2a , tm90c2b , and tm91c235 are isolates from thailand and all are chloroquine and mefloquine resistant . tm91c235 was isolated from a patient that failed mefloquine twice , whereas tm90 - c2a and tm90 - cb are admission and recrudescent isolates , respectively , of the first patient who failed treatment with atovaquone ( alone ) in thailand . subsequent susceptibility testing demonstrated that the recrudescent isolate ( 2b ) was approximately 2000 fold resistant to atovaquone , when compared with the admission isolate and other atovaquone - susceptible isolates from thailand . cloning of the fabi gene . the fabi gene from plasmodium falciparum is located on chromosome 4 and codes for a 432 amino acid protein . the fabi gene from gdna of the 3d7 strain of p . falciparum was amplified using pfu turbo polymerase ( stratagene ) and two primers ( 5 ′- ggtggtgaattcatgaataaaatatcacaacgg - 3 ′ and 5 ′ ggtggtgtcgacttattcattttcat - tgcgatatatatc - 3 ′). the resulting amplicon was digested with ecori and sail endonucleases and gel purified using the qiaquick gel extraction kit from qiagen . the digested ampicon was ligated with t4 dna ligase ( boehringer mannheim ) into the pmal - c2x vector ( new england biolabs ) which had previously been digested with the same endonucleases and treated with shrimp alkaline phosphatase ( usb ). a second construct , lacking fabi residues 1 - 84 , was prepared in the same way using the following two primers : 5 ′- ggtggtgaattctcaaacataaa - caaaattaaagaag - 3 ′ and 5 ′- ggtggtgtcgact - tattcattttcattgcgatatatatc - 3 ′. this truncated construct was called fabit . overexpression of fabit in bacterial culture . the pmal - c2x vector containing the fabit construct was transformed into bl21 - codonplus ( de3 ) cells ( stratagene ). bacterial cultures were grown in shaker flasks at 37 ° to an od600 of 0 . 6 and then induced with iptg ( sigma ) to a final concentration of 0 . 4 mm . induced cultures were transferred to a 20 ° shaker and incubated for an additional 12 hours . after this period , the cells were harvested by centrifugation at 5 , 000 × g for 15 minutes and the cell pellet was frozen at − 20 °. purification of recombinant fab it fusion protein . cell lysis buffer ( 20 mm na / k phosphate ph 7 . 5 , 1 mg / ml lysozyme ( sigma ), 2 . 5 dg / ml dnase i ( sigma ), 200 mm nacl ) was added to the frozen cell pellets ( 20 ml per liter of original culture ) and gently vortexed . resuspended cells were incubated on ice for 10 minutes followed by 30 seconds of sonication . cell lysate was clarified by centrifugation at 20 , 000 × g for 15 minutes at 4 ° and applied to a 10 ml amylose column ( new england biolabs ) equilibrated in 20 mm na / k phosphate ph 7 . 5 , 200 mm nacl . the column was washed with 5 column volumes of 20 mm na / k phosphate ph 7 . 5 , 500 mm nacl followed by elution with 20 mm na / k phosphate ph 7 . 5 , 200 mm nacl , 100 mm maltose . cleavage of fabit fusion protein and purification of fabit . purified fabit fusion protein was digested with factor xa ( new england biolabs ) at ratio of 1 mg factor xa per 500 mg of fusion protein . calcium chloride was added to the reaction mixture at a final concentration of 1 mm and the mixture was incubated at 4 ° for 24 hours . the reaction mixture was desalted with a hiprep 26 / 10 desalting column ( pharmacia ) equilibrated in 20 mm na / k phosphate ph 8 . 0 , iodm nad +. desalted protein was applied to a sp sepharose cation exchange column ( phaimacia ) equilibrated in 20 mm na / k phosphate ph 8 . 0 , 10dm nad + and washed for 10 column volumes with the same buffer . adsorbed proteins were eluted from the column with a linear gradient to 20 mm na / k phosphate ph 8 . 0 , 10dm nad +, 500 mm nacl in 20 column volumes . fractions containing pure fabit protein were pooled for further analysis . overexpression of recombinant fab i . the fabi gene was amplified from cdna of the 3d7 strain of p . falciparum and inserted into the pmal - 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