USOO6699654B1 (12) United States Patent (10) Patent No.: US 6,699,654 B1 McLeod et al. (45) Date of Patent: Mar. 2, 2004 (54) ANTIMICROBIAL AGENTS DIAGNOSTIC (51) Int. Cl." ........................... C12O 1/68; G01N 33/53 REAGENTS, AND WACCINES BASED ON (52) U.S. Cl. ................................. 435/4; 435/6; 435/7.1 UNIQUE APICOMPLEXAN PARASITE (58) Field of Search .................................. 435/4, 6, 7.1 COMPONENTS Primary Examiner Sean McGarry (76) Inventors: Rima L. W. McLeod, 5729 Kimbark, (74) Attorney, Agent, or Firm-Alice O. Martin; Barnes & Chicago, IL (US) 60637; Craig W. Thornburg Roberts, 17 Kirklee Circus, Kirklee, Glasgow, G12 OTW Scotland (GB); (57) ABSTRACT Fiona Roberts, 17 Kirklee Circus, This invention relates uses of components of plant-like Kirklee, Glasgow, G12 OTW Scotland metabolic pathways not including psbA or PPi phosphof (GB); Jennifer J. Johnson, 13 Penny ructokinase and not generally operative in animals or Ct., Bolingbrook, IL (US) 60440; encoded by the plastid DNA, to develop compositions that Laurens Mets, 814 Sheridan Rd., interfere with Apicomplexan growth and Survival. Compo Wilmette, IL (US) 60091 nents of the pathways include enzymes, transit peptides and nucleotide Sequences encoding the enzymes and peptides, or (*) Notice: Subject to any disclaimer, the term of this promoters of these nucleotide Sequences to which patent is extended or adjusted under 35 antibodies, antisense molecules and other inhibitors are U.S.C. 154(b) by 0 days. directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors. (21) Appl. No.: 09/103,331 (22) Filed: Jun. 23, 1998 9 Claims, 16 Drawing Sheets U.S. Patent Mar. 2, 2004 Sheet 1 of 16 US 6,699,654 B1 2 FLASD Giullamate 4 RNAGlu Giutamate - RNAGlu Glycine . Succinyl CoA COg Glutamate 1-Semialdehyde CoA GSA am no. transferasc At - Porphobilinogen (Plants, Algae and Bacteria) (Mannals Protoporphyrin X HEME -71 via/ /fs GOOOO -- - SCOOO 4OOOO 3OOOO 2OOOO OOOO Gabaculine (mM) U.S. Patent Mar. 2, 2004 Sheet 2 of 16 US 6,699,654 B1 54000 8000 -O- Mediurn 42000 - "" ....... Gabaculine 36000 30000 24000 8000 2000 6000 Days Post infection PtCS FA Y ACS ACETY COA re- 2 A -1N We P N MALATE OXAOACEATE CoA. Sh CoA.S + Yr 1. GLYOXYATE CRATE SOCRATE C1 AC OS (SUCCtrl ATE) U.S. Patent Mar. 2, 2004 Sheet 3 of 16 US 6,699,654 B1 4OOOO . 3OOOO i. 2OOOO OOOO Nitochondri On RR Cytochiotic b Cytochfornc ef Oehydrogenase Cytochromc c oxidase ther the Succinale Oehydrogenase y I7-y mAid H. At SUCCIf Ata FUMARATE CRC ACC CYCE MARX could also be in 1ho plasid --- U.S. Patent Mar. 2, 2004 Sheet 4 of 16 US 6,699,654 B1 70000 GOOOO 50000 40000 30000 2OOOO OOOO THE SHKMATE pAHWAY posphoenolpy fit WATE t gray trios. 4. Pics. Phi Are credo t SHKATE SKAEkou.S.E(EPsF) gets Costa Of p- -2. SY-ASE Nz. 2/. A CHORSATE 6. Yoo Yi EThe Y Yo RoPTE Rt Hor Hospha E .A. a. ooe NZOATE ..okyor oxYBEZOATE -1 -- fotate C-ry of Oa als fied JCASE O ET TER f:YOROFOCATE U3, U(OE U.S. Patent Mar. 2, 2004 Sheet S of 16 US 6,699,654 B1 CPM --- n a e co C s o s o C C) d D g C RH-SEESSEESS - P M S. Mcdium E.SE SESSEE No R - J. ~2. A3 U.S. Patent Mar. 2, 2004 Sheet 6 of 16 US 6,699,654 B1 2.2. 62d co OO 3CO tood thrup (relrwto) 2%. 42 120 u so O s 5 60 AO A. O-5 10-4 10-3 10-2 10-1 100 101 PABA or FA (uM) U.S. Patent Mar. 2, 2004 Sheet 7 of 16 US 6,699,654 B1 ufco 4 3 2. PLASTD 2 s la e.scAf Henle Synthesis Patil'? - sk?: Shikiniate Pathway Kisse GLAA Seanese SKA.OST: elena ) SY AS2 SAMCSSE GABACULINE C wiAMNK s moduce CSA. EROEach SYASE 8-O-Witc. CEAN Ris Auxis-a-AOMAC AMOACOS scos, ATE n-AMOBENZOATE YASE POPOLOGE YfoxY -- DYoore it 3-DIYonoxY(Elizon PHOSPA 4 Y(XY2O: OO offs FOLAre Ori' (ii. AAS CPAREANC f 3.Craft -a. YOOXO, ties Yode Alternative Generation Of Energy TRAMBANE --- SPACE (Goautott)----- - SNN: I? Y -- Cytochroric in a Tritta cities Cytocifolie y AO succring rut. A? Alf ARX 4 CYCLES )f M S- M TO CHOND RON SUCCNAE Glyoxylate Cycle NA SOCTRAELYASE CONCAC M tory GLYCXYATE CTRATE CoA.S. MAAEk OXALOACETAEA COASE { NN- -/ SACTY CoA-a- FATY ACOS ---POS U.S. Patent Mar. 2, 2004 Sheet 8 of 16 US 6,699,654 B1 s - E - - - - - - - - SHAM (1g/ml) - 8-hydroxyquinoline NPMG Gabaculine E. O.2% etoh added (ig/ml) (ug/ml) (g/ml) U.S. Patent Mar. 2, 2004 Sheet 9 of 16 US 6,699,654 B1 32OOO -- Mediurn -- 28000 - - - - ... ... GabaCuline 24000 ----O---- P/S / 2OOOO M-1 7 6000 - -- ufc2. -- g 12000 - 8OOO 4OOO g. O't!-------............. -O O T- - 5 6 7 S Days Post infection OO -- CO-RO SO MG 3. FYIR/PMG c 60 r-y S. vf?2.r 5 of 40 SS 2O O O 2 4 6 8 O 12 4 16 DAYS POS INFECTION U.S. Patent Mar. 2, 2004 Sheet 12 of 16 US 6,699,654 B1 fa. 7O (7) T. gondii MSSYGAALRI Synechocystis . -- - - - - - -a - - - - - - - - - - - - - - - - - - - - - - - 1 1- - - MGNTFGSLFRIT 2 S. lycopersicura MASS4LTKOFLGAPFSS FGSGQQPSKLCSSNLRFPTE:RSOPKRLEIOAAGNTFGNYFRVT O . ar. r. r or - w w a ser ---a w- (- www or r r a ur or - a u a 1- - - - - - - - - - - we a MSTFGHYFRVT ... crassa H. influenza - - - - - - - - - - - - - - - - - - - - - MAGNTIGOLFRVF 3. S. cerevisae . - - - - - as a ex a- a - a - w - - - - - -s-- - - - - - as all as an as - a- or -- a-- a-- a--- we - MSTFGKLFRVT T. gondii TFGESGSAWGCIDGLPPRLPLSWEEVOPOLNRRRPGQGPLSTQRREKDRVNLSGVEd Syechocystis TFGESHGGGVGVIIDGCPPRLEISPEEICVDLDRRRPGOSKITTPRKEADOCEILSGWFE 72 S. lycopersicum TFGESHGGGWGCIDGCPPRLPLSESDMCWELDRRRPGOSRITTPRKETDTCKSSGTA 20 N. crassa TYGESHCKSVGCIVDGVPPGMELTEDDIQPQ-TRRRPGQSAITTPRDEKDRVIIOSGTEF H. influenza IFGESHGIALGCIVDGVPPNLELSEKDIOPDLDRRKPGTSRYTTPRREDDEVOILSGVFE 3 S. cerevisae TYgESHCKSVGCIVDGVPPGMSLTEADIQPQLTRRRPGQSKLSTPRDEKDRVEIQSGTEF T. gondii GYTLGTPA-LV.NEDRRPODYH- -AAWPRPGHGDFTYHAKYHIHAKSGGGRSSARET 129 Syne chocystis GKTLGTPIAILVRNKDARSODYN -- EMAWKYRPSHADATYEAKYGIRNOGGGRSSARET 130 ... lycopersicum, GLTTGSPIKVEVPNTDQRGJDYS- - EMSI.AYRPSHADATYDFKYGVRSVQGGGSRSSARET 8 aSs a GvTLGTPIGMLVMNEDQPPKDYGNKT-1DIY PRPSHADWTYLEKYGVKASSGGGRSSARET 3. influenza GKTTGTSIGM 1 INGOORSQDYG-- DIKORFRGHADFTYQQKYGIRDYRGGGRSSARET 3. ... cerevisae gKTLGTPIAMMIKNEDQRPHDYS--DMDKFPRPSHADFTYSEKYGIKASSGGGRASARET 29 T. gondii LARWAAGAVWEKLG(4YGTSFTAWCQVGDWSWPRSLRRKEROPPTRODVDRLGVWRV 89 Synechocystis IGRWAAGAIAKKLAOFNGWEIWAYWKSIOOIEA - - - - - - - - - - - - - - - - - - - - - - - - - - 6 4. S. lycoper sic util IGRWAAGAVAKKLKLYSGTEILAWSOVWWLP- - - - - - - - - - - - - - - - - - - - - - - - - 23 is classa IGRVAGAIAEKY LKPRYGVEIWAFVSSVGSEHLFPPTAEHPSPST - - - - - - - - - - - - - - 7 ... influenza AMRWAAGAACKYLREHFGEWRGFLSOIGIKAP- - - - - - - - - - - - - - - - - - - - - - - - 67 S. cere visa e IGRVAsgi, IAEKFAQNSNVEIVAFVTOIGEIKMNR - - - - - - - - - - - - - - - - - - - - - - - - 65 T. gondii SpgTTFLDANRLYDERGEELVEEEDKNRRRLFGVDNTPGETWETRCPCPSTFWRM 249 Syne chocystis - - - - - -w TVSNTVTLEOVES - - - - - - - - - - - - - - - - - - - - - - - - - IWRCPDEECAE-i 92 S. lycopersicum - - - - a EDLVDtNOWLEOESN - - - - - - - - - - - - - - - - - - - - - - - - - WRCPNPEYAEK, 243 N. crassa - - - NEF.K.WSTRETWOSFL - - - - - - - - - - - - - - - - - - - - - - - - PWRCPOENKR 20 H. influenza . m . QKVGQIoteKWNSN- - - - - - - - - - - - - - - - - - - - - - - - - PFFCOESRVEKF 94 S. cerevisa e DSFDPEFQHLLNTITREKVDSMG - - - - - - - - - - - - - - - - - - - - - - - - PIRCEDASVAGL 2. T. gondii AWKIOTRSLGDSIGGCISGAIWRPLGGEPCFDKWEAELAKAM-1SLPATKCFEIGOGF 9 Synechocystis IERIDQVLROKDSIGGVWECAIRNAPKGLGEPWFDKLEADLJAKAME4SLPATKGFEFGSGF 2S2 S. lycopersicut IGADWRWRGOSWGGVWTCIWRNWRGLGTPWFDKLEAELAKACMSLPATKGFEFGSGF 303 ... crassa EDLI TKFRDSDSIGGTWCVIRWPSGGEPAFDKLEAMLAE-SIAKGFEWGSGF 20 H. in fuerza CEL REKKEGOS GAKLWAE:WWGGEWFORADAA-GINAWKGWE GOCSF 25.4 S. cerevisae 2 sl O. F. G. O. (2) - - - U.S. Patent Mar. 2, 2004 Sheet 13 of 16 US 6,699,654 B1 To FG 1 O ( ) . T. gondii ASWTLRGSEHNDRFIPFERASCSFSESAASTIKHERDGCSAFTLSRERASDGRTTSRHEE 369 Syne chocyst is AGTLLTGSQHNDEYYLDEAGER- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 27 S S. lycopers cut AGTFMTGSEHNDEFFIDEHDOIR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 326 ... Crassa (SGCEWPGSHNDPFWSAENTEIPPSWAAS GAARGI. - - - - - - - - - - - - - - - - - - - - - - - 3 O 6 i. influenza AWWEORGSEHRDEMTPNGFESN- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 27 7 S. cerevisa e QGVSVPSSKHNDPFYFEKETNR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 283 T. gondii EVERGRERIORDTLHVTGVDOONG SEDSVRYTSKSEASI TRLSGNAASGGAPVCRIPLG 429 Syne chocystis S. lycopersicum N. crassa H. influenza S. cerev is ae T. gondii EGWRIRCGSNNAGGTLAGITSGENIFFRVAFKPWSSIGLECIETADFA-GE-1OLAVKGRE 488 Synechocystis . TRTNRSGGWOGGESNGEP2-1RAFKPTATIGOEOTWSNI-GEETTLAAKGRH 32 S. lycopersicum - - - - - - TKTNRSGGIOGGISNGEI IN-RWAFKPTSTIARKOHTVSRD - KHETELIARGRI 379 ... crassa PRPKLTTKTNFSGGIQGGISNGAPIYFRVGFKPAATIGQEQTTATYDGTSEGVLAAKGRH 366 H. influenza ... - - - - - - - AGGILGGISSGOp. IATIALKPTSSITIPGRSINLN-GEAVEVVTKGRH 325 S. cerevisae - - - - LRTKTNNSGGVOGGISNGENIYFSVPFKSVATISQEQKTATYD-GEEGILAAKGRH 33B T. gondii DipCVLPRAPPLWESMAALVIGDLCLRQRAREGPHPLLVLQHSGCPSC - - - - - - - - - - - - S35 Syne chocystis DipCVLPRAWMVEAMAALVLCDHLLRFQAQCKTI - - - - - - - - - - - - - - - - - - - - - - - - - - 362 S. lycopersicum DPCVVPRAWPMVEA-IVALVI,VDQLMTOYAQCi-LFPVNLTLOEPLOPSTTKSA- - - - - - - - 43. N. crassa DpSwVPRAVPEVEAMAALVIMDAVLAHEARVTAKSLLPPLKQTINSGKDTVGNGVSENVQ 426 H. influenza DpCVGIRAWPIAEAMVAIVLLDHLLRFKAOCK- - - - - - - - - - - - - - - - - - - - - - - - - - - - 3S) S. cerevisa e ppAvT PRAIPIVEA4TALVLARALLIOKARDFSRSVV):- - - - - - - - - - - - - - - - - - - - - - 36 T. gondii