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United States Patent 19 11 Patent Number: 5,780,253 Subramanian Et Al

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United States Patent 19 11 Patent Number: 5,780,253 Subramanian Et Al III USOO5780253A United States Patent 19 11 Patent Number: 5,780,253 Subramanian et al. (45) Date of Patent: Jul. 14, 1998 54 SCREENING METHOD FOR DETECTION OF 4.433.999 2/1984 Hyzak ....................................... 71.03 HERBCDES 4.6–552 2/1987 Anoti et al. if O3. 4,802,912 2/1989 Baker ........................................ 7/103 Inventors: Wenkiteswaran Subramanian Danville: Anne G. Toschi. Burlingame. OTHERTHER PPUBLICATION CATIONS both of Calif. Heim et al. Pesticide Biochem & Physiol; vol. 53, pp. 138-145 (1995). 73) Assignee: Sandoz Ltd., Basel. Switzerland Hatch. MD.: Phytochem. vol. 6... pp. 115 to 119, (1967). Haworth et al. J. Agric. Food Chem, vol. 38, pp. 1271-1273. 21 Appl. No.:752.990 1990. Nishimura et al: Phytochem: vol. 34, pp. 613-615. (1993). 22 Filed: Nov. 21, 1996 Primary Examiner-Louise N. Leary Related U.S. Application Data Attorney, Agent, or Firm-Lynn Marcus-Wyner: Michael P. Morris 63 Continuation of Ser. No. 434.826, May 4, 1995, abandoned. 6 57 ABSTRACT 51 Int. Cl. ............................... C12Q 1/48: C12Q 1/32: C12Q 1/37; C12O 1/00 This invention relates to novel screening methods for iden 52 U.S. Cl. ................................. 435/15:435/18: 435/26: tifying compounds that specifically inhibit a biosynthetic 435/23: 435/4, 536/23.6:536/23.2:536/24.3 pathway in plants. Enzymes which are specifically affected 536/26.11:536/26.12:536/26.13 by the novel screening method include plant purine biosyn 58 Field of Search .................................. 435/15, 8, 26, thetic pathway enzymes and particularly the enzymes 435/23 4: 536/23.6, 23.2, 24.3, 26.1, involved in the conversion of inosine monophosphate to 26.12, 26.13 adenosine monophosphate and inosine monophosphate to guanosine monophosphate. Further the invention relates to 56) References Cited compositions and method of use comprising herbicidally U.S. PATENT DOCUMENTS effective amounts of the identified compounds. 4,400.529 8/1983 Chin .......................................... 7/103 18 Claims, 5 Drawing Sheets OPOC OCC v'sC GAR Ny CAIR ... ." N's M THF- GAF transformylase H.N. R-5-P ROQse-5-P - ATP + asp T SACAiR synthetase RP PP kinase -N ADP + P, AMP a s & FGAR or N HOOCs-cooh ofoo R-5-P N N o24. N HC – OHOP-O. ATP+Gin-i-HC FGAff N ) SACAIR PRPP ADP-Glu-P- Synthetase " Asp y Gin + HO anidophospho- N Fumarate - Adenyicsuccinate lyase Giu + PP, - ribosyl transferase r FGAM in 1N s N CPCCvoy NH F-5-P /-Ny AICAR , A HN - TP- AiR synthetase R5-P HC O- ADP P, - N-FTHF 3-5'-Phosphoroosylamine AICAR transformylase AP-gly N.y Sator,s. iridazole- THF - GAR synthetase Ribotice (AIR 9 ADP + P,- R-5-p HM N t -NH. | y FACAR f - e- N CPOC , CC AIF cartoxylase o2 N. R-5-P -, a? HOOC N - IMP cyclohydrolase -, CAIR H.O HO C- - ^ N GAR * R-5-p N y MP s'ss ----- A-5-P U.S. Patent Jul. 14, 1998 Sheet of 5 5,780,253 OPOC GAR A \ OH HO OH NO-FIHF RibOSe-5-P GAR transformylase THF 2 AiP N RP PP kinase 1 N AMP FGAR O of N R-5-P ATP+Gln-HO FGAM Synthetase PRP ADP+Glu-P, Gin -- H2O amidophospho Glu + PP, ribosyltransferase Y O.POCP NH AP AIR Synthetase HO OH B-5'-Phosphoribosylamine ADP + P. ATP+gly 5-aminoimidazole GAR synthetase Ribotide (AIR) ADP- P. HN NH ol. AIR Carboxylase OPOC N HO OH GAR FIG. 1A U.S. Patent Jul. 14, 1998 Sheet 2 of 5 5,780.253 HOOC N y CAR /NN HN R-5-P ATP + asp N SACAIR synthetase ADP + P. HOOC-rucooh N O N ) SACAR HN N R-5-P Fumarate Adenylosuccinate lyase O HN*" Ny ACAR H2N A 5-P No-FTHF AICAR transformylase THF O HN Ny FAICAR orn H2O IMP cyclohydrolase O N N tryS-SN ne FIG. 1C R-5-P U.S. Patent Jul. 14, 1998 Sheet 3 of 5 5,780.253 isRib-5-P e NA ) + H2O IMP Dehyd- NADH + H" rogenase Rib-5-P AdenvloSuCCinate MP Adenylo GLN + ATP + H2O succinate GMP synthase Fu - lyase GLU + AMP + PP, NH2 O AMP FIG. 2 U.S. Patent Jul. 14, 1998 Sheet 4 of 5 5,780.253 Control "... (-) - (-)- Probe Compound *() () ()(3)(3) (3) 5ul 1041 25 Lal 50Lil 100Lll 150L Columns l 2 3 4. 5 6 FIG. 3 U.S. Patent Jul. 14, 1998 Sheet S Of S 5,780.253 FIG. 4 5 780. 253 1 1. SCREENING METHOD FOR DETECTION OF FIG. 2 depicts the pathway for the conversion of IMP to HERBICDES AMP and GMP. FIG. 3 illustrates schematically the lethal concentration This is a CONTINUATION of application Ser. No. step of the claimed assay procedure. Row A is the control 08/434.826. filed on May 4, 1995 now abandoned. 5 and does not contain a probe compound. Row B contains 6 concentrations or doses of an aqueous solution of the probe BACKGROUND OF THE INVENTION compound (5, 10, 25, 50. 100 and 150 ul). In this figure the In recent years, a number of herbicides have been found lethal concentration is 50 ul. to inhibit enzymes in specific biosynthetic pathways, for FIG. 4 illustrates schematically the reversal step of the example glyphosate inhibits enzymes in the aromatic amino O claimed assay procedure. acid pathway and imidazolinones and sulfonylureas inhibit enzymes in the branched amino acid pathway. Rapid screen DETALED DESCRIPTION OF THE ing methods have been devised to identify potential herbi INVENTION cidal compounds which target specific enzymes or metabolic To assist in interpreting the means and scope of the pathways. Since typically a test or probe compound is 15 present invention, the following terms and abbreviations are determined to be herbicidal by spraying the compound on a intended to have the meaning described herein: whole plant or plant part or applying the compound to the ADSS=adenylosuccinate synthetase. soil prior to seedling emergence, with chemical effect deter AMP=adenosine monophosphate mined at a specific time interval after chemical application, ADP-adenosine diphosphate the screening methods utilizing the inhibition of enzymes in 20 AICAR=5-aminoimidazole-4-carboxamide ribotide a metabolic pathway provide an efficient and rapid method AICARP=5-aminoimidazole-4-carboxamide ribotide 5' for assessing the herbicidal properties of probe compounds. phosphate We have discovered that the herbicidal activity of the AIR-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl known compound hydantocidin and at least some of its 5-monophosphate derivatives is a result of their inhibition of AMP biosynthesis 25 AS=adenylosuccinate in the plant purine biosynthetic pathway. We have further ASL=adenylosuccinate lyase discovered a rapid means for screening potential herbicidal ATP-adenosine triphosphate compounds acting on the enzymes in the plant purine GAR=glycinamide ribotide biosynthetic pathway. GA:glycinamide 30 GDP=guanosine diphosphate SUMMARY OF THE INVENTION GMP=guanosine monophosphate This invention relates to novel screening methods for GTP-guanosine triphosphate identifying compounds that specifically inhibit a metabolic IMP-inosine monophosphate target site or pathway in plants. Enzymes which are specifi XMP-xanthosine monophosphate 35 NAP-H=5'-Phospho-N-acetyl-hydantocidin also known as cally targeted by the novel screening method include plant N-acetylphosphohydantocidin purine biosynthetic pathway enzymes and particularly PRPP-phosphoribosyl pyrophosphate enzymes that inhibit AMP and GMP biosynthesis. R5P-ribose-5-phosphate Therefore, one of the main objectives of the invention was The term "IMP biosynthesis” means the conversion of to develop a screening assay for identifying inhibitory RSP into IMP. “AMP biosynthesis” means the conversion of compounds of the plant purine biosynthetic pathway that IMP into AMP. Specifically the term "AMP biosynthetic could potentially act as herbicides. inhibitory effective amount" means an amount of a probe Accordingly, the present invention comprises a method of compound which causes a significant decrease in the ability identifying potential inhibitors of the plant purine biosyn of ADSS or ASL to convert the substrate. IMP into product. thetic pathway which are potential herbicides comprising 45 AMP in the presence of GTP and aspartate as measured by testing a probe compound, in a lethal concentration and quantified enzymatic activity as generally known to those reversal assay. This two-step procedure includes determin skilled in the art. ing the lethal concentration of the probe compound and "GMP biosynthesis" means the conversion of IMP into reversion of the inhibition caused by the probe compound at GMP Specifically the term “GMP biosynthetic inhibitory the lethal concentration in the presence of antidote com 50 effective amount” means an amount of a probe compound pounds described hereinbelow. The invention further relates which causes a significant decrease in the ability of IMP specifically to novel compounds which inhibit IMP, AMP dehydrogenase or GMP synthase to convert the substrate, and GMP biosynthesis, and specifically the target enzymes IMP into product, GMP in the presence of ATP and adenylosuccinate synthetase (ADSS), adenylosuccinate glutamine as measured by quantified enzymatic activity as lyase (ASL), IMP dehydrogenase or GMP synthase. 55 generally known to those skilled in the art. Another aspect of the present invention is a method for the A "probe compound" is a compound used in the methods control of undesirable plant growth which comprises apply described herein which potentially inhibits either directly or ing to a locus where control is desired a herbicidally indirectly one or more of the plant purine biosynthetic effective amount of an inhibitory compound identified pathway enzymes. Unless indicated otherwise, the term according to the method disclosed herein. "plant purine biosynthetic pathway enzyme" as used herein According to still another aspect, the invention is a refers to any enzyme which is involved in the purine herbicidal composition comprising an inhibitor compound biosynthetic pathway as depicted in FIGS.
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