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Wo 2012/058621 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 3 May 2012 (03.05.2012) WO 2012/058621 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/527 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/US201 1/058432 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 28 October 201 1 (28.10.201 1) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, (26) Publication Language: English RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (30) Priority Data: ZM, ZW. 61/408,4 18 29 October 2010 (29.10.2010) U S (84) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): SHIRE kind of regional protection available): ARIPO (BW, GH, HUMAN GENETIC THERAPIES INC. [US/US]; 300 GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Shire Wau, Lexington, MA 02421 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, (72) Inventor; and DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, (75) Inventor/Applicant (for US only): WIEDERSCHAIN, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, Gherman [US/US]; 63 Bradwood Street, Roslindale, MA SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, 0213 1 (US). GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (74) Agent: TRE ANNIE, Lisa, M.; Morse, Barnes-brown & Published: Pendleton, P.C., Reservoir Place, 1601 Trapelo Road, Suite 205, Waltham, MA 0245 1 (US). — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) (54) Title: ASSAYS AND KITS TO DETERMINE UREA CYCLE ENZYME ACTIVITY ON SOLID SUPPORT © - (57) Abstract: The present invention discloses compositions, methods, kits and assays which facilitate the rapid, high-throughput and sensitive detection of at least one enzyme in a test sample. Also disclosed are assays performed on a solid support which are o useful for the detection of urea cycle enzymatic activity. Inventor(s): Gherman Wiederschain Attorney's Docket No.: SHIR-002-WO1 ASSAYS AND KITS TO DETERMINE UREA CYCL E ENZYME ACTIVITY ON SOL ID SUPPORT REL ATED APPL ICATIONS This application claims the benefit of U.S. Provisional Application No. 61/408,418, filed October 29, 2010. The entire teachings of the above application are incorporated herein by reference. BACKGROUND OF THE INVENTION The urea cycle represents a series of metabolic processes which occur in mammals that produce urea from ammonia. A deficiency in any of these metabolic processes, or more particularly the enzymes involved in these processes, may result in the accumulation of excess ammonia which may be toxic. Specifically, the urea cycle consists of a series of five biochemical reactions and serves two primary functions: the elimination of nitrogen as urea and the synthesis of arginine. Defects in the urea cycle result in the accumulation of ammonia and its precursor amino acids (glutamine, glutamic acid, aspartic acid, and glycine). The resulting high levels of ammonia are neurotoxic, and the triad of hyperammonemia, encephalopathy, and respiratory alkalosis frequently characterize the urea cycle metabolic disorders. As a result, such deficiencies may result in extensive ammonia accumulation, which in turn may lead to extensive liver damage and death if not properly treated. Advances in molecular biology have supported the development of recombinant biological agents {e.g., recombinant proteins and/or enzymes) which are useful for modulating protein or enzyme activity or nucleic acid expression. For example, such recombinantly prepared proteins and/or enzymes may be therapeutically administered to humans who demonstrate an endogenous deficiency of such protein and/or enzyme. The diagnosis of such endogenous deficiencies, as well as the in vitro production of recombinant biological agents both require means of rapidly assessing the enzymatic activity of the recombinantly prepared enzyme. Generally, assays used to assess enzymatic activity involve contacting a biological sample or a recombinantly prepared enzyme with a substrate with which the enzyme of interest is known to predictably react. Enzymatic activity may be subsequently measured by either evaluating the depletion of the substrate and/or the yield of an enzymatic product over time. Different methods of measuring the concentrations of substrates and/or products exist and many enzymes can be assayed in several different ways, many of which are time-consuming. Assays available to analyze enzymatic activity are frequently performed in a test tube which may prove especially cumbersome and time consuming, for example due to the handling of hazardous waste and/or the immobilization of substrates onto solid supports. Such analyses may be further complicated by the performance of multiple steps or reactions, each often performed on a different solid support. A number of time consuming assays for determining the enzymatic activity of the enzyme L-arginine amidinohydrolase (ARG) have been previously described. (See, Chinard, FP., J . Biol. Chem. 199: 91-95 (1952); Konarska, L ., et al Clin. Chim. Acta, 154: 7-17 (1986); Mellerup, B., Clin. Chim. 13 (10): 900-908 (1967); Jung, D., et al., Clin. Chim. 2 1 (8): 1136-1 140 (1975); and Levinson, S Clin. Chim. 24 (12):2199-2202 (1978)). Most of these assays are based on quantification of either ornithine or urea as the products of the ARG enzymatic reaction and are performed in a test tube. There is therefore a need for improved methods and high-throughput assays for the routine analysis of enzymatic activity, preferably which are performed in a quick and accurate manner, in a single solid support. The methods, assays and kits of the present invention are useful in carrying out such methods. SUMMARY OF THE INVENTION The present invention relates to methods, assays and kits which are useful for the high-throughput determination of enzymatic activity of a test sample. Generally, such methods, assays and kits comprise the steps of contacting a test sample with a substrate and using routine means (e.g., absorption or fluorescence spectroscopy techniques) to quantify the product of an enzymatic reaction, or alternatively the depletion of a substrate. The methods, assays and kits of the present invention contemplate the use of substrates which are known to predictably react with an enzyme whose presence is suspected in a test sample. In some embodiments the methods, assays and kits of the present invention provide useful tools to measure the presence of urea cycle enzymes (i.e., carbamoyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase and L- arginine amidinohydrolase) in a test sample, or alternatively the principles presented herein can be applied generally to determine the presence of any particular enzyme in a test sample. The methods, assays and kits of the present invention provide tools which are useful to visually, colorimetrically, fluorometrically and/or chemically distinguish the presence or absence of a predicted enzymatic reaction, and are preferably capable of quantitatively determining the presence of an enzyme in a test sample using routine means (e.g., absorption or fluorescence spectroscopic techniques). For example, in one embodiment of the present invention, enzymatic activity, such as the hydrolysis of a substrate by an enzyme, may yield a fluorescently-detectable signal which can be measured using fluorescence spectroscopy and thus provide means of quantitatively assessing the presence of such enzyme. The methods, assays and kits may optionally be useful for the detection of an enzyme as a diagnostic marker, predictor or identifier of disease (e.g., diagnosis of particular urea cycle enzyme deficiencies by evaluating a test sample obtained from a human). One embodiment of the present invention relates to methods and assays which are useful for determining and/or measuring the presence and/or activity of the enzyme ornithine transcarbamylase (OTC) in a solid support. OTC is the second enzyme in the mammalian urea cycle and is responsible for catalyzing the transfer of a carbamoyl group of carbamyl phosphate to ornithine, thus producing citrulline. Such methods and assays comprise the steps of determining OTC activity by reacting a test sample with ornithine and a suitable substrate (e.g., carbamyl phosphate). The reaction is subsequently halted by contacting the reactants with an anti-catalyst or stop buffer (e.g., phosphoric acid and/or sulfuric acid) to enable quantification of the applicable reactants. The present invention contemplates determining and/or quantifying the presence of OTC in the test sample by, for example, quantifying the production of citrulline during the preceding reaction. n a preferred embodiment, citrulline production is determined by contacting the reactants with a chromogenic reagent (e.g., 2, 3 butanedione monoxime) under suitable conditions (e.g., in acidic media at about 95-100° C) such that a detectable signal is produced which enables a colorimetric quantification of the citrulline present in the reaction media, if any. OTC activity may accordingly be measured as a function of citrulline production in the solid support, for example using known spectroscopic techniques. In a preferred embodiment, the methods, assays and kits of the present invention are performed in a single solid support (e.g., a 96-well microplate).
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