WO2000018950A2 - Assay method for ntp hydrolising enzymes - Google Patents

Abstract

The present invention is drawn to a high throughput assay method of monitoring the activity of enzymes which hydrolyze NTPs as cosubstrates by forcing the reaction to run in the metabolically reverse direction and quantitating the NTP synthesized by the target enzyme. The present invention is further drawn to assay systems which use the assay method.

Description

TITLE OF THE INVENTION

HIGH THROUGHPUT ASSAY METHOD FOR ENZYMES

WHICH METABOLICALLY HYDROLYZE NUCLEOSIDE

TRIPHOSPHATES AND AN ASSAY SYSTEM THEREFOR

FIELD OF THE INVENTION

The present invention is directed to a method of assaying enzymes which metabolically hydrolyze nucleoside triphosphates and further to an assay system for carrying out the disclosed method.

BACKGROUND OF THE INVENTION

All known organisms contain enzymes which hydrolyze nucleoside triphosphates (NTPs) as cosubstrates of biochemical reactions. The hydrolysis of the phosphoanhydride bonds of NTPs is very exergonic, i.e. the hydrolysis of the bonds releases free energy and the reactions are thus thermodynamically favored. The energy released upon hydrolysis of NTPs is used by an organism to drive many endergonic reactions, reactions which are thermodynamically unfavored. Because of their essential role in organisms, NTP hydrolyzing enzymes are attractive candidates for drug targeting. However, one of the limiting steps in drug design is the necessity for a high throughput assay which can be used to quickly screen potential inhibitors (or activators) of specific enzymes. Enzyme screening assays are typically based on a method of quantitating the metabolic formation of enzyme products. The specific nature of the products generated by each specific enzyme reaction that hydrolyzes an NTP as a cosubstrate dictates that individualized assay methods for quantitating the specific enzyme product produced have to be developed for each different enzyme of interest. Individualized assay design is both time-consuming and expensive. In addition, it is often the case that while a particular enzyme may be considered a good potential target for drug therapy, because it is not possible to easily assay the enzyme activity in drug screening, the enzyme is not pursued as a therapeutic target.

The present invention provides a high throughput robust assay method for studying enzymes, which hydrolyze NTPs. The present assay method further provides a universal reporter system such that diverse enzymes which hydrolyze NTPs can be screened with the same assay format. This will allow for easy roboticization of the assay and high throughput screening for compounds, particularly inhibitors, of interest.

SUMMARY OF THE INVENTION

The present invention is drawn to a method of screening for inhibitors or activators of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises,

(i) contacting (a) a target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate with

(b) a compound of the formula X-Nuc-P, wherein X- Nuc-P is a product produced by the target enzyme m a metabolically forward reaction wherein a nucleoside triphosphate is hydrolyzed,

(c) NDP, where NDP is a nucleoside diphosphate,

(d) a reporter enzyme system which is thermodynamically favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the metabolically reversed direction, and

(e) a potential inhibitor or activator compound; and (ii) determining whether said target enzyme is inhibited or activated by the potential inhibitor or activator compound. The present invention further encompasses a method of screening for inhibitors or activators of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises ,

(i) contacting (a) a target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate with

(b) a compound of the formula X-NDP, wherein X-NDP is a product produced by the target enzyme in a metabolically

forward reaction wherein a nucleoside triphosphate is hydrolyzed,

(c) PPi, wherein PPi is inorganic pyrophosphate,

(d) a reporter enzyme system which is thermodynamically favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the

metabolically reversed direction, and

(e) a potential inhibitor or activator compound; and (ii) determining whether said target enzyme is inhibited or activated by the potential inhibitor or activator compound.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents only) .

Figure 2. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and PPi cosubstrate) . Figure 3. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and ADP-D-glycero-D-mannoheptose target substrate) .

Figure 4. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and ADP-heptose synthetase target enzyme) .

Figure 5. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi and ADP-D-glycero-D-mannoheptose) .

Figure 6. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi and ADP-heptose synthetase) .

Figure 7. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, ADP-D-glycero-D-mannoheptose and ADP- heptose synthetase) . Figure 8. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi, ADP-D-glycero-D-mannoheptose and ADP- heptose synthetase) .

Figure 9. ADP-heptose synthetase demonstrates Michaelis- Menton kinetics with respect to the target substrate, ADP-D- glycero-D-mannoheptose .

Figure 10. Linear relationship between v_. and the concentration of the target enzyme, ADP-heptose synthetase.

Figure 11. Demonstration that the reaction catalyzed by the enzyme CT236 can be run in reverse and the rate of the reaction can be monitored using a coupled enzyme assay.

Figure 12. Demonstration that the CT236 catalyzed reaction is

linear at enzyme concentrations between 1.4 - 5.6 μg/ml .

Figures 13A and B. Figure 13A shows that the velocity of the luciferase reaction (defined as RLU/min from 5-40 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, CDP-choline. Figure 13B shows that a Lineweaver-Burk plot of 1/ [S] vs. l vi demonstrates a linear correlation.

Figure 14. Demonstration that GlmU uridylyltransferase reaction can be reversed to synthesize UTP .

Figure 15. Demonstration that GlmU uridylyltransferase activity is linear with respect to light production in the coupled assay at concentrations of enzyme between

0.28 μM and 1.12 μM.

Figure 16. Demonstration that the velocity (V_J.) of the luciferase reaction demonstrates saturation kinetics with respect to the concentration of the target substrate, UDP-GlcNAc.

Figure 17. Demonstration that inhibition of PKA activity by a PKA specific inhibitor peptide can be measured with a reverse assay.

DETAILED DESCRIPTION OF THE INVENTION

The direction of an enzymatic reaction is defined based on the metabolic pathway in which a specific enzyme participates in a cell. The metabolic forward direction is the ultimate target for inhibition or activation by compounds identified with the present invention. Theoretically, all biochemical reactions are reversible with the reverse reaction occurring at the same reaction site as the forward reaction. When a reaction is run in reverse the metabolic products of the forward direction become the substrates for the reverse reaction. Thus, enzymes which hydrolyze NTPs as cosubstrates in the metabolic forward direction will synthesize the respective NTP as a product if the reaction is thermodynamically pushed in the reverse reaction.

The present invention is drawn to a high throughput assay method of monitoring the activity of enzymes which hydrolyze NTPs as cosubstrates by forcing the reaction to run in the metabolically reverse direction and quantitating the NTP synthesized by the target enzyme. The NTPs synthesized by the reverse reaction can be universally quantitated by monitoring the activity of a coupled enzyme, known as a reporter enzyme, which uses ATP as a cosubstrate to produce light, color or some other measurable readout . An important aspect of the present assay method is that the method of ATP quantification be enzyme-dependent . When the reporter enzyme is saturated with all substrates other than ATP, the enzyme reaction catalyzed by the reporter enzyme will be thermodynamically favored such that the reaction catalyzed by the target enzyme will be pulled in the metabolic reverse direction to synthesize, rather than hydrolyze NTPs.

Since most inhibitors target the active site of the target enzyme and the reverse reaction of the target enzyme utilizes the same site, inhibitors of the forward reaction will also inhibit the reverse reaction. In addition, activators of an enzyme may shift the substrate binding site of an enzyme into a catalytically active conformation for either the forward or reverse direction. Thus, the present invention provides a single format high throughput robust assay method for screening for inhibitors or activators with all enzymes which metabolically hydrolyze NTPs.

The reporter enzyme system of the present invention is an enzyme read out system, which uses ATP as a cosubstrate in the enzymatic generation of a signal. From a kinetic perspective, the reporter enzyme reaction is only rate limited by the production of ATP by the target enzyme system. As a result the reporter enzyme thermodynamically pulls the target enzyme in the reverse direction while at the same time providing a read out system for quantitating target enzyme activity.

The general design of an assay of the present invention will involve first identifying a target enzyme. The target enzyme may be any enzyme of interest which hydrolyzes an NTP in the metabolic forward direction. The product or products, which are produced by the target enzyme reaction in the metabolic forward direction also need to be identified. The product (s) produced by the metabolic forward reaction, or analogues thereof, will be used as substrates and cosubstrates in reverse reaction. In addition, optimum reaction conditions can be determined as detailed below.

The present invention can be adapted to create a universal high throughput method for use with all target enzymes which hydrolyze NTPs, regardless of the NTP cosubstrate, by coupling the reverse reaction of the target enzyme to a nucleoside diphosphate kinase driven reaction. The nucleoside diphosphate kinase catalyzes the transfer of a phosphate group from the NTP produced by the reverse target enzyme reaction to ADP, producing ATP, which can be quantitated by the reporter enzyme as described above.

Thus, if the target enzyme hydrolyzes ATP as a substrate, the reporter enzyme reaction can be directly coupled to the reverse target enzyme assay. This reaction scheme may be exemplified as Assay Scheme (I) as follows: Assay Scheme (I) :

target X + ATP ■<— » target product (substrate of reverse enzyme reaction)

r«-_nnrt-pr reporter rep ^J- ^ reporter products (signal) substrate _enzyme Alternatively, if the target enzyme hydrolyzes an NTP other than ATP the reverse target enzyme reaction is itself first coupled to nucleoside diphosphate kinase. The nucleoside diphosphate kinase will transfer a phosphate group from the NTP synthesized in the target enzyme reverse reaction, to ADP, generating ATP. The ATP can then be quantitated by the reporter enzyme system. The reaction scheme for the assay method of the present invention with target enzymes which produce NTPs other than ATP is depicted as Assay Scheme (II) , as follows:

target

X + NTP — > target product (substrate of reverse enzyme ,_ . .

¹ reaction)

nucleoside + ADP -^ NDP + ATP diphosphate

_PDDT"i"_PT t-^JJ-L -ti- f , , ^ reporter products (signal) substrate _enzyme

Enzymes which metabolically hydrolyze NTPs can be classified into four general groups based on which bond within the NTP is cleaved. Group I enzymes are protein, lipid, carbohydrate, amino acid, or other organic molecule kinases which catalyze the following general reaction (I) :

kinase -_. X-Nuc + NTP > X-Nuc-(P) + NDP In reaction (I) , X is a protein, lipid, carbohydrate, amino acid or other organic molecule having a nucleophilic hydroxyl group. Nuc : represents the nucleophilic functionality of X. Typically, the nucleophilic functionality of X is a hydroxyl moiety. Lipids, proteins, carbohydrates and amino acids may be naturally occurring or synthetic and have their standard chemical definition as found, for example, in Stenesh, J. , Dictionary of Biochemistry and Molecular Biology, John Wiley & Sons, Inc. (1989) . This reaction involves the nucleophilic

attack by the protein, lipid or carbohydrate substrate at the γ

phosphate of NTP with the subsequent loss of NDP. Thus, the Group I enzymes use X-Nuc-P as the target substrate in the reverse reaction, with the appropriate NDP as a cosubstrate.

Examples of Group I enzymes, which use ATP as a cosubstrate and thus may be used in Assay Scheme (I) , above, include protein kinases as generally disclosed in Hunter T, eth. in Enzymology, 200: 3-37 (1991), such as protein kinase C and protein kinase A; lipid kinases such as ethanolamine kinase, Weinhold and Rethy, Biochem. 13: 5135-5141 (1974) and choline kinase, McCaman et al . , Anal. Biochem. 42: 171-177; ca'rbohydrate kinases such as hexokinase, Schwartz and Basford, Biochem. 6:1070-1079 (1967), phosphofructokinase , Layzer et al . J. Biol . Chem. 244:3823-3831 (1969) and galactokinase , Blum and Beutler, J. Biol . Chem. 246:6507-6510; amino acid kinases such as homoserine kinases, Miyaj ima and Shiio, J. Biochem. (Tokyo) 71:219-226(1972) and aspartate kinase, Biswas et al. J. biol. Chem. 243:3655-3660 (1968); as well as other kinases such as phosphoglycerate kinase, Scopes Biochem. J. 113:551-554(1969); glycerol kinase, Hayashi and Lin, J. Biol. Chem. 242:1030-1035(1967); mealonate-5-phosphotransferase and phosphomevalonate kinase, Bazas and Beytia, Biochem. 19:2300- 2304 (1980) ; and flavokinase, Mayhew and Wassink, Biochim. Biophys. Acta 482:341-347 (1977).

When the NTP hydrolyzed by the target enzyme is not ATP, but rather GTP, CTP or UTP, Assay Scheme II may be used to measure enzyme activity and screen for inhibitors or activators. With Assay Scheme II, the NTP produced by the reverse target enzyme reaction is utilized as a substrate in a reaction with ADP and the enzyme nucleoside diphosphate kinase. The nucloeside diphosphate kinase transfers the phosphate group from the NTP to the ADP, producing ATP which, in turn, can be monitored by the reporter enzyme. When using Assay Scheme II, the nucleoside diphosphate kinase is saturated with ADP such that the amount of ATP produced by the nucleoside diphosphate kinase from ADP is directly proportional to the amount of NTP produced by the reverse target enzyme reaction. Examples of Group I target enzymes which may be used with Assay Scheme II include: when NTP is GTP; phosphoenolpyruvate carboxykinase (PEPCK) , Ballard and Hanson, J. Biol. Chem. 244:5625- 5630(1969) and nucleoside diphosphate kinase, Edlund, Acta Chem. Scand. 25:1370-1376 (1971);

- when NTP is CTP; dolichol kinase, Rip and Canal, Can. J. Biochem. 58:1051-1056 (1980) and nucleoside diphosphate kinase, Edlund, Acta Chem. Scand. 25:1370-1376 (1971); and - when NTP is UTP; nucleoside diphosphate kinase, Edlund, Acta Chem. Scand. 25:1370-1376 (1971).

Group II enzymes are nucleotide-sugar synthetases or nucleotide lipid synthetases, nucleotide transferases, or pyrophosphorylases which metabolically catalyze the following reaction (II) :

synthetase X-Nuc + NTP NDP-X + PPj

In reaction (II) , X-Nuc is typically a phosphorylated carbohydrate or lipid molecule. Reaction (II) involves a

nucleophilic attack by a phosphate oxygen on X-Nuc at the α

phosphate of NTP with the subsequent loss of pyrophosphate

(PPi) . The substrate of the reverse reaction of Group (II) enzymes is NDP-X, with PPi as a cosubstrate. Examples of Group (II) target enzymes include:

- when NTP is ATP; nucleotide sugar sythetases such as ADP-heptose synthetase, which is encoded by the rfaE gene of gram negative bacteria, Ding et al . J. Biol. Chem. 269: 24384- 24390 (1994) , and FAD pyrophosphorylase, which synthesizes FAD from riboflavin, Gomes and McCormick, Proc . Soc . Exp. Med. 172 :250-254 (1983) ;

when NTP is GTP; GDP-α-D-mannose pyrophosphorylase,

Smoot and Serif, Eur . J. Biochem. 148:83-87 (1985); when NTP is CTP; CTP :phosphocholine cytidyl transferase, Nelson and Srisney, Can. J. Biochem. 148:83-87

(1985) and CTP :phosphatidate cytidyl transferase, Vorbeck and

Martin, Biochem, Biophys . Res. Commun. 40:901-908 (1970); and - when NTP is UTP; glucose pyrophosphorylase, Aksamit and

Ebner, Biochim. Biophys. Acta 268:102-112 (1972).

Of particular importance are the bacterial Group (II) enzymes, for example, rfaE enzyme, as discussed above, of Helicobacter pylori , which provide ideal targets for drug therapies.

Group (III) enzymes are cyclases which catalyze the following reaction (III) : cyclase NTP « -» CNMP + PP j

With Group (III) enzymes, the substrate for the reverse reaction is cyclic NMP (cNMP) , with PPi as a cosubstrate. Groups III enzymes include adenylate cyclase, Tao and Lipman, Proc. Natl. Acad. Sci. USA 63:86-92 (1969), which uses cAMP in the reverse reaction as a substrate; guanylate cyclase, White and Zenser, Meth. in Enzymol . 38:192-195 (1979), which uses cGMP as a substrate in the reverse reaction.

Group IV enzymes transfer the adenosyl group of ATP in the following general reaction (IV) :

enzyme X ₊ ATP ₄ >» X-adenosyl + P± + PPJ

Group IV enzymes are responsible for the synthesis of coenzyme B₁₂ (where X is Co⁺) and S-adenosylmethionine (where X is methionine) . Group IV enzymes include, cobalamin synthetase,

Lawrence and Roth, J. Bacterol . 177:6371-6380 (1995) and S- adenosylmethonine synthetase, Green, Biochem. 8:2255-2265

(1969) . As indicated above, the reporter enzyme system is based on an enzyme which uses ATP as a cosubstrate and which produces some measurable signal upon hydrolysis of the ATP.

Examples of suitable reporter enzymes include a luciferase/luciferin based system or a phosphoglycerate kinase and glyceraldehyde 3 -phosphate dehydrogenase system, as detailed in Hasen et al . Meth. in Enzymol . 8:248 (1966). The latter system utilizes the conversion of NADH to NAD+ by the dehydrogenase and NAD+ production may be monitored spectrophormetrically .

Firefly luciferase catalyzes the following reaction.

luciferin, Mg 2+ lucifenn + ATP » oxyluciferin +AMP + PP.

⁺⁰2 + CO ₂ + light

As indicated in the reaction scheme, one of the products produced upon the hydrolysis of ATP is light. The amount of light produced can be easily quantitated using a luminometer and is directly related to the amount of ATP hydrolyzed by the luciferase when the luciferin substrate is present at saturating amounts. Since the Km of luciferin for luciferase

is approximately 7-20 μM in a pH range of 6.8-8.0, see Denburg

et al . Arch. Biochem. Biophys. 134:381-394, luciferase will be greater than 90% saturated at the minimum concentration of 63-

180 μM luciferin. As such, in using luciferase as a reporter

enzyme, the following general reaction conditions will pull the thermodynamics of the target enzyme in the reverse reaction so that ATP is synthesized. General reaction conditions for the assay in which luciferase/luciferin is the reporter enzyme include a buffered solution between pH 6.5 - 8.5, with peak activity at pH 7.8 (Lundin et al . , Anal. Biochem. 75:611-620 (1976); 5-10 mM Mg⁺²;

minimum luciferin concentration of 63 μM depending on pH used;

1-50 μg/ml luciferase. The reaction may also optionally

include 1-5 mg/ml serum albumin, which helps stabilize luciferase activity.

The buffer conditions will be adjusted as appropriate for each target enzyme tested. It will be readily apparent to one skilled in the art how to modify the buffer conditions, with regard to buffer composition, pH and serum albumin as necessary to accommodate the reaction conditions of the target enzyme. Ideally, care should be taken to keep the luciferase reaction as near pH 7.8 as possible since at this pH it will be more active and will therefore thermodynamically pull the reaction of the target enzyme in reverse. Changes in luciferin and luciferase concentrations can alter the sensitivity of the assay. To validate that the concentrations of luciferin and luciferase used are behaving properly as a reporter system, each assay should be evaluated to ensure that the velocity of the luciferase reaction (i.e. light produced per unit time) is directly proportional to the target enzyme concentration . Exemplified reaction conditions are as follows: Gylcine - 100 mM

MgS0₄ - 10 mM Human Serum Albumin - 2 mg/ml

Luciferin - 130μM

luciferase - 20μg/ml

pH 7.8

In addition, ImM EDTA may optionally be included in the reaction. To quantify the amount of NTP produced by the target enzyme, and thus properly assess, the effects of potential inhibitors or activators on the target enzyme, the reaction mixture will ideally be substantially free from exogenous NTPs. "Substantially free from exogenous NTPs" means that the components of the reaction mixture, i.e. the target enzyme, target cosubstrate, target substrate, reporter enzyme system etc., does not need to be completely pure, however, no more than negligible amounts (i.e. no more than 1

μM, preferably no more than 10 nM) of contaminating, exogenous

NTPs should be present. As such, the reaction should be evaluated for exogenous contamination of NTPs. This may be easily done by separately introducing each target substrate or cosubstrate into the reporter enzyme reaction mixture in the absence of the target enzyme, to assess for contamination of the target substrate or cosubstrate. To assess for possible contamination of the target enzyme, the target enzyme should be added to the reaction mixture in the absence of substrate and cosubstrate. In addition, the target enzyme must be saturated with the appropriate cosubstrates such that the reverse reaction rate is linear with respect to the target substrate concentration. A general reaction mixture for the present inventive method using luciferase as reporter enzyme with a target enzyme that metabolically hydrolyzes ATP, includes in an appropriate buffer: a) target enzyme or target substrate; b) saturating concentrations of appropriate cosubstrates;

c) luciferase and luciferin at approximately 20μg/ml

and 130μM, respectively.

The reaction can be initiated with the addition of target substrate or target enzyme and the rate of ATP synthesis, which is directly correlated to the reaction catalyzed at the active site of the target enzyme, measured with a luminometer as light generated per unit time.

In using the present method as a high throughput screening assay for inhibitors of the target enzyme, the assay should be run under conditions where the concentration of the target substrate is less than the K_M of the target substrate for the target enzyme under given reaction conditions. The K_M of the target substrate for the target enzyme can be determined experimentally by evaluating the amount of light produced per unit time (initial velocity) as a function of target substrate concentration. See Fersht , Enzyme Structure and Mech . , 2^nd Ed, 1985, W.H. Freeman and Co. NY. In addition, at concentrations of target substrate that are significantly lower than the Km, the initial velocity of the reaction will be proportional to the concentration of target substrate. As

such, the initial velocity of, for example, lμM versus 2μM

target substrate can be compared. If the initial rate

doubles, the reaction rate is linear and lμM is a valid

concentration of target substrate to use in screening inhibitors. To validate the concentrations of each target enzyme and target substrate, the linear response of the luciferase reporter enzyme should be demonstrated to be directly proportional to the amount of target substrate and target enzyme used in the screening assay. This can be done, by simply doubling or halving the target substrate or target enzyme concentration and evaluating the initial velocity.

When the target enzymes utilizes an NTP other than ATP as a substrate, the reporter system will be coupled to nucleoside diphosphate kinase to produce ATP from the target enzyme NTP product. When the nucleoside diphosphate kinase coupled reporter system is used, ADP and nucleoside diphosphate kinase are additionally added to the reaction system. The ADP

concentration will typically be 20-200 μM with 1-100 U/ml

nucleoside diphosphate kinase. In particular, the ADP

concentration should be kept below 200 μM since high

concentrations of ADP may inhibit the luciferase reaction, Denberg et al . , Arch. Biochem. Biophys. 134:381-394(1969).

In addition, the reverse reaction catalyzed by Group I enzymes can be further encouraged thermodynamically by the addition of the enzyme inorganic pyrophosphotase to either Assay Scheme I or II when the reporter enzyme is luciferase. Inorganic pyrophosphotase catalyzes the hydrolysis of PPi into 2Pi. Since this involves the hydrolysis of a phosphoanhydride bond in PPi there is significant free energy released upon cleavage. PPi is a product of the luciferase reaction, hence coupling the hydrolysis of this product to Assay Scheme I or II will contribute additional thermodynamic "pull" at driving the reverse reaction of the target enzyme. Typically the addition of 1-100 U/ml of inorganic pyrophosphotase will be used in this design. This strategy is not appropriate for Group II -IV enzymes since PPi is a cosubstrate of the reverse reaction. EXEMPLIFIED EMBODIMENTS OF THE INVENTION

The following are exemplified embodiments of the present invention and are in no way intended to limit the scope of the present invention.

1) Assay for screening for inhibitors of Group II enzymes

The present examples embody assays for screening for inhibitors or activators of Group II enzymes. Although, the following examples are directed to evaluating the enzymatic activity of an ADP-heptose synthetase, a CTP :phosphocholine cytidylyltransferase, and an N-acetylgluocosamine-1-phosphate uridylyltransferase, the present formats may be used for any Group II enzymes.

a) Method of screening- for inhibi tors or activators of the enzyme ADP-heptose synthetase The present invention is of particular use for screening for potential antibiotics. Bacteria typically contain unique enzymes which hydrolyze nucleoside triphosphates as cosubstrates and which are involved in biosynthetic pathways which are critical to bacterial survival. As such, these unique enzymes provide an ideal target for drug therapy. Of particular interest as a drug target are the biosynthetic pathways which produce the lipopolysaccharide (LPS) outer membrane of gram negative bacteria. The LPS membrane of gram negative bacteria is made from lipopolysaccharide, lipid and protein. This outer coat makes gram negative bacteria particularly refractory to standard antibiotics. The LPS has been shown to be directly related to the ability of the outer membrane to serve as a functional barrier to antibiotics, bile salts and other hydrophobic molecules. An essential component of the LPS is L-glycero-D-mannoheptose. L-glycero-D- mannoheptose is unique to gram negative bacteria. The unique structure of the L-glycero-D-mannoheptose suggests that the enzymes which synthesize the molecule are likely also unique, making them ideal drug targets. Mutations in the biosynthetic enzymes responsible for inner core polysaccharides of the gram negative bacteria E. coli , S . typhi urium and H. influenza demonstrate hypersensitivity to numerous antibiotics, bile salts, serum killing and temperature. These finding support that inhibition of the biosynthetic enzymes involved with LPS formation increases the sensitivity to hydrophobic antibiotics and macrolides such as vancomycin for use against gram negative bacteria.

ADP-heptose synthetase metabolically catalyzes the transfer of AMP to D-glycero-D-mannoheptose-lP generating ADP- D-glycero-D-mannoheptose as shown in the following reaction.

ADP-heptose D-glycero-D-mannoheptose ► ADP-D-glycero-D-mannoheptose + ATP synthetase _+ppi In Helicobacter pylori this ADP-heptose synthetase is encoded by the rfaE gene. H. pylori is a gram negative, spiral bacteria that is a common bacteria of the human gastrointestinal tract. Infection with H. pylori is associated with most duodenal and gastric ulcers and is a risk factor in the development of gastric adenocarcinoma. The present invention is therefor useful for screening potential inhibitors of ADP-heptose synthetase of H. pylori . The reverse enzyme reaction of rfaE was developed as follows.

Recombinant ADP-heptose synthetase encoded by the rfaE gene of H. pylori was expressed in E. coli as a fusion protein with glutathione S-transferase (GST, Pharmacia, Piscataway, NJ) . The fusion protein was purified by chromatography on a glutathione column and the ADP-heptose synthetase enzyme was removed from the GST fusion partner by specific proteolytic cleavage between the two proteins using the enzyme PreScission Protease (Pharmacia, Piscataway, NJ) . To further purify ADP- heptose synthetase from GST, the products of the protease reaction were separated on a glutathione column since both the GST and the PreScission protease (which has GST at its N- terminus) will bind with high affinity and the ADP-heptose synthetase will elute. ADP-heptose synthetase purified in this manner is greater then 80% pure and contains no exogenous ATP. The concentration of purified ADP-heptose synthetase was estimated by quantitating the protein concentration of the purified enzyme and calculating molarity using a molecular weight of 52,656 g/mole for the enzyme.

Experiment 1 -Demonstration that the ADP-heptose synthetase reaction can be reversed to synthesize ATP.

ATP synthesis was evaluated by the reporter enzyme system consisting of luciferase/luciferin. In this embodiment of Assay Scheme I using a Group II enzyme the cosubstrates of the reverse reaction are NDP-X (in the case of ADP-heptose synthetase, NDP-X is ADP-D-glycero-D-mannoheptose) and PPi. The concentrations of each of the assay components were as

follows: 130 μM luciferin, 20 μg/ml luciferase, 98 mM glycine,

163 mM Tris, 9.75 mM MgS0₄ , 0.975 mM EDTA, 2 mg/ml human serum

albumin, 194 μM PPi, 0.97 μM ADP-D-glycero-D-mannoheptose, and 68 nM ADP-heptose synthetase, pH 7.8. Light was quantitated using a Dynex luminometer in a 96 -well plate format using the parameters shown below in Table I . Table I. Parameters for ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme.

Calculation mode Linear Regression

Start mode Immediate

Read Time/Well 0 ₂0 Sees Gain Setting Autogain (0-10000 RLU)

RAW Data Handling Average Readings Scaling Factor Scale data by 1

Shaking Diabled Heating Plate Temp Disabled

Dispense Volume A 0 μL After Read Cycle 0

Dispense Volume B 0 μL After Read Cycle 0

Dispense Volume C 0 μL After Read Cycle 0

No of Readings 40 Reading range 1 40

Duration of kinetic 00 19 30 Time interval ₃0 seconds

Cut-off 1 00e-004

Resul ts and discussion . ATP is synthesized by ADP- heptose synthetase under conditions where the reaction is run m reverse with both cosubstrates of the reaction and the enzyme present (Fig. 8) . Figures 1-7 demonstrate the lack of ATP synthesis as reported by the luciferase/luciferin system when any of the components of the reaction are not added to the reaction mixture. In addition, figures 1-7 demonstrate the absence of contaminating ATP m each component of the coupled assay reaction mix. Each figure shown reports ATP synthesis as directly related to the amount of light formed per unit time. RLU represents relative light units. The components in each reaction mixture are indicated above each relevant figure.

Experiment 2 - The velocity (v of the luciferase reaction (v^ defined as RLU/min over the initial 4 mm of the reaction) demonstrates Michaelis-Menton (or saturation) kinetics with respect to the concentration of the target substrate, ADP-D- glycero-D-mannoheptose .

The reaction conditions were similar to those reported in Experiment 1 with the exception that the concentration of ADP- D-glycero-D-mannoheptose was evaluated at the following

concentrations: 19.5 μM, 9.75 μM, 4.88 μM, 2.44 μM, 1.2 μM and

0.6 μM. The v^ of the luciferase reaction at each

concentration of ADP-D-glycero-D-mannoheptose was determined in duplicate and the mean rate was graphed vs. concentration (Fig. 9) .

Resul ts and discussion . Table II shows the vi at each experimental concentration evaluated. The low C.V.s reported demonstrate the excellent well to well reproducibility of this assay. Figure 9A shows that the velocity of the luciferase reaction (v defined as RLU/min over the initial 4 min of the reaction) demonstrates saturation kinetics with respect to the concentration of the target substrate, ADP-D-glycero-D- mannoheptose. Figure 9B shows that a Lineweaver-Burk plot of 1/ [S] vs. 1/ demonstrates an excellent linear correlation (R² = 0.9998). The combination of the data in Fig. 9A and 9B is consistent with the target enzyme demonstrating typical Michaelis-Menton kinetics with respect to the target substrate at saturating levels of PPi. The K_M of the target substrate for the target enzyme is 6.2 μM as determined by the

assumption that in a Lineweaver-Burk plot the x-intercept is equal to -I/KM- The linear portion of the curve appears to be

at concentrations of ADP-D-glycero-D-mannoheptose below 2.0 μM.

This demonstrates that the luciferase reporter system is behaving as an appropriate reporter for the activity of the target enzyme, ADP-heptose synthetase. In addition, this experiment demonstrates the excellent sensitivity of this assay since the lowest concentration of ADP-D-glycero-o- mannoheptose evaluated still had considerable activity above background (vi = 2.48 RLU/min for 0.6 uM substrate as compared to vi = 0.0012 RLU/min in the absence of substrate). This is very important for the economics of a high throughput assay design since the lower amount of substrate required for the assay would considerably reduce the cost of screening for inhibitors.

Table II. Mean initial velocity {v ) at varying concentrations of ADP-D-glycero-D-mannoheptose.

Location (RLU/min) (RLU/min) S.D. C.V. μMxlOO Data Mean

E8 19.68₃2 20.2828 0.876 4.320% 0.195 F8 20.9024

E7 17.9684 17.8993 0.098 0.546% 0.098 F7 17.8₃02

ES 12.6585 12.8567 0.000 0.002% 0.049 F6 12.8589

E5 7.8231 7.8090 0.259 3.314% 0.024 F5 7.8889

E4 4.4477 4.5285 0.114 2.524% 0.012 F4 4.6094

E3 2.4245 2.4826 0.082 3.312% 0.006 F3 2.5407 Experiment 3 - Demonstration that the initial velocity (v^) of the luciferase reaction is linear with respect to the concentration of the target enzyme, ADP-heptose synthetase. The reaction conditions were similar to those reported in Experiment 1 with the exception that the concentration of ADP- heptose synthetase was evaluated at the following concentrations: 68 nM, 34 nM, 17 nM, and 8.5 nM. The v^ of the luciferase reaction at each concentration of ADP-D-glycero-D- mannoheptose was determined and the mean rate with standard deviation indicated was graphed vs. concentration (Fig. 10).

Resul ts and discussion . Figure 10 demonstrates that the Vi of the luciferase reaction is linear with respect to the concentration of the target enzyme. Consistent with the results of Experiment 2 , these results demonstrate that the luciferase reporter system is behaving as an appropriate reporter for the activity of the target enzyme, ADP-heptose synthetase. As discussed in Experiment 2, the sensitivity of this assay design is further demonstrated in this experiment since the lowest enzyme concentration evaluated reported ( i = 0.0135 RLU/min at 8.5 nM) had a i that was significantly greater than background (0.0002 RLU/min). This high sensitivity is very important for the economics of high throughput assay design since the lower amount of enzyme required for the assay will considerable reduce the cost of screening for inhibitors. The RLU/min reported m this assay does not represent lower activity then experiments 1 and 2 but represent a change m the relative scale of RLU.

b) Experimental design for Group II enzymes that utilizes NTP other than ATP

The present examples embody assays for screening inhibitors or activators of enzymes that hydrolyze an NTP other than ATP. As demonstrated m the Assay Scheme (II) , the reverse reaction of a target enzyme which qualifies for this scheme will result m the synthesis of a specific NTP other than ATP as a product of the reverse reaction. By coupling the target enzyme reverse reaction to the activity of the enzyme nucleoside diphosphate kinase (NDK) , NTP will be converted to ATP. The ATP can than be quantitated using an appropriate reporter enzyme system. The following exemplified assays evaluate the activity of enzymes which hydrolyze CTP (CTP :phosphocholme cytidyltransferase (CCT) ) or UTP (N-acetylglucosoamme-1- phosphate uridylyltransferase) . However the format of these assays may be used for any enzyme which hydrolyzes an NTP other than ATP as a cosubstrate. i ) Experimental design for a Group II enzyme that hydrolyzes CTP as a cosubstrate

Although the following example is directed to evaluating the activity of the enzyme CTP :phosphocholιne cytidyltransferase (CCT) , the present format may be used for any enzyme hydrolyzmg a CTP as the cosubstrate or, more generally, any enzyme hydrolyzmg an NTP other than ATP as a cosubtrate .

The enzyme CTP rphosphocholme cytidyltransferase is a critical enzyme m the biosynthesis of phosphatidylcholme, an essential component of many prokaryotic and eukaryotic membranes. Inhibition of the activity of this enzyme has been demonstrated to be responsible for the anti-neoplastic effect of the drugs edelfosme (Vogler, et al . 1996, Leukemia Research, 20: 947-951) and hexadecylphosphocholme (Boggs, et al . 1998, Biochim Biophys Acta, 1389: 1-12). This enzyme catalyzes the transfer of a CMP group from CTP to phosphocholme to generate CDP-cholme, with the subsequent loss of pyrophosphate (PP as shown below (We hold et al . 1986, 261: 5104-5110):

CCT

CTP + phosphoc oline > CDP-c oline ₊ PP ± The enzyme CCT has been shown to be composed of a number of functional domains (Kent 1997, Biochim Biophys Acta 1348: 79- 90) . The full-length CCT enzyme requires lipids for in vi tro activity. The necessity for lipid activation has been explained by the observation that the C-termmal domain of CCT behaves as an inhibitor of enzyme activity in the absence of lipids (Wang and Kent, 1995, J. Biol. Chem. 270: 18948-18952). A C-termmal truncated form of CCT, known as CT236 (composed of the N-termmal 236 ammo acids) , has enzyme activity similar to the full-length molecule but does not require exogenous lipids for in vi tro activity (Wang and Kent, 1995, J. Biol. Chem. 270: 18948-18952). The truncated CT236 form of the CCT enzyme was used m the present studies. To evaluate the activity of CT236 m an assay of Scheme II format the following enzyme coupled reaction was designed:

pvh.osphocih-o1l ■ e CT236> CDP-choline ₊ PP + CTP

luciferase + luciferin ^ *-: oxyluciferin + AMP

+0 2 + PP i + CO 2 + light A product of the reverse reaction of CT236 is CTP. In the reaction, CTP is converted to ATP by the action of the enzyme nucleoside diphosphate kinase. ATP synthesis is reported using the enzyme luciferase, which produces light relative to the amount of ATP. The amount of light generated is quantitated using a luminometer. In the design of coupled enzyme reactions it is important that the activity of the enzyme of interest, in this case CT236, is in fact the rate- limiting step in the coupled reactions. To insure that this is the case, the substrates of both NDK (i.e. ADP) and luciferase (i.e. luciferin) must be at or near saturating conditions .

Experiment 1 - Demonstration that the CTP :ϋhosphocholine cytidyltransferase reaction can be reversed to synthesize CTP. CTP synthesis was measured by coupling the production of CTP to the synthesis of ATP using the enzyme NDK as depicted above. ATP synthesis was evaluated by quantitating the amount of light produced by the luciferase/luciferin reporter enzyme system. The concentrations of each of the assay core

components were as follows: 130 μM luciferin, 20 μg/ml

luciferase, 98 mM glycine, 163 mM Tris, 9.75 mM MgS0₄, 0.975 EDTA, 2 mg/ml human serum albumin, 2 units diphosphate

nucleoside kinase, 100 μM ADP, 1 mM sodium pyrophosphate. The pH was approximately 7.8. The concentrations of the target substrate, CDP-choline, and the target enzyme, CT236 (generous gift from Dr. Claudia Kent, University of Michigan) , were varied in each experiment as specified.

Resul ts and Discussion . Figure 11 demonstrates that the reaction catalyzed by the enzyme CT236 can be run in reverse under the conditions described and the rate of that reaction can be monitored using a coupled enzyme assay as described above. To evaluate the activity of CT236, the reaction was evaluated with the core components described above and 1 mM CDP-choline as the target substrate in the absence (solid

circles) and presence (open squares) of 7 μg/ml of CT236

enzyme. It is clear from the data presented that the production of light by the coupled luciferin/luciferase reporter assay is only observed in the presence of the target enzyme, CT236.

Experiment 2 - CT236 activity is linear with respect to light production in the coupled assay at concentrations of enzyme

between 1.4 μg/ml and 5.6 μg/ml.

When evaluating enzyme activity in a coupled assay format it is critical that the reaction catalyzed by the target enzyme be the rate-limiting step in the ultimate production of product by the reporter enzyme system. Therefore, to establish that the reverse reaction of CT236 (i.e. synthesis of CTP) is consistent with this assumption the initial velocity of the luciferase reaction was evaluated with respect to the concentration of CT236 enzyme. Each reaction contained the core components at the concentrations described above, along with 1 mM CDP-choline as the target substrate. The enzyme concentrations evaluated were 0, 1.4, 2.8, 4.2,

5.6, and 7.0 μg/ml. The initial velocity was determined

by evaluating the slope of each progress curve from 5-40 sec and reported as RLU/sec. This region of the progress curve was chosen since the reaction was clearly linear (R² > 0.97) .

Table III. Relationship of enzyme concentration with initial velocity.

[E] μg/ml v,- (RLU/sec)

1.4 0.022

2.8 0.030

4.2 0.043

5.6 0.052

7.0 0.057 Resul ts and Discussion . Table III shows V at each concentration evaluated. Figure 12 demonstrates that the CT236 catalyzed reaction is linear at enzyme

concentrations between 1.4 - 5.6 μg/ml. Therefore under

conditions of the assay within this range, the production of light by the coupled reporter system is linear with respect to the target enzyme. In addition, this experiment demonstrates the sensitivity of this coupled

assay system, since 1.4 μg/ml of purified CT236 is

approximately equal to 50 nM of the enzyme.

Experiment 3 - The velocity (vy) of the luciferase reaction demonstrates Michaelis-Menton (or saturation) kinetics with respect to the concentration of the target substrate, CDP-choline.

Experiment 2 defined the concentration range in which the reaction catalyzed by the target enzyme is linear with the production of light by the luciferase reporter enzyme system. For inhibitor screening assays it is important that the concentration of target substrate be below K_M, in a range such that activity is linear with respect to substrate concentration. This is important in the identification of competitive inhibitors of the target substrate at low concentrations of inhibitor. To define the relationship between v₂ and target substrate concentration, the velocity of the luciferase reaction (RLU/sec) was evaluated with respect to differing amounts of the target substrate CDP-choline. Each reaction contained the core components at the concentrations

described above, along with 2.8 μg/ml CT236 as the target

enzyme. The CDP-choline concentrations evaluated were 0,

100, 200, 400, and 800 μM The initial velocity was

determined by evaluating the slope of each progress curve from 5-40 sec and reported as RLU/sec. This region of the progress curve was chosen since the reaction was clearly linear (R² > 0.99).

Table IV. Relationship of substrate concentration with initial velocity.

[S] μM ^vι (RLU/sec)

100 0.017

200 0.025

400 0.028

800 0.032

Resul ts and discussion . Table IV shows v₂ at each concentration evaluated. Figure 13A shows that the velocity of the luciferase reaction (defined as RLU/mm from 5-40 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, CDP- choline. Figure 13B shows that a Lineweaver-Burk plot of 1/ [S] vs. l/v demonstrates a linear correlation. The combination of data in Figures 13A and 13B is consistent with the target enzyme demonstrating typical Michaelis- Menton kinetics with respect to the target substrate at saturating levels of pyrophosphate (1 mM) . The K_M of the target substrate, CDP-choline, for the target enzyme,

CT236, is 120 μM. Therefore for the reverse reaction of

CT236 using the coupled assay presented, concentrations

of CDP-choline less than approximately 40 μM will be

within the linear range of the enzyme activity and can be used to screen inhibitors.

ii ) Experimental design for a Group II enzyme that hydrolyzes UTP as a cosubstrate

Although the following example is directed to evaluating the activity of the enzyme N-acetylglucosamine-1-phosphate uridylyltransferase, the present format may be used for any enzyme hydrolyzing a UTP as the cosubstrate or, more generally, any enzyme hydrolyzing an NTP other than ATP as a cosubtrate .

Peptidoglycan is the primary structural polymer of the bacterial cell wall and is essential for the structural integrity of both gram negative and gram positive bacteria. Many of the most successful broad-spectrum antibiotics (e.g. penicillins, cephalosporms, vancomycm, cycloserme) inhibit peptidoglycan synthesis. UDP-N-acetylglucosamme (UDP-GlcNAc) is a critical precursor of both bacterial peptidoglycan and lipid A biosynthesis. In bacteria, synthesis of UDP-GlcNAc is catalyzed by the protein product of the GlmU gene. In E. coli , the GlmU protein is a 456 ammo acid bifunctional enzyme that contains both acetyltransferase and uridylyltransferase activities (Mengm-Lecreulx and van Hei enoort, 1993, J. Bacteriol . 175- 6150-6157, Mengm-Lecreulx and van Heijenoort, 1994, J. Bacteriol . 176: 5788-5795) . This enzyme catalyzes the following overall reaction:

GlmU glucosamine-1-phosphate ^ UDP-GlcNAc ₊ CoA

+ acetyl-CoA + UTP + p ^

The essential role for UDP-GlcNAc m bacteria make inhibition of GlmU enzyme activity an attractive target for new antibiotics. It has been demonstrated that the acetyltransferase reaction precedes the uridylyltransferase reaction at separate active sites on the enzyme (Gehrmg, et al., 1996, Biochemistry, 35:579-585). Therefore, using Assay Scheme II a coupled assay system was developed for monitoring the uridylyltransferase activity of the GlmU enzyme. The assay presented is therefore useful for screening potential inhibitors of UDP-GlcNAc synthesis and peptidoglycan synthesis. The uridylyltransferase activity catalyzes the following reaction:

GlmU N-acetylglucosamine-1-P ^ UDP-GlcNac + PP ±

+ UTP

Recombinant protein encoded by the GlmU gene of the gram negative pathogen, Helicobacter pylori , was expressed in E. coli as a fusion protein with glutathione-S- transferase (GST, Pharmacia, Piscataway, NJ) . The fusion protein was purified by chromatography on a glutathione-sepharose column and the GlmU enzyme was removed from the GST fusion partner by specific proteolytic cleavage between the two proteins using the enzyme PRESCISSION Protease (Pharmacia, Piscataway, NJ) . To further purify the GlmU enzyme activities from the GST, the products of the protease reaction were separated on a glutathione column since both the GST and the PRESCISSION protease (which contains GST at its N-terminus) will bind with high affinity and the GlmU enzyme activity will elute. GlmU enzyme purified in this manner is greater then 85% pure and contains no exogenous ATP. The concentration of the purified GlmU enzyme was estimated by absorption at 280 nm using the extinction coefficient 15,360 M^cm^"1 (estimated by the method of Gill and von Hippel, 1989, Anal . Biochem. , 182:319-326). To evaluate the uridylyltransferase activity of the purified GlmU enzyme the following enzyme coupled reaction was designed:

UTP + N-acetylglucosamme-1-P ^GlmU UDP-GlcNAc + PP.

luciferase

+ lucerferih + O 2 ■_«, oxyluciferm + AMP +PP _±

+ CO 2 + light

A product of the reverse reaction of the GlmU uridylyltransferase activity is UTP In the reaction shown above, UTP is converted to ATP by the action of the enzyme nucleoside diphosphate kinase (NDK) . ATP synthesis is reported using the enzyme luciferase, which produces light relative to the amount of ATP. The amount of light generated is quantitated using a lummometer. In the above couple assay design, the target enzyme is the GlmU uridylyltransferase and the target substrate is UDP-GlcNAc. Therefore, to insure that the target reaction is the rate limiting reaction, the concentration of PP^ ADP and luciferin must be at or near saturating conditions. Experiment 1 - Demonstration that the GlmU uridyltransferase reaction can be reversed to synthesize UTP.

UTP synthesis was measured by coupling the production of UTP to the synthesis of ATP using the enzyme NDK as depicted above. ATP synthesis was evaluated by quantitating the amount of light produced by the luciferase/luciferin reporter enzyme system on a luminometer. The concentration of each of the

assay core components were as follows: 130 μM luciferin, 20

μg/ml luciferase, 98 mM glycine, 163 mM Tris, 9.75 mM MgS0 ,

0.975 EDTA, 2 mg/ml human serum albumin, 2 units diphosphate

nucleoside kinase, 100 μM ADP, 1 mM sodium pyrophosphate. The

pH was approximately 7.8. The concentrations, of the target substrate, UDP-GlcNAc, and the target enzyme were varied in each experiment as specified.

Resul ts and discussion . Figure 14 demonstrates that the GlmU uridylyltransferase reaction can be reversed to synthesize UTP using the coupled assay design shown above. The activity shown is dependent on the presence of both the target substrate and target enzyme in the assay (closed triangles) . In the absence of either target substrate (open triangles) or target enzyme (closed circles) , no light is observed in the coupled assay. Experiment 2 - GlmU uridylyltransferase activity is linear with respect to light production m the coupled assay at

concentrations of enzyme between 0.28 μM and 1.12 μM.

When evaluating enzyme activity m a coupled assay format it is critical that the reaction catalyzed by the target enzyme be the rate-limitmg step m the ultimate production of product by the reporter enzyme system. Therefore, to establish that the reverse reaction of the GlmU uridylyltransferase activity is consistent with this assumption the initial velocity of the luciferase reaction was evaluated with respect to the concentration of GlmU enzyme. Each reaction contained the core components at the

concentrations described above, along with 400 μM UDP-GlcNAc

as the target substrate. The enzyme concentrations evaluated

were 0.28, 0.56, 0.84, and 1.12 μM. The initial velocity was determined by evaluating the slope of each progress curve from 4-64 sec and reported as RLU/sec. This region of the progress curve was chosen since the reaction was clearly linear (R² > 0.99 ) . Table V. Relationship of enzyme concentration with initial velocity.

[E] μM Vi (RLU/sec )

0 . 28 0 . 07

0 . 56 0 . 14

0 . 84 0 . 23

1.12 0.30

Resul ts and Di scussion . Table V shows Vi at each concentration evaluated. Figure 15 demonstrates that the GlmU uridylyltransferase catalyzed reaction is linear at

enzyme concentrations between 0.28-1.12 μM. Therefore

under conditions of the assay within this range, the production of light by the coupled reporter system is linear with respect to the target enzyme. In addition, this experiment demonstrates the sensitivity of this coupled assay system, since even at 280 nM enzyme the activity is well above background.

Experiment 3 - The velocity (v__j_) of the luciferase reaction demonstrates Michaelis-Menton (or saturation) kinetics with respect to the concentration of the target substrate, UDP-GlcNAc. Experiment 2 defined the concentration range in which the reaction catalyzed by the target enzyme is linear with the production of light by the luciferase reporter enzyme system. For inhibitor screening assays it is important that the concentration of target substrate be below K_M, m a range such that activity is linear with respect to substrate concentration. This is important the identification of competitive inhibitors of the target substrate at low concentrations of inhibitor. To define the relationship between v_. and target substrate concentration, the velocity of the luciferase reaction (RLU/sec) was evaluated with respect to differing amounts of the target substrate UDP-GlcNAc. Each reaction contained the core components at the concentrations

described above, along with 0.56 μM purified GlmU protein

as the target enzyme. The UDP-GlcNAc concentrations evaluated were 5, 10, 20, 40, 80, 130, 160, 320, and 520

μM. The initial velocity was determined by evaluating

the slope of each progress curve from 4-64 sec and reported as RLU/sec. This region of the progress curve was chosen since the reaction was clearly linear (R² > 0.99). To generate a broad concentration range, two stocks of UDP-GlcNAc were used. Overlapping data points

were thus generated at 20, 40 and 80 μM substrate. All

data was plotted m Figure 16 and used to generate the curve fit. Table VI. Relationship of substrate concentration with initial velocity.

[S] ,μM

(RLU/sec)

5 0.022

10 0.037

20 0.062

20 0.067

40 0.101

40 0.105

80 0.149

80 0.167

130 0.200

160 0.231

320 0.290

520 0.331

Results and discussion. Table VI shows v at each concentration evaluated. Figure 16 shows that the velocity of the luciferase reaction (defined as RLU/min from 4-64 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, UDP- GlcNAc. Using SigmaPlot curve fit, the above data was fit to a rectangular hyperbola:

y ax b + x

The curve fit reported a = 0.4 RLU/sec and b = 118 μM

with an r² of 0.996. The data is consistent with the target enzyme demonstrating typical Michaelis-Menton kinetics with respect to the target substrate at saturating levels of pyrophosphate (1 mM) with a V_max for the reaction of 0.4 RLU/sec and a K_M of the target

substrate, UDP-GlcNAc, for the target enzyme of 118 μM.

Therefore for the reverse reaction of GlmU uridylyltransferase activity using the coupled assay presented, concentrations of UDP-GlcNAc less than

approximately 40 μM will be within the linear range of

the enzyme activity and can be used to screen inhibitors.

2) Experimental design for group I enzymes

Although the present example is directed towards evaluating the enzymatic activity of Protein kinase A, the format used in the experiment is applicable to any Group I enzyme .

Protein kinases catalyze the transfer of the γ-phosphoryl

group of ATP to an acceptor protein or peptide substrate.

Protein kinase A (also known as cAMP-dependent protein kinase)

is a serine kinase. As such, Protein kinase A transfers the γ-

phosphoryl group from ATP to the hydroxyl group of a serine residue on the protein or peptide substrate. Protein kinase A is an intracellular enzyme containing two domains, a regulatory and a catalytic domain. The regulatory domain is controlled by the binding of cyclic AMP (cAMP) . This enzyme is critical to many signal transduction events in eukaryotic cells and, therefore, is the target of drug development for cancer and inflammatory diseases. Traditionally, assay methods for protein kinases demand the use of radioactively

labeled [γ-³²P]ATP substrate and the evaluation of the

incorporation of the label into a peptide substrate. This is a very slow assay and not easily amenable to high-through put design. Additionally, traditional assays create significant amounts of radioactive waste.

A specific peptide substrate of Protein kinase A has the sequence Leu-Arg-Arg-Ala-Ser-Leu-Gly and is referred to as "kempeptide" (Mailer et al . , Proc. Natl. Acad. Sci. USA 75:248 (1978) ) . Therefore, the protein kinase A reaction using kempeptide as a substrate can be drawn:

PKA LRRASLG + CTP ₄ > LRRA(pS)LG + ADP

Where (pS) respresents a phosphorylated serine residue. Hence for the reverse reaction, LRRA(pS)LG is the target substrate and ADP the cosubstrate. Experiment 1 - Demonstration that the PKA reaction can be reversed to synthesize ATP

When the reaction is run in reverse ATP will be a product and can be quantitated using the luciferase-luciferin reporter system. The concentrations of each of the assay components

were as follows: 130 μM luciferin; 20 μg/ml luciferase; 98 mM

glycine; 163 mM Tris; 9.75 mM MgS0₄ ; 0.975 mM EDTA; 2 mg/ml human serum albumin; 10 units protein kinase A (catalytic

subunit, ICN Biomedicals, Aurora, OH) , and 100 μM ADP.

In the presence of all the above reagents except the peptide substrate, LRRAS(P)LG, the coupled assay reports background activity, Fig. 17, closed circles. This background activity is due to minor ATP contamination of the ADP reagent since when the assay is with all reagents except PKA and peptide substrate the activity is identical (data not shown) . There is a substrate dependent increase in ATP synthesis when 100 uM of the peptide substrate is included in the above assay, Fig. 17, open circles. Table VII shows that the velocity of the PKA reverse reaction (ATP synthesis as reported by the coupled luciferase enzyme is 2.7-fold greater than background levels observed in the absence of the peptide substrate (0.19 vs 0.07, respectively). Table VII. Velocity of the PKA reverse reaction

Sample Initial velocity (RLU/sec)

All reagent, no peptide 0.07 (•)

All reagents, plus peptide 0.19 (O)

All reagents, plus peptide, 0.07 (■) plus PKA inhibitor

Experiment 2 - Demonstration that PKA activity can be inhibited by a PKA specific inhibitor peptide ATP levels are reduced to those observed in the absence of LRRAS(P)LG when a PKA specific peoptide inhibitor (PKI(6- 22) amide, GIBCO-BRL, Rockville, MD, final concentration in

assay = 10 μM) is added to the assay containing peptide

substrate. As shown in both Figure 17 and Table VII, the velocity of the reaction in the presence of inhibitor is identical to that observed when no substrate is added. This experiment also supports the conclusion that the background activity observed is not due to PKA activity and is the result of contamination of the ADP. Resul ts and Discussion . The data presented demonstrate two important concepts: (i) PKA can be driven to synthesize ATP in the presence of the substrates ADP and LRRAS(P)LG when coupled to a luciferase/luciferin reporter assay; and (ii) this activity can be completely inhibited by the addition of a PKA specific inhibitor molecule. Together, this data demonstrates the utility of the present invention as a high- throughput assay for inhibitor screening.

Claims

1. A method of screening for inhibitors or activators of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises,

(i) contacting (a) a target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate with

(b) a compound of the formula X-Nuc-P, wherein X- Nuc-P is a product produced by the target enzyme in a

metabolically forward reaction wherein a nucleoside triphosphate is hydrolyzed and wherein X of X-Nuc-P is a

protein, lipid, carbohydrate, amino acid or other organic molecule having a nucleophilic hydroxyl group and Nuc represents a nucleophilic functionality of X,

(c) NDP, where NDP is a nucleoside diphosphate,

(d) a reporter enzyme system which is thermodynamically favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the metabolically reversed direction, and

(e) a potential inhibitor or activator compound; and (ii) determining whether said target enzyme is inhibited or activated by the potential inhibitor or activator compound.

2. The method of claim 1, wherein said target enzyme is free of exogenous NTP.

3. The method of claim 1, wherein said target enzyme is purified.

4. The method of claim 1, wherein step (ii) comprises comparing said reporter enzyme activity to a baseline activity in the absence of said potential inhibitor.

5. The method of claim 1, wherein said potential inhibitor compound is a potential drug compound.

6. The method of claim 1, wherein the nucleoside triphosphate cosubstrate of said target enzyme is ATP, GTP, UTP or CTP.

7. The method of claim 1, wherein NDP is ADP, UDP, CDP or GDP.

8. The method of claim 1 wherein NDP is ADP.

9. The method of claim 1, wherein NDP is ADP and said reporter enzyme system comprises luciferin and luciferase.

10. The method of claim 1, wherein said reporter enzyme system comprises nucleoside diphosphate kinase, ADP luciferin and luciferase .

11. The method of claim 10 wherein said reporter enzyme system further comprises inorganic pyrophosphotase.

12. The method of claim 1, wherein said target enzyme is a kinase .

13. The method of claim 12, wherein said kinase is selected from the group consisting of protein kinases, lipid kinases, carbohydrate kinases, amino acid kinases and other organic molecule kinases.

14. A method of screening for inhibitors of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises, (i) contacting (a) a target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate with

(b) a compound of the formula X-NDP, wherein X-NDP is a product produced by the target enzyme in a metabolically forward reaction wherein a nucleoside triphosphate is hydrolyzed,

(c) PPi, wherein PPi is inorganic pyrophosphate, (d) a reporter enzyme system which is thermodynamically favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the metabolically reversed direction, and (e) a potential inhibitor or activator compound; and

(ii) determining whether said target enzyme is inhibited or activated by the potential inhibitor or activator compound.

15. The method of claim 14, wherein said target enzyme is free of exogenous NTP .

16. The method of claim 14, wherein said target enzyme is purified.

17. The method of claim 14, wherein step (ii) comprises comparing said reporter enzyme activity to a baseline activity in the absence of said potential inhibitor.

18. The method of claim 14, wherein said potential inhibitor compound is a potential drug compound.

19. The method of claim 14, wherein the nucleoside triphosphate cosubstrate of said target enzyme is ATP, GTP, UTP or CTP.

20. The method of claim 14, wherein NDP of X-NDP is ADP, UDP, CDP or GDP.

21. The method of claim 14, wherein NDP of X-NDP is ADP.

22. The method of claim 14, wherein NDP of X-NDP is ADP and said reporter enzyme system comprises luciferin and luciferase.

23. The method of claim 14, wherein said reporter enzyme system comprises nucleoside diphosphate kinase, ADP, luciferin and luciferase.

24. The method of claim 14, wherein said target enzyme is selected from the group consisting of nucleotide sugar synthetases and nucleotide lipid synthetases.

25. The assay of claim 14, wherein said target enzyme is ADP- heptose synthetase and X-NDP is ADP-D-glycero-D-mannoheptose.

26. The assay of claim 25 wherein the reporter enzyme system comprises luciferin and luciferase.

27. An assay system for enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate comprising, (a) a compound of the formula X-NDP, wherein X-NDP is a product produced by the target enzyme in a metabolically forward reaction wherein a nucleoside triphosphate is hydrolyzed, (b) PPi, wherein PPi is inorganic pyrophosphate, and

(c) a reporter enzyme system which is thermodynamically favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the metabolically reversed direction.

28. The assay system of claim 27, wherein said assay system is for screening for inhibitors of the target enzyme.

29. The assay system of claim 27, wherein said assay system is for screening for activators of the target enzyme.

30. The assay system of claim 27, further comprising a purified target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate in the metabolically forward direction.

31. An assay system for enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate comprising, (a) a target enzyme which hydrolyzes a nucleoside triphosphate as a cosubstrate, (b) PPi, wherein PP is inorganic pyrophosphate, and

(c) a reporter enzyme system which is thermodynamically

favored to hydrolyze nucleoside triphosphates and which forces the target enzyme in the metabolically reversed direction.

32. The assay system of claim 31 further comprising a compound of the formula X-NDP, wherein X-NDP is a product produced by the target enzyme in a metabolically forward reaction wherein a nucleoside triphosphate is hydrolyzed.

33. The assay of claim 31, wherein the nucleoside triphosphate cosubstrate of said target enzyme is ATP, GTP,

UTP or CTP.

34. The assay of claim 32, wherein X-NDP is X-ADP, X-UDP, X- CDP or X-GDP.

35. The assay of claim 32, wherein X-NDP is X-ADP and said reporter enzyme system comprises luciferin and luciferase.

36. The assay of claim 32, wherein said reporter enzyme system comprises nucleoside diphosphate kinase, ADP luciferin and luciferase .

37. The assay of claim 31, wherein said target enzyme is selected from the group consisting of nucleotide-sugar synthetases and nucleotide lipid synthetases.

38. The assay of claim 31, wherein said target enzyme is rfaE and X-NDP is ADP-D-glycero-D-mannoheptose.

39. The assay of claim 38, wherein the reporter enzyme system comprises luciferin and luciferase.