Background to the Invention The ability to screen compounds for inhibitory effects on a particular biological target is an increasingly important requirement in the pharmaceutical industry. The availability of many thousands of compounds and the possibility of synthesising many thousands more by combinatorial synthesis techniques have meant that the screening of these compounds for activity is both costly and time-consuming. For example, in a drug discovery programme, there may be much known about the biological system under study, including the activity of the particular target which needs to be controlled; however, it may be necessary to screen many hundreds, possibly thousands, of compounds to identify one with the requisite level of activity. It is therefore important that the screening techniques used can be carried out efficiently without the necessity for complicated or lengthy procedures.

Many target systems that are desirable to control comprise enzymes. If an enzyme itself is the target there is usually the requirement for its isolation and purification prior to assay in the presence of chemical compounds. In many cases, the enzyme is only one of a number involved in a particular biological pathway. The aim of a drug development programme may ultimately be the controlled inhibition of the pathway achieved through inhibition of any one (or several) of a number of enzymes common to the pathway. Screening for activity in these pathways is difficult, since there are different reactants and products for each enzyme; consequently, it is more

usual to isolate a single enzyme and screen for activity against it, normally using a measurement of product formation as the basis for the screen.

In Webb, Proc. Nat. Acad. Sci. (USA), 1992; 89: 4884- 4887, there is described an assay for the measurement of phosphate using the chromogenic substrate 2-amino-6- mercapto-7-methylaminopurine ribonucleoside (methyl- thioguanosine). Phosphate was found to cleave this nucleoside in a reaction catalysed by purine nucleoside phosphorylase, generating ribose 1-phosphate and the corresponding free base, 2-amino-6-mercapto-7- methylaminopurine (methylthioguanine; MTG). Conversion of the nucleoside to MTG generates a readily monitored spectrophotometric signal at 360 nm. This assay was later extended by Lloyd et al, Nucleic Acids Research, 1995; 23: 2886-2892, to measure inorganic pyrophosphate (PPi) by incorporating inorganic pyrophosphatase in the assay to generate inorganic phosphate (Pi) which can then be assayed as described above.

This assay has been used previously in the measurement of activity of isolated enzymes.

Summary of the Invention According to the present invention, a method for determining an inhibitory effect of a compound on an enzyme, comprises the step of determining the difference in the amount of phosphate released in the absence and presence of the compound; in particular, the assay is conducted when the enzyme is present in a system comprising two or more enzyme-coupled reactions, wherein phosphate is generated as a final product of the system.

According to one aspect of the invention, the method comprises the addition of pyrophosphatase to an enzyme- coupled reaction producing PPi, and measuring the amount of phosphate generated. This can then be used to quantify how much pyrophosphate was generated in the system. The phosphate may be measured by the addition of a substrate that reacts with phosphate to generate a chromophoric

substance. For example, the phosphate-reactive substrate may be 2-amino-6-mercapto-7-methylaminopurine ribonucleoside.

According to a further aspect of the invention, if inhibition is detected, the enzyme that is inhibited in the system may be identified by the separate addition of the substrates for each of the enzymes present in the system and subsequently determining the amount of phosphate generated. If an increase in phosphate is detected, this indicates that the substrate acted downstream of the inhibited enzyme and this therefore helps to identify the particular enzyme that has been inhibited.

The present invention therefore provides a method for assaying different enzymes in a pathway in an efficient manner, whereby inhibition of any one enzyme is reflected in the downstream inhibition of phosphate or PPi production by the final enzyme.

Description of the Drawings Figure 1 illustrates the co-enzyme A biosynthetic pathway and the measurement of phosphate; Figure 2 illustrates the folate biosynthetic pathway and the measurement of the final product, phosphate; and Figure 3 illustrates the shikimate pathway and the measurement of the final product, phosphate.

Description of the Invention A major advantage of the present method is that it allows two or more different enzymes to be screened together thereby reducing the necessity for separate assay screens for each enzyme. Chemicals of interest can then be used in the screens and an inhibition event detected. This allows the easy identification of chemical compounds that may have interesting inhibitory properties. The specific target enzyme may then be identified in later experiments for further study.

The skilled person will appreciate how to couple enzyme reactions to achieve sequential enzymatic reactions resulting in phosphate (or pyrophosphate) release. The

coupled system does not have to be one found in nature, but may be apparent due to a shared product/substrate between two enzymes.

A description of three specific enzyme coupled systems follows, each of which illustrate the invention with reference to the accompanying drawings.

(1) Coenzyme A (CoA) synthesising enzymes: Pantothenate kinase (PtK) and 4-phosphopantetheine adenylyltransferase (PPAT).

The two enzymes PtK and PPAT catalyse non-sequential reactions in the pathway of CoA biogenesis in vivo. The reaction catalysed by PtK involves the transfer of the y- phosphate of ATP to pantothenate, yielding 4- phosphopantothenate. The reaction catalysed by PPAT is the transfer of adenosine monophosphate (AMP) moiety of ATP to 4-phosphopantetheine, generating dephosphocoenzyme A (dCoA) and PPi. Clearly, no product of either enzyme reaction forms the substrate of the other. However, PtK is able to phosphorylate pantetheine (I) (or other suitable analogs or derivatives) as efficiently as pantothenate, yielding 4- phosphopantetheine (II) rather than 4-phosphopantothenate.

4-phosphopantetheine is a substrate for PPAT which, on addition of ATP, produces dCoA (III) and PPi (see Figure 1). Therefore, although the reactions catalysed by PtK and PPAT are not sequential in vivo, the activities of PtK and PPAT can be linked in an assay sequence which culminates in PPi release, the latter may then (optionally) be cleaved to release phosphate.

The production of phosphate or PPi in the final enzyme step may be measured by spectrophotometric means. Any increase in absorbance at 360 nm is dependent on ATP, PtK, PPAT and pantetheine (or suitable analog or derivative) and indicates that the combined activities of PtK and PPAT are being measured.

The addition of an inhibitor of PtK will result in a decrease in the measured production of phosphate due to the lack of substrate production for PPAT. The reduction in

the rate of phosphate production may also indicate an inhibitory effect on PPAT.

The following Example illustrates this system.

Example A reaction was carried out to measure phosphate release from the enzyme-coupled system, in the presence and absence of inhibitors previously shown to be specific for either PtK or PPAT.

The reaction assay comprised 0.02 units of PtK and 0.076 units of PPAT (where 1 unit is sufficient to catalyse 1XM of substrate per minute). The substrate for PtK was the pantetheine derivative S- [3' (N- propyl) succinamidyl] pantetheine (NPS-pantetheine) (60ßM), and ATP was also present in the reaction mix at a concentration of lmM.

Phosphate release was measured using the spectrophotometric assay as disclosed in Webb, supra.

The compound [N-acetyl (O-t-butyl) D-serinyl] -alanyl- 3,3-dimethyl-1-butamine was used as the inhibitor of PtK, and 2,4,3', 5'-tetrachlorobenzanilide was used as the inhibitor of PPAT (both at 50ßm).

Inhibition of PtK: The rate of phosphate release in the absence of inhibitor was 1.35 ßm/min/ml. In the presence of inhibitor the rate was determined as 0.22 ßm/min/ml, giving an effective rate of inhibition of approximately 80%.

Inhibition of PPAT: The rate of phosphate release in the absence of inhibitor was measured as 0.735 Um/min/ml.

In the presence of inhibitor, the rate was 0.500 ßm/min/ml, giving an inhibition rate of approximately 30%.

(2) Folate biosynthetic enzymes: Dihydroneopterin aldolase (DHNA), hydroxymethyl dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS).

The above three enzymes catalyse a naturally-occurring sequence of reactions in the pathway of microbial folate synthesis (illustrated in Figure 2). Dihydroneopterin (IV) is cleaved by DHNA to generate glycoaldehyde (V) and

hydroxymethyldihydroneopterin (VI). HPPK then catalyses the attack of the hydroxyl oxygen of hydroxymethyl dihydropterin on the ß-phosphorous atom of ATP to generate hydroxymethyl dihydropetrin pyrophosphate (VII) and AMP as a byproduct. PPi is then displaced from dihydroxymethyl dihydropterin pyrophosphate by para-aminobenzoic acid (VIII) in a reaction catalysed by DHPS, the final enzyme in the folate biosynthetic sequence, generating dihydropteroate (IX). The PPi generated by this sequence of three enzymes may be measured, for example using the inorganic pyrophosphatase/purine nucleoside pyrophosphorylase/methylthioguanosine detection system to generate an increase in absorbance at 360 nm. The time course of product (PPi) release is linear for at least 10 minutes and is dependent upon the presence of each component of the system.

The addition of the HPPK inhibitor,-y-methylene adenosine triphosphate, decreases the observed rate of the coupled enzyme reaction. As the inhibitor has no effect on the other enzymes (DHNA or DHPS), this illustrates that the coupling of the activities of DHNA, HPPK and DHPS so as to generate PPi (and ultimately phosphate) provides a simple and practical means for simultaneous screening of each and every one of the three enzymes for inhibitors of microbial folate synthesis.

(3) The enzymes: dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase and enoylpyruvoyl shikimate 3-phosphate synthese.

The above enzymes catalyse steps (shown in Figure 3) in the so-called shikimate pathway that results ultimately in the production of enoylpyruvoylshikimate 3-phosphate.

Dehydroquinate (X) is first converted into dehydroshikimate (XI) by dehydroquinate dehydratase.

Shikimate dehydrogenase then converts the dehydroshikimate to Shikimate (XII), and shikimate kinase then catalyses the formation of Shikimate 3-phosphate (XIII). This compound is then catalysed by 5-enolpyruvoyl Shikimate 3-phosphate synthase into 5-enoylpyruvoyl shikimate 3-phosphate (XX), with the additional conversion of phosphoenol pyruvate (IXX) into phosphate.