GB2378246A

GB2378246A - Detection of enzymatically active hydrolases

Info

Publication number: GB2378246A
Application number: GB0211258A
Authority: GB
Inventors: David Buttle; Tim Higgenbottam; Birgit Helm; Alan Spivey; Tin Htut
Original assignee: University of Sheffield
Current assignee: University of Sheffield
Priority date: 2001-05-16
Filing date: 2002-05-16
Publication date: 2003-02-05
Also published as: GB0211258D0; GB0111987D0

Abstract

An assay for assessing the presence or absence of an enzymatically active hydrolase in a sample, particularly an allergenic enzyme such as a proteinase, such as those found in house dust mites which are implicated in allergic reactions, in which the assay involves the use of a hydrolase binding moiety which binds specifically to only the active form of the enzyme. Preferred hydrolase binding moieties for use in the method are substrates of the enzyme under study. Compounds used in the assay and kits containing them are also provided.

Description

Method The present invention relates to methods of detecting enzymatically active hydrolases (preferably proteinases and lipases) using hydrolase binding moieties which selectively bind to active but not inactive forms of said hydrolases, particularly to detect allergenically active hydrolases, particularly derived from house dust mites, in which the methods preferably involve the use of small molecule substrates and leaving groups which provide visually detectable signals. Methods of determining the allergenicity of a sample are also provided.

A great deal of work has been undertaken to increase our understanding of the mechanisms underlying the development of an allergic reaction. Exposure to allergens causes sensitisation and the development of atopic diseases including asthma. The atopic disease is multifactorial with a major genetic factor, but there is evidence that non-atopic persons can also become sensitised if the insult is continuous as may occur in occupational exposures.

Common sources of allergens related to asthma are derived from indoor sources such as household pets, cockroaches, house dust mites and allergens from fungal spores such as mould allergens. Outdoor sources such as grass, ragweed or tree pollen allergens and fungal spore allergens may also cause allergic responses which may represent asthma risk factors.

The house dust mite (HDM) is one of the most important indoor allergens, causing sensitisation and provoking asthma symptoms in > 70% of the 1.5 million children with asthma in the UK. It is therefore a major cause of morbidity in the developed world, and an important medical and economic drain on society, due to

the cost of treatment estimated to be E672 million for the UK national health service in 1995/1996, and millions of lost working days per annum.

There are four species of HDM, which share common allergen groups. Ten groups of allergens have been isolated from HDM, most of which are hydrolases. In common with many HDM allergens, many allergens from other sources are proteinases (e. g. pollen and mould allergens) (Platts-Mills et al. , 1997, J. Allergy Clin.

Immunol. , 100, Sl-24). Of the HDM allergen groups, four are proteinases; the product of the Der p 1 gene is a cysteine proteinase and the products of the Der p 3,6 and 9 genes are serine proteinases. Other HDM allergens include amylases, glucoamylases and molecules that exhibit homology to glutathione S-transferase. Two of the proteinase allergen groups are particularly important and react with anti-mite IgE antibodies in > 80% of sensitised subjects (Platts-Mills, 1997, supra).

One of these is the product of the Der 1 gene.

We have coined the name acaripain for the Der p 1 gene product, due to the taxonomic classification of the house dust mites in the Acariformes superorder, and the known evolutionary relationship of this proteinase to papain. The major allergen from D. farinae, the product of the Der f 1 gene is 84% identical in amino acid sequence and thus is also considered to be an acaripain.

These and other papain-like enzymes are phylogenetically classified as Subfamily A of family Cl in clan CA of the proteinases (Rawlings & Barrett, 1999, Nucl. Acids Res., 27, p325-331).

Acaripain is associated with faecal particles that collect in bedding, carpets, clothes and other materials in infested buildings. The cDNA has been cloned and sequenced (Chua et al. , 1988, J. Exp. Med. , 167, pl75- 182) and a model of acaripain structure has been proposed based on the structure of papain (Topham et al. , 1994, Protein Eng. , 7, p869-894). This model

predicted on the surface of acaripain a highly immunogenic region of overlapping T cell epitope sites.

However, the model incorrectly predicted the substrate specificity of acaripain and no structure for acaripain has been determined.

Proteolytically active allergens are also known to be present in other sources such as an aspartic protease (Bla g 2) and a glutathione transferase (Bla g 5) from cockroach and alkaline and/or vacuolar serine proteinases from mould, e. g. from the genera Pencillium or Aspergillus. Other allergens with enzyme activity, such as hydrolase activity are also known to exist, notably lipases (such as bee venom phospholipase A2) and other hydrolases in parasitic secretions.

Stimulation of allergic sensitisation to HDM, occurring by an unknown mechanism, is required for the development of symptoms and appears to be crucially dependent on the enzymatic activity of the offending allergen (Machado et al. , 1996, Eur. J. Immunol. , 26, p2972-2980 ; Gough et al. , 1999, J. Exp. Med. , 190, pl897-1902 ; King et al. , 1998, J. Immunol., 161, p3645- 3651 and Dudler et al. , 1995, J. Immunol. , 155, p2605- 2613). Similarly, the enzymatic activity of bee venom phospholipase A2 is linked to its efficacy as an allergen (Dudler et al., 1995, J. Immunol. , 155, p2605-2613).

Primary sensitisation is followed by the production of allergen-specific IgEs, which attach to the receptors of mast cells in skin and mucous membranes and trigger an acute or primary allergic response when the sensitised person is challenged with the allergen. This results in the degranulation of mast cells releasing histamine and other chemical mediators of the inflammatory response such as the pro-inflammatory cytokines IL-4 and IL-13, which have a central role in the induction of IgE response, and IL-6 which further enhances the IgE response induced by IL-4 (Helm, 1994, Adv. Exp. Med. Biol., 347, pl-10). These in turn

produce cellular and humoral responses resulting in a secondary allergic reaction. With subsequent exposures, a chain-reaction develops leading to a chronic inflammation and eventually, the atopic disease.

Acaripain has the ability, through its enzymatic activity to modify the profile of type 2 responses. It has been proposed that the enzymatic action of acaripain also enhanced the sensitisation process and the induction of IgE responses by increasing mucosal permeability and mast cell degranulation. The proteolytic activity of acaripain has been implicated as a major contributor to its allergenicity (Gough et al., 1999, supra and King et al. , 1998, supra). Similar hypotheses have been put forward in respect of the allergenic lipases (Machado et a !., 1996, supra and Dudler et al., supra).

There are traditionally three methods of estimating exposure to HDM: mite counts, immunoassay for mite allergens and measurement of guanine in dust samples.

Immunologically determined levels of acaripain have been used to quantify HDM exposure in the UK. The measurements are made by ELISA, but are costly and timeconsuming and measure the amount of acaripain as protein. The assay can not discriminate between active and inactive enzyme as this usually depends on the oxidation state of the active site thiol rather than alterations in the epitopes identified by the immunoassays.

Thus existing techniques fail to take into account the level of the allergenic hydrolase which is present but is inactive thus providing results which do not accurately reflect the allergenic potential of a sample.

Furthermore, prior art methods are not sufficiently safe and easy to use by non-technical people without access to appropriate equipment and/or expertise. Thus rapid and easy techniques to allow for example allergy sufferers to monitor their environment for allergenic

hydrolases without recourse to laboratory testing remains an unmet need in the art.

Surprisingly it has now been found that enzymatically active hydrolases, particularly proteinases, e. g. papain-like enzymes such as some of the allergens from house dust mites, lipases (preferably phospholipases) and amylases can be assessed by a simple and convenient assay. In particular, the level of allergenic house dust mite antigens can be determined by assessing the levels of enzymatically active proteinase antigens which are present in a sample, e. g. a dust sample, by using reagents which specifically and selectively bind only to the active form of the proteinase. Similarly, the level of other allergenic hydrolases, e. g. lipases may be detected.

Further, convenient means for putting this assay and similar assays into effect have been found in which the enzymatic function of the enzyme in question is used to cleave and thereby release a chromogenic product which may be assessed visually thus providing an assay which may be readily performed by the non-skilled person, conveniently in their own home, without the use of expensive or technically demanding equipment. Small molecule substrates which may be used in the assay of the invention or for other purposes such as to form inhibitors or for binding and optional retrieval of the enzymes to which they bind are also provided.

Thus, in a first aspect the present invention provides an assay for assessing the presence or absence of an enzymatically active hydrolase, preferably an allergenic hydrolase, especially preferably a proteinase or lipase, in a sample, comprising at least the steps of: a) contacting said sample with a hydrolase binding moiety which specifically and selectively binds to the active form but does not bind to the inactive form of said hydrolase, wherein said moiety preferably contains

or comprises a reporter means; and b) assessing the presence or absence of said hydrolase by determining the extent of binding of said hydrolase binding moiety to an enzymatically active hydrolase in said sample, preferably by determining the presence or absence of the signal present on, or generated by, said reporter means in which case said method may include the step of generating a signal from said reporter means.

Alternatively stated, the present invention provides a method for determining, or a method of determining the allergenicity of a sample (or for assessing the amount'of HDM) by determining the presence or absence of an enzymatically active allergenic hydrolase, preferably as described above.

The term"assessing"as used herein includes both quantitation in the sense of obtaining an absolute value for the presence, e. g. level of an (allergenic) hydrolase in a sample, and also obtaining a semiquantitative or other indication, e. g. an index or ratio, of the amount of said hydrolase in the sample under study. Assessment may also involve the generation of further molecules for detection, for example by generating a signal from the reporter means.

As used herein"presence"refers to the existence or level or amount of hydrolase in said sample as determined relative to background levels."Absence" refers to a failure to detect the presence of said hydrolase relative to background levels under the conditions used in the assay.

An"enzymatically active"hydrolase refers to an enzyme which exhibit hydrolytic activity on a natural substrate of said enzyme, or on a small molecule substrate. In the case of for example Der p 1 (acaripain), active enzymes are considered to be those which catalyze the cleavage of Val-Ala-Leu-Ser at a

kc/Km 2 1 1 104 M-Is', preferably : > 3 x 10"Ms under

the conditions described by Billson et al., 1998, Bioorganic and Medical Chemistry Letters, 8, p993-998.

A"small molecule substrate"refers to a moiety recognized by, ie. capable of binding to and being cleaved by, said enzyme. Whilst such small molecule substrates may take a variety of forms, preferably such substrates are peptides derived from one of the natural substrates, e. g. constitutes a portion of a natural substrate which comprises the active site binding region and a site of cleavage. As is usual in the art however, the small molecule substrate may be modified relative to the naturally occurring sequence, e. g. to improve binding providing binding to the enzyme and cleavage by that enzyme is still possible.

As used herein"allergenic"refers to the ability of the molecule in question, when present at appropriate levels, to induce an allergic response in a host after presentation of said allergen, e. g. as manifest by stimulation of IgE production or mast cell degranulation above established threshold levels (e. g. as detected by histamine release, tryptase release or production of IL- 4 in analogous experiments to those conducted by Machado et al. , 1996, supra). Whilst not wishing to be bound by theory, it is believed that allergenic hydrolases may bring about allergenicity by IgE-independent activation of the IgE receptor, e. g. through proteolytic cleavage or damage to the phospholipid layer.

As used herein a"hydrolase"is an enzyme which catalyzes the addition or removal of a water molecule.

Such hydrolases include proteinases, lipases and amylases.

As used herein a"proteinase"is a hydrolase capable of cleaving the amide bond (ie. a peptide bond) between two amino acids and is classified according to its activity and functional group (s). Preferably proteinases assessed according to the assay of the invention are cysteine, aspartic, serine or metallo

proteinases with specificity for a defined stretch of amino acids as substrate. Especially preferably, the proteinase is a cysteine proteinase such as the Dermatophagoides spp. Group 1 proteins.

The proteinases which may be identified are preferably allergenic proteinases and include allergens which have been identified in house dust mites and other insects, or in mammals, plants, e. g. pollen or fungi, e. g. in Penicillium or Aspergillus. In a preferred feature however the proteinases are derived from house dust mites, particularly from Dermatophagoides ssp. (for example Dermatophagoides pteronyssinus and

Dermatophagoides farinae), Euroglyphus maynei, Blomia tropicalis, Lepidoglyphus destructor and Tyrophagous putrescentiae. Preferably the proteinases are derived from Dermatophagoides ssp. , particularly preferably the Group 1, Group 3, Group 6 and Group 9 allergens. In an especially preferred feature the allergens to be identified are Group 1 allergens, such as Der p 1 (acaripain). As described herein, such allergens include variants which exist within species, e. g. polymorphic variants (e. g. as described in Robinson et al. , 1997, Clin. Exp. Allergy, 27, plO-21), or in other species as well as naturally or non-naturally occurring functional derivatives thereof, ie. derivatives which exhibit the same enzymatic and allergenic functions, e. g. genetically modified molecules.

As mentioned above, the invention extends to the assessment of the presence or absence of non-allergenic proteinases. In this respect, the assessment of proteinases which are papain-like enzymes and are phylogenetically classified as Subfamily A of family Cl in clan CA of the proteinases is preferred.

As referred to herein a"lipase"is an enzyme which hydrolyses fatty acid esters. Particularly preferred are phospholipases, which are divided into phospholipases Al, A2, C and D dependent on their

preferred cleavage sites, which are capable of cleaving phosphatidyl inositol 4,5-biphosphate to give rise to specific products dependent on the cleavage site of the phospholipase.

"Amylases"as referred to herein are enzymes which hydrolyze starches or glycogen and include a-and amylase and glucoamylase.

As defined herein, said"sample"refers to any sample derived from the local environment in which said hydrolases might be present. Thus for example in the case of allergenic hydrolases from organisms (e. g. house dust mites), said sample is derived from any solid, gas or liquid with which said organisms or parts or produce (e. g. waste products such as faeces) thereof have had contact and may thus have been contaminated, e. g. an environmentally derived sample such as a solid, e. g. dust or material, air or liquid sample which may preferably be assayed directly or used to prepare a sample for assay. Allergenic hydrolases may also be found in other sources such as products containing the naturally occurring form of that hydrolase or a synthetic copy thereof, e. g. products containing all or part of the source organism, e. g. biological washing powders or meat tenderisers. Preferably however, the sample is environmentally derived or is a manufactured product and comprises an untreated sample of air, liquid or solid, e. g. dust, especially preferably however, said sample is prepared by dissolution or distribution of said environmental sample or product in a solution and is preferably filtered prior to use.

As used herein"contacting"refers to providing suitable contact between the hydrolases in the sample and the hydrolase binding moiety to achieve selective and specific binding thereto. Where necessary, said contacting conditions also provide a suitable environment for cleavage of a scissile bond in the hydrolase binding moiety, e. g. to release a leaving

group.

As used herein said"hydrolase binding moiety"is any entity which is capable of specific and selective binding to the active form of the hydrolase, relative to binding to the inactive form of that enzyme. In a preferred feature, the hydrolase binding moiety binds to the active site of said hydrolase, e. g. is an inhibitor or substrate of said enzyme, although moieties which bind to the remainder of the molecule or only a portion of the active site are also contemplated. The"active site"of the enzyme is the site involved in enzymatic activity, e. g. proteinase activity, under study and provides the critical spatial and charge configuration necessary for the activity by virtue of the arrangement of several amino acid residues.

As used herein"specific"binding refers to association with the target hydrolase at levels significantly above background levels, preferably providing a signal: noise ratio of at least 3 under the assay conditions which are used. Preferably however, where said binding moiety is a substrate it has a Km < 10-4M, preferably about 10-6M. In the case of reversible inhibitors preferably the Kl is greater than 10-6, e. g.

10-6-10-9. In the case of irreversible inhibitors a rate > 105M-1s-1 is preferred.

"Selective"binding refers to binding which is selective insofar as the binding moiety exhibits a preference for binding to the enzymatically active target hydrolase relative to other hydrolases or molecules in the sample. This is of course required to allow assessment of the levels of target hydrolase alone. Selectivity however, may be achieved by choice of a binding moiety with appropriate specificity used at an appropriate concentration.

"Binding"as used herein refers to the formation of an association between the target hydrolase and the hydrolase binding moiety. Such association may for

example involve interactions such as covalent bonds, hydrogen bonds, van der Waal's interactions, ionic interactions or hydrophobic interactions.

"Determining the extent"of binding of the hydrolase binding moiety to the hydrolase refers to quantitatively, qualitatively or semi-quantitatively assessing how many hydrolase molecules are bound to the hydrolase binding moieties at any time during the assay.

Conveniently and preferably this is achieved using hydrolase binding moieties with reporter means. In an alternative embodiment however, molecules which do not specifically recognize the active site may be used before or after use of the hydrolase binding moieties. e. g. an assay may additionally use other molecules which bind to the hydrolase and which carry a reporter means, e. g. one may perform capture of the target molecules with the hydrolase binding moieties and the molecules which are captured may then be identified with second hydrolase binding moieties (not specific for the active binding site) which carry reporter means.

The"reporter means"as referred to herein is any moiety capable of direct or indirect detection by the generation or presence of a signal. The signal may be any detectable physical characteristic such as conferred by radiation emission, scattering or absorption properties, magnetic properties, or other physical properties such as charge, size or binding properties of existing molecules (e. g. labels) or molecules which may be generated (e. g. gas emission etc. ). Techniques are preferred which allow signal amplification, e. g. which produce multiple signal events from a single active binding site, e. g. by the catalytic action of enzymes to produce multiple detectable products.

Conveniently the reporter means may be a label which itself provides a detectable signal, such as a radiolabel, chemical label, for example chromophores or fluorophores (e. g. dyes such as fluorescein and

rhodamine), or reagents of high electron density such as ferritin, haemocyanin or colloidal gold. In such cases direct detection of the reporter means may be possible.

Preferred labels for use according to the invention are chromophores and fluorophores.

Alternatively indirect detection may be achieved, e. g. by cooperation with or binding of the reporter means to a complimentary agent to produce a detectable effect directly or indirectly, e. g. interaction with an enzyme to produce a signal such as gas evolution, light emission, colour change, turbidity, precipitation, etc.

This interaction may occur directly between the reporter means and the complimentary agent or may be mediated by one or more further molecules, which depends on the nature of the reporter means. For example, the hydrolase binding moiety (optionally together with the hydrolase to which it binds) may comprise the reporter means to which a further molecule is bound which is capable of generating a signal directly or indirectly, e. g. it may be recognized by an affinity binding partner such as an antibody which itself may be labelled or carry a moiety capable of generating a signal (e. g. carrying an enzyme as described above).

Other forms of indirect signal generation are however also contemplated, such as interaction of the reporter means with a biological molecule or chemical agent (e. g. a reducing or oxidizing agent) to alter the form of the reporter means to a readily detectable form.

Such moieties are well known within the field of diagnostic assays.

As mentioned above, the hydrolase binding moiety preferably contains or comprises a reporter means or part of said reporter means. Thus, the reporter means, or part thereof, may comprise all or only a part of the hydrolase binding moiety. When the reporter means comprises all of the hydrolase binding moiety, the signal may be the physical characteristics of that

moiety such as its charge, size or other characteristic which may be detected directly or indirectly. Where the reporter means comprises only a part of the hydrolase binding moiety, conveniently that part is a label (e. g. one or more radiolabels, fluorophores or chromophores) or can generate a signal (e. g. has enzymatic function or may be converted into a detectable form or may be used for association with a complimentary molecule to allow signal generation).

As mentioned above, the hydrolase binding moiety may comprise or contain only a part of the reporter means. In such cases the other part of the reporter means may be provided by one or more further molecules, e. g. the hydrolase binding moiety together with the hydrolase to which it binds may make up the reporter means which provides the site for selective and specific binding of a molecule which allows the generation of a signal.

Depending on the form of the reporter means it may be conjugated post-synthesis to the hydrolase binding portion of the hydrolase binding moiety or may be synthesized at the time of making that moiety.

As used herein"determining the level" of the signal generated refers to quantitatively, qualitatively or semi-quantitatively assessing the amount or presence of signal above background levels. Since this level correlates to the amount of enzymatically active hydrolase bound to the hydrolase binding moiety, this level is indicative of the amount of said enzymatically active hydrolase. In order to obtain qualitative assessment, this may be compared to signal generated in controls and/or standards (developed using the enzyme under study) to assess the presence or absence of the enzymatically active target hydrolase. To provide semiquantitative or quantitative assessment, the signal generated may similarly be assessed relative to controls and/or standards to provide an estimate of the amount of

enzymatically active target hydrolase which is present.

In such cases it is necessary to take into account the form of the hydrolase binding moiety and the means of signal generation to account for amplification effects which particularly may result through the catalytic generation of entities which are directly or indirectly responsible for signal generation.

As used herein,"determining the allergenicity" refers to relating the level of signal and hence target hydrolase to a threshold level or levels considered to be indicative of the likely allergenicity of a sample.

Thus, the level of signal observed for a particular sample is compared to a signal generated in controls and/or standards developed using the enzyme under study wherein the control/standard is quantified in terms of allergenicity attributable to a specific amount of enzyme (using for example the allergenicity tests described hereinbefore). By comparison to the standards/controls, a reading of allergenicity may be obtained. This allows a determination to be made of whether a sample is likely to cause allergenicity in susceptible individuals, ie. if levels over threshold allergenicity causing levels are surpassed.

Generally, for performance of the invention, suitable extracts or source material containing the enzyme of interest is provided in a buffer suitable to maintain its activity. Other sample preparations may however be employed. The enzyme may be provided in purified or semi-purified form. Conveniently the enzyme is provided in solution, although in some embodiments of the invention it may be appropriate to attach the enzyme to a solid support, e. g. via antibodies to the enzyme, providing these are not directed to the active site.

Conveniently, to mimic physiological conditions to which the enzymes would be exposed on contact with an animal body, a reducing agent may be added to convert reversibly inactivated enzymes to their activated

counterparts, where such inactivation occurs through oxidation. That portion of the enzyme population that is irreversibly inactive will of course remain inactive.

Conveniently reducing agents are used at mM concentrations and include for example cysteine base (e. g. at a final concentration of 4mM), dithiothreitol (e. g. at 2mM) or ascorbate.

The enzyme is then contacted with the hydrolase binding moiety (as described herein) which is allowed to bind to the enzyme, e. g. for up to 2 hours.

Conveniently the hydrolase binding moiety is attached to a solid support (e. g. via the group X as described hereinafter).

The hydrolase binding moiety optionally contains or comprises a reporter means or part thereof from which a signal can be generated. Thus for example, where the enzyme has been bound to a solid support, a hydrolase binding moiety may be used which carries a label or from which a signal can be generated. In that case, the entire hydrolase binding moiety may remain associated with the enzyme and after washing the solid support the amount of label or signal associated with the support may be determined. In an alternative embodiment the hydrolase binding moiety which has bound may be eluted from the enzyme by appropriate means, e. g. by use of a reducing agent, and then quantified. In a further alternative, the binding moiety can be cleaved by the action of the enzyme and the signal generated directly or indirectly by the products of cleavage, detected.

Alternatively the hydrolase binding moiety may itself be attached to a solid support to which the enzyme may then be bound. A signal generated through that binding, e. g. by release of a cleavage product which can be detected directly or indirectly can then be assessed.

In a further alternative, the hydrolase binding moiety may be used to selectively capture the active

hydrolases which may then be eluted from said hydrolase binding moiety and the amount of eluted enzyme may be determined by appropriate means, e. g. by use of antibodies or other affinity binding molecules which bind specifically to the hydrolase, but not necessarily to its active site.

The signal thus generated can then be compared to standard and/or control samples generated using the same protocol with standard amounts of the enzyme under study to estimate the level of the active enzyme which is present.

Numerous suitable supports for immobilization of molecules such as peptides, and methods of attaching them, are well known in the art and widely described in the literature. Thus for example, supports in the form of sheets, gels, filters, membranes, microfibre strips, plates, microtitre wells, tubes, dipsticks, particles, fibres or capillaries may be used, made of a polymeric material for example of agarose, cellulose, alginate, teflon, latex or polystyrene.

The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde, epoxide or amino groups for the attachment of the moiety to capture the enzyme or to attach the hydrolase binding moiety. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, eg. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups. US patent No. 4,654, 267 describes the introduction of many such surface coatings.

Alternatively, the support may carry other moieties for attachment, such as avidin or streptavidin, DNA binding proteins or antibodies or antibody fragments.

In a preferred embodiment an extract of the source

of the antigen (e. g. a HDM extract) is prepared, (e. g. as described by Machado et al. , 1996, supra) or more preferably an environmentally derived sample, e. g. dust, is solubilized in an appropriate buffer, e. g. PBS, and then filtered. The filtrate is contacted with the hydrolase binding moiety. Preferably this moiety takes the form of a small molecule substrate as described in formula I', especially preferably formula I, attached to, or comprising, a reporter means. Preferably that reporter means is as defined in formula II.

Conveniently this substrate is attached to a solid support (e. g. absorbent paper) and the enzyme then becomes bound to the small molecule substrate and cleaves the amide (or ester) bond releasing the reporter means. Depending on the form of the reporter means, the signal can be developed by appropriate means and the signal generated interpreted as before.

Preferably the hydrolase binding moiety is a substrate or inhibitor which binds to the active site of the target hydrolase. Preferably however the use of substrates is preferred in which more than one binding moiety may be bound during any single incubation step of the assay due to cyclic steps of binding, cleavage and release of said moieties.

Clearly the choice of molecule binding to the active site will depend on the active site of the enzyme to be detected. Preferably however, the inhibitor or substrate comprises a portion (e. g. a peptide portion in the case of a proteinase) which corresponds to or mimics the natural substrate, in which the portion may comprise naturally or non-naturally occurring components (e. g. amino acids in the case of peptides), providing they retain the appropriate charge and shape for binding, and where necessary, cleavage. In a preferred feature, (particularly preferably when the proteinase under study is a papain-like enzyme, e. g. Dermatophagoides ssp.

Group 1 proteins such as Der p 1 (acaripain) ), the

substrate/inhibitor has the general formula I: X-Y-Y-Yi-Z (I) wherein X is an N-terminal group such as one or more hydrogen atoms, an amino acid residue, peptide or a linker or blocking group or derivatives thereof; Y are amino acid residues each of which may be the same or different; and Z is a C-terminal group such as a hydroxyl group or an amino acid residue, peptide or a linker or a blocking group or derivatives thereof, preferably comprising all or part of a reporter means, especially preferably a leaving group containing or comprising all or part of a reporter means.

When the compounds of formula I do not act as substrates, e. g. are inhibitors, Z is not a leaving group and on binding remains associated with the target proteinase, ie. the bond between Y1 and Z is not scissile. However, in a preferred feature compounds of formula I are substrates and Z represents a leaving group containing or comprising all or part of a reporter means. Preferably such leaving groups are as described hereinbelow.

As referred to herein amino acid residues which appear in formula I are in the form in which they would appear if contained within a peptide, ie. lacking one or more N-terminal hydrogen atoms and C-terminal hydroxyl groups. However, where the residue appears at the end of a molecule of formula I, the residue is completed to provide the required hydroxyl group or hydrogen atoms.

Where reference is made to particular residues, e. g.

glycine, the atoms/groups appearing at the N or Cterminal ends depend on the position of the residue in formula I.

The amino acid residues or peptides of X and Z, where present, may be or comprise any amino acids which do not adversely affect binding to the active site to the extent that selective and specific binding is no longer achieved. Thus modifications may be contemplated such as the use of non-naturally occurring amino acids, e. g. amino acids which have been derivatized or chemically modified, e. g. by the introduction of blocking groups or labels. Thus, X and/or Z may comprise an amino acid residue or peptide which is blocked or protected or which has a linker attached to it. Alternatively X and/or Z may simply comprise a blocking or protecting group.

X and Z includes derivatives of the hydroxyl group, amino acid residue, peptide, linker or blocking group.

Such derivatives include non-naturally occurring amino acids as described above, in particular derivatized to include all or part of a reporter means or a leaving group containing or comprising the same. Thus Z may comprise a derivatized hydroxyl group which is a leaving group (which in effect corresponds to a leaving group attached to Y1) or a modified amino acid or peptide (which has an attached reporter group) which may act as a leaving group as described in more detail hereinafter.

Where X is or comprises an N-terminal protecting group this may be an acyl group having 1-20 carbon atoms, e. g. a lower alkanol group having 1-5 carbon atoms such as an acetyl group, or an aroyl or aralkanoyl group having 7 to 20 carbon atoms such as a benzoyl or phenylacetyl group. Such groups may be added during or post-synthesis. Alternatively, amino protecting groups may be added during synthesis and several suitable protecting groups are known and exemplified in the art, e. g. in Schrder, E. , and Lbke, K. , The Peptides, Vols.

1 and 2, Academic Press, New York and London, 1965 and 1966; Pettit, G. R. , Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970,1971, 1975 and 1976; Houben-Weyl, Methoden der Organischen Chemie, Synthese von Peptiden, Band 15, Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides, Analysis, Synthesis, Biology 1- 7; Ed: Erhard Gross, Johannes Meienhofer, Academic Press, NY, San Francisco, London; Solid phase peptide synthesis 2nd ed. , John M. Stewart, Janis D. Young, Pierce Chemical Company.

Thus, for example amino protecting groups which may be employed include protecting groups such as carbobenzoxy (Z-), t-butoxycarbonyl (Boc-), 4-methoxy- 2,3, 6-trimethylbenzene sulphonyl (Mtr-), 9fluorenylmethoxycarbonyl (Fmoc-), acetyl, benzoyl and tosyl.

Appropriate carboxyl protecting groups include benzyl (-OBZl), p-nitrobenzyl (-ONB) and t-butyl (-tOBu) groups. Side-chain protecting groups comprise further derivatized forms of the amino acids which may be used to produce the hydrolase binding moiety, which particularly may be introduced during synthesis of the molecules.

Similarly the amino acids of Yl-3 may be derivatized or modified e. g. non-natural amino acids or analogs thereof (e. g. comprising a label or all or part of a reporter means) providing this does not prevent binding, and where necessary cleavage to release the leaving group.

The group X or Z may consist of or comprise a linker group, e. g. to facilitate binding to a solid support. Such linker groups may be for example a polyethylene glycol or aminohexanoyl group.

Peptides as described above for use in accordance with the invention may be prepared by conventional modes of synthesis including genetic or chemical means.

Chemical synthesis may be performed by methods well

known in the art involving cyclic sets of reactions of selective deprotection of the functional groups of a terminal amino acid (e. g. using the protecting groups described above) and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups. Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art and as described above.

The identity of T-Yi (optionally including surrounding amino acids) will depend on the specificity of the hydrolase to be determined. Where appropriate, substrates/inhibitors containing sequences which bind to multiple enzymes may be used, for example a substrate may be based on casein or azocasein which may be cleaved by a variety of proteinases.

In a preferred feature, for binding to Der p 1 (acaripain), Y3 is any amino acid, Y2 (P2 when the binding moiety is a substrate) is an aliphatic

hydrophobic residue and Y1 (Pi when the binding moiety is a substrate) is a charged residue. Preferably Y1 is selected from the group consisting of: Arginine, Leucine, Asparagine and Glycine, especially preferably Arginine. Preferably Y2 is selected from the group consisting of: Alanine, Valine, Serine, Leucine, Tyrosine and Glycine, especially preferably Alanine.

Preferably Y3-Y2-Y1 is selected from the group consisting of: Gln-Ala-Arg Phe-Val-Arg Asp-Ser-Leu Leu-Leu-Asn Trp-Tyr-Gly Ala-Ala-Leu Val-Gly-Gly Z as described above may comprise a hydroxyl group

(which comprises the hydroxyl group of the amino acid residue Y1), an amino acid residue or a peptide or a linker or blocking group or derivatized versions thereof. Such amino acid residues or peptides may themselves comprise all or part of the reporter means, where present, or these amino acids or peptides or the hydroxyl group at this position may be derivatized to provide all or part of the reporter means. Particularly preferred derivatives include those which comprise reporter means which are not based on a peptide structure, ie. a derivatized hydroxyl group. Z is preferably an appropriate leaving group depending on the enzyme under study. Preferably Z is attached by a scissile bond and thus Z is a leaving group which is released by the activity of the target proteinase, ie. is thus an amide or ester bond. This leaving group, where present, contains or comprises all or part of a reporter means.

Examples of irreversible inhibitors which may be derivatized for use as described herein are disclosed in Billson et al., 1998, supra.

When the hydrolase under study is not a proteinase, appropriate substrates or inhibitors may be developed based on the above mentioned considerations, but taking into account the particular enzyme under study. Thus in general terms the substrate or inhibitor has the formula I' : X'Y'-Z'in which X'Y' (which corresponds to X-Y3-Y2-

Y1 when the hydrolase to be detected is a proteinase) binds to the active site of the enzyme and Z'which may also bind to the active site, is preferably attached to X'Y'via a scissile bond and preferably comprises all or part of a reporter means, and especially preferably is a leaving group containing or comprising all or part of a reporter means. In the case of lipases said scissile bond is an ester and Z'comprises a fatty acid (or an analog thereof), to which may be attached a reporter means. In the case of amylases, said scissile bond is a

glycosidic linkage (preferably an a-1, 4-linkage), and Z' and X'Y'comprise saccharides (or analogs thereof) at their terminal ends which through covalent linkage form the scissile bond.

Especially preferably, a substrate is used which the hydrolase cleaves to generate a signal (directly or indirectly). In this instance the portion which is released constitutes the leaving group. Thus for example, the reporter means may be released on cleavage which then may be used to generate a signal directly or indirectly. Alternatively, cleavage may allow the generation of a signal by release of a part of the reporter means such as in instances in which signal generation is inhibited or impaired by the presence of a component which can be released on cleavage, e. g. quench fluorescence. Suitable quench fluorescence pairs for inclusion in said hydrolase binding moiety (e. g. by derivatization of an amino acid side chain) include oaminobenzoyl (Abz) and nitrophenol or a 2,4dinitrophenyl group or 3-nitrotyrosine, tryptophan and dansyl or a 2,4-dinitrophenyl group, and 7methoxycoumarin derivatives and a 2,4-dinitrophenyl group (see Knight, 1995, Methods Enzymol. , 248, pl8-34).

In a preferred feature however, the reporter means is released on cleavage.

Particularly preferably, said leaving group comprises, or preferably consists of, a reporter means which may be detected by its fluorometric, colorimetric or spectrophotometric properties. Preferably attachment of said leaving group is by an amide or ester bond which can hence be cleaved by a proteinase.

Suitable fluorometric groups include those in the art and in particular 7-amino-4-methylcoumarin (AMC), 3cyano-7-hydroxycoumarin, fluorescein, 5- (pentafluorobenzoylamino) fluorescein, 6-aminonapthalenesulfonamides and Rhodamine 110.

Spectrophotometrically detectable leaving groups include

nitroaniline leaving groups which can be assayed at Azol Thus preferred reporter means are those which produce a signal which is electromagnetic radiation with a wavelength of 200 to 800nm, preferably 490 to 700nm, especially preferably 500 to 650nm ie. preferably produces visible radiation, ie. is or forms a colorimetric or chromogenic compound. Such reporter means may for example use chromophores such as rhodamine 110 which is essentially colourless in its bis-amide form (ie. when conjugated to a peptide via both its amino functions) but is liberated as an intense blue-

green leaving group detectable at Asoo-Other suitable visibly detectable compounds include 7-hydroxy-9H- (1, 3- dichloro-9, 9-dimethylacridin-2-one, resorufin, indigotin disulfonate sodium and indigoidin (Molecular Probes Europe BV, Leiden, The Netherlands). Especially preferred however is a leaving group such as a 3aminoindolyl, 3-aminobenzo[b]furyl or 3- aminobenzo[b]thienyl group. On cleavage, this releases

a 3-aminoindole (or 3-aminobenzo[b]furan or 3aminobenzo[b] thiophene) which is readily oxidatively dimerized to give an intense colour, e. g. in the case of dimerized 3-aminoindole an indigo-purple dye.

Thus in a preferred feature the leaving group has the formula II:

wherein X is a sulfur or oxygen atom or the group NR4 ; Rl which may be the same or different represents a hydrogen atom or a lower alkyl group, e. g. Cl, an aryl group or preferably an electron withdrawing group such as a nitro group or a halogen such as a

fluoride, chloride, bromide or iodide atom; n is # 1, preferably 1, 2 or 3; R2 is the group -NRs or -0 ; R3 is a hydrogen atom or an optionally substituted alkyl group, preferably an alkyl group substituted with a heteroaromatic aryl group which itself may be substituted, especially preferably pyridin-4- ylmethyl or N-methylpyridinium-4-ylmethyl ; R4 is one or more hydrogen atoms or a lower alkyl, e. g. Cl-6, aryl, amido or cyano group, preferably a hydrogen atom; and Rs is a hydrogen atom or a lower alkyl, e. g. C-, aryl, amido or cyano group, preferably a hydrogen atom.

In a preferred feature, X is the group NR4, R2 is the group-NRg, n = 1 and R3, R4 and R5 are each a hydrogen atom.

As used herein, the term "alkyl" includes any long or short chain, straight-chained, branched or cyclic aliphatic saturated or unsaturated hydrocarbon group optionally mono or poly substituted by hydroxy, alkoxy, acyloxy, nitro, alkoxycarbonyloxy, amino, aryl, oxo or halo groups. Thus unsaturated alkyl groups may be monoor polyunsaturated and include both alkenyl and alkynyl groups. Such groups may contain up to 10 carbons but are preferably C. g. The term"acyi"as used herein includes both carboxylate and carbonate groups.

Preferred aryl groups include phenyl and monocyclic 5-7 membered heteroaromatics, especially phenyl and such groups may themselves optionally be substituted.

Especially preferably leaving groups of formula I are indolyl groups, e. g. a 3-amino-4-chloro-5-bromo- indo-3-yl group.

Compounds as described above may be synthesized by known methods.

On cleavage, the above 3-aminoindolyl (or 3- aminobenzo[b]furyl or 3-aminobenzo[b]thienyl group) is

released from the enzyme as a 3-aminoindole (or 3- aminobenzo[b]furan or 3-aminobenzo[b] thiophene group).

In order to generate a chromogenic substrate, this 3aminoindole is dimerized by oxidation. This oxidation is achieved by use of an oxidising agent. Whilst any appropriate oxidizing agent may be used, conveniently said oxidising agent is K3FeCN6, H202, iodoacetate, household bleach or nitroblue tetrazolium. To produce a stable colour, an acidic polymer capable of stabilizing the cleaved product can be used, e. g. may be incorporated in the solid support if one is used.

Suitable compounds include compounds such as methylvinyl ether and maleic acid.

The amount of the chromogen which is formed can then be assessed to provide an indication of the amount of the indole that has been formed and thus an indication of the amount of target, active, enzyme that is present.

Molecules comprising the formula X-Y-Y-Yi-Z as described above, in which Z is a C-terminal group which is attached via a non-scissile bond may also be used for binding to target proteinases, particularly papain-like enzymes which are phylogenetically classified as Subfamily A of family Cl in clan CA of the proteinases, e. g. in their purification and thus form a further aspect of the invention, as do proteins isolated using affinity purification in this way.

In addition, molecules comprising the formula X-Y3- YZ-Y1-Z as described above, in which Z is a C-terminal group which is attached via a scissile bond may also be used for binding to target proteinases as described above to perform the assay as described herein.

Furthermore, leaving groups as described above in formula II wherein the free bond of the R2 NRg-group is replaced with a hydrogen atom to form an amino group, or R2 is removed or replaced in its entirety (e. g. with oxygen or sulfur analogs to the NRg-group, e. g. a

hydroxyl group) form further aspects of the invention as do molecules in which such compounds are attached as leaving groups which may be released by the action of a variety of enzymes, including naturally occurring or non-naturally occurring proteinases, esterases, lipases (particularly allergenic lipases such as Phospholipase A2) etc. when attached to appropriate substrates via appropriate R2 groups.

In a particularly preferred feature the present invention provides an assay for assessing the presence or absence of an enzymatically active hydrolase, preferably a proteinase, especially preferably Der p 1 (acaripain), in a sample, comprising at least the steps of: a) contacting said sample with a hydrolase binding moiety (preferably a proteinase binding moiety, especially preferably a Der p 1 binding moiety) which specifically and selectively binds to the active form but does not bind to the inactive form of said hydrolase, wherein said hydrolase binding moiety has the general formula I' (preferably general formula I) as described above in which Z'is a leaving group containing all or part of a reporter means ; b) allowing said enzyme to cleave said leaving group from said hydrolase binding moiety ; c) determining the presence or absence of the signal present on, or generated by, said reporter means to assess the presence or absence of said hydrolase (preferably said proteinase, especially preferably Der p

1).

Especially preferably said leaving group has the general formula II as defined above and after cleavage of said leaving group at least the following steps are performed: c) effecting oxidative dimerization of said leaving group to produce a chromogenic substance ; and

d) determining the presence or amount of said chromogenic substance to assess the presence or absence of said hydrolase.

The present invention also extends to kits for performing the methods of the invention, comprising at least the following :a hydrolase binding moiety as defined hereinbefore.

Preferably, said hydrolase binding moiety may be provided on a solid support or have means for immobilization or alternatively a solid support suitable for immobilizing the enzyme of interest may be provided.

Additionally, one or more appropriate buffers or solutions optionally containing one or more compounds or agents of use in the assay of the invention, e. g. a solution containing an oxidizing agent and/or a solution containing a reducing agent and/or a solution containing a stabilizing compound (such as an acidic polymer), and/or standard or control samples and/or a package insert describing how the method should be performed, optionally providing standard graphs for interpretation of results obtained when performing the invention, may be provided.

The following Examples are given by way of illustration.: Example 1: Assay to detect Der p 1 (acaripain) activity A HDM extract or environmentally derived sample, e. g. dust is solubilized in an appropriate buffer, e. g. PBS, and then filtered. Conveniently a reducing agent is added at mM concentrations to activate reversibly inactivated enzymes. The filtrate is contacted with a solid support (e. g. absorbent paper) to which is attached the proteinase binding moiety, for example the 3-aminoindolyl prochromogen-tagged synthetic peptide substrate. Colour fixative properties are also provided

on the solid support by pre-coating the solid support with methylvinyl ether and maleic acid (commercially available as Gantrez-AN-119, GAF Corporation, New York, USA). The solid support is then dipped into a solution of oxidant which has the duel effect of immediately inactivating the enzyme via oxidation of the active site thiol and dimerisation of the 3-aminoindole derivative.

Alternatively, if the prochromogen which is released by enzyme activity does not remain associated with the solid support, e. g. by hydrophobic interactions, the solid support is optionally removed from solution and oxidant is added to that solution. The intensity of the resultant colouration is then compared with a chart of standards prepared using known quantities of the dimerized indigo-type dye standardised in terms of the amount of enzymatic activity required to generate them.

Claims

Claims: 1. An assay for assessing the presence or absence of an enzymatically active hydrolase in a sample, comprising at least the steps of: a) contacting said sample with a hydrolase binding moiety which specifically and selectively binds to the active form but does not bind to the inactive form of said hydrolase ; and b) assessing the presence or absence of said hydrolase by determining the extent of binding of said hydrolase binding moiety to an enzymatically active hydrolase in said sample.
2. An assay as claimed in claim 1 wherein said hydrolase is an allergenic hydrolase, preferably a proteinase or lipase.
3. An assay as claimed in claim 1 or 2 wherein said proteinase is derived from a house dust mite.
4. An assay as claimed in claim 3 wherein said house dust mite is Dermatophagoides ssp. and said proteinase is preferably a Group 1 allergen.
5. An assay as claimed in claim 4 wherein said Group 1 allergen is Der p 1 (acaripain).
6. An assay as claimed in any one of claims 1 to 5 wherein said hydrolase binding moiety contains or comprises a reporter means or part thereof and the extent of binding of said moiety is assessed by determining the presence or absence of the signal present on, or generated by, said reporter means.
7. An assay as claimed in any one of claims 1 to 6 for determining the allergenicity of a sample.

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8. An assay as claimed in any one of claims 1 to 7 wherein said hydrolase binding moiety is a substrate or inhibitor of said target hydrolase.
9. An assay as claimed in claim 8 wherein said substrate or inhibitor has the formula I' : X'Y'-Z' (I') wherein X'Y'is a moiety which binds to the active site of the hydrolase; and Z'is a moiety attached to X'Y'preferably via a scissile bond and preferably comprises all or part of a reporter means.
10. An assay as claimed in claim 8 or 9 wherein said hydrolase is a proteinase and said hydrolase binding moiety is a small molecule substrate or inhibitor which has the general formula I: X-Yg-Y-Yi-Z (I) wherein X is an N-terminal group such as one or more hydrogen atoms, an amino acid residue, peptide or a linker or blocking group or derivatives thereof; Yl-3 are amino acid residues each of which may be the same or different; and Z is a C-terminal group such as a hydroxyl group or an amino acid residue, peptide or a linker or a blocking group or derivatives thereof, preferably comprising all or part of a reporter means.
11. An assay as claimed in claim 10 wherein X is a

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blocking group, preferably selected from carbobenzoxy (Z-), t-butoxycarbonyl (Boc-), 4-methoxy-2,3, 6trimethylbenzene sulphonyl (Mtr-), 9fluorenylmethoxycarbonyl (Fmoc-), acetyl, benzoyl and tosyl.
12. An assay as claimed in claim 10 or 11 wherein said hydrolase is Der p 1 and Y3 is any amino acid, Y2 is an aliphatic hydrophobic residue and Y1 is a charged residue.
13. An assay as claimed in claim 12 wherein Y1 is selected from the group consisting of: Arginine, Leucine, Asparagine and Glycine, and Y2 is selected from the group consisting of : Alanine, Valine, Serine, Leucine, Tyrosine and Glycine.
14. An assay as claimed in claim 13 wherein Y3-Y2-Y1 is selected from the group consisting of: G1n-Ala-Arg, Phe-Val-Arg, Asp-Ser-Leu, Leu-Leu-Asn, Trp-Tyr-Gly, Ala-Ala-Leu, and Val-Gly-Gly.
15. An assay as claimed in any one of claims 9 to 14 wherein Z'or Z is a leaving group containing or comprising all or part of a reporter means, and said enzymatically active hydrolase cleaves said leaving group from said hydrolase binding moiety.
16. An assay as claimed in any one of claims 1 to 15 wherein said reporter means is detected by its fluorometric, colorimetric or spectrophotometric properties.

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17. An assay as claimed in claim 15 or 16 wherein said leaving group has the formula II :

wherein X is a sulfur or oxygen atom or the group NR4 ; Ri which may be the same or different represents a hydrogen atom or a lower alkyl group, an aryl group or preferably an electron withdrawing group such as a nitro group or a halogen such as a fluoride, chloride, bromide or iodide atom; n is 11 preferably 1,2 or 3; R2 is the group-NRs or-0 ; R3 is a hydrogen atom or an optionally substituted alkyl group, preferably an alkyl group substituted with a heteroaromatic aryl group which itself may be substituted, especially preferably pyridin-4- ylmethyl or N-methylpyridinium-4-ylmethyl; R4 is one or more hydrogen atoms or a lower alkyl, amido or cyano group, preferably a hydrogen atom; and R5 is a hydrogen atom or a lower alkyl, aryl, amido or cyano group, preferably a hydrogen atom.
18. As assay as claimed in claim 17 wherein X is the group NR4, R2 is the group-NRg, n = 1 and R3, R4 and Rs are each a hydrogen atom.
19. An assay as claimed in claim 17 or 18 wherein said leaving group is a 3-aminoindolyl, 3-aminobenzo[b]furyl or 3-aminobenzo[b]thieny1 group.

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20. An assay as claimed in any one of claims 15 to 19 wherein after cleavage of said leaving group by said enzymatically active hydrolase at least the following steps are performed: c) effecting oxidative dimerization of said leaving group to produce a chromogenic substance; and d) determining the presence or amount of said chromogenic substance to assess the presence or absence of said hydrolase.
21. An assay as claimed in any one of claims 1 to 20 wherein said hydrolase binding moiety is attached to a solid support.
22. A hydrolase binding moiety as defined in any one of claims 10 to 14 wherein Z is a leaving group as defined in any one of claims 17 to 19, containing or comprising all or part of a reporter means.
23. A kit for performing a method as described in any one of claims 1 to 21, comprising at least the following :- a hydrolase binding moiety as defined in any one of claims 10 to 15 or 17 to 19, preferably provided on a solid support or having means for immobilization.