WO1996006357A1

WO1996006357A1 - Screen for potential therapeutic compounds

Info

Publication number: WO1996006357A1
Application number: PCT/GB1995/001912
Authority: WO
Inventors: Denis Raymond Stanworth; Ian Victor Lewin; Sarita Maman; Valerie Jones
Original assignee: Peptide Therapeutics Limited
Priority date: 1994-08-24
Filing date: 1995-08-14
Publication date: 1996-02-29
Also published as: AU3227495A; GB9417086D0

Abstract

The present invention provides a screen which utilises a direct biochemical interaction between moieties to determine whether or not a potential therapeutic compound (PTC) may be useful as a drug. It will be particularly useful in combinatorial approaches to drug design and testing. The screen of the invention allows determination of the binding ability of a PTC with the three amino acid motif of a decapeptide within the Cε4 region of IgE, or a similar amino acid motif. Any such binding should be indicative of a potential therapeutic effect. The screen comprises: (i) a binding compound which includes a recognition site and a binding site having the sequence of amino acid residues Xaa1-Xaa2-Xaa3; and (ii) a mono or polyclonal wholly, semi- or non-synthetic antibody or fragment thereof (A1) which is able to bind selectively to the binding site; wherein Xaa1 and Xaa3 are the same or different positively charged amino acid residues such as Lys, Arg or His, preferably Lys or Arg, most preferably Lys; and Xaa2 is any other amino acid residue, preferably a neutrally charged amino acid residue, more preferably Thr, Pro, Gly or Ala, most preferably Thr.

Description

Screen for Potential Therapeutic Compounds

The present invention relates to a screen, and a method for screening for potential therapeutic compounds to provide drugs for the treatment of allergies or allergic asthma.

Amino acids and amino acid residues are represented herein by their standard codes as identified by IUPAC-IUB Biochemical Nomenclature Commission and represent D and L amino acids, their analogues or derivatives.

A potential therapeutic compound which may be used effectively in drugs for the treatment of allergies or allergic asthma is represented herein by the abbreviation PTC.

The term PTC used herein includes but is not limited to new chemical entities, known chemical entities, peptides, peptido mimetics, biochemicals and any combination or mixture thereof.

Current methods of screening for PTCs all use animal or human cells as a basis for test systems. These can be split into two main types; (i) in vitro and (ii) in vivo.

The most common types of in vitro screens for detecting asthma PTCs are based on: (i) human mast cells (ii) rat peritoneal mast cells as in WO 90/15878 and (iii) rat basophilic leukaemia cells.

The most common types of in vivo screens for detecting asthma PTCs are based on: (i) a primate Ascaris model (ii) a mouse asthma model. The most common types of in vivo screens for detecting allergy PTCs are based on: (i) a rat Passive Cutaneous Anaphylaxis (PCA) model (ii) a mouse skin sensitivity assay using Trimellitic Anhydride (TMA) and (iii) a rat model which involves active immunisation with allergen and Bordatella pertussis to induce a high level of production of anti-allergen IgE as in WO 90/15878.

There are several problems associated with the known in vi tro screens for detecting PTCs which include: poor reproducibility, cross-species variability, the possibility of false positive results, time-consumption and cost. There is also the possibility of false negative results i.e. a valid compound might not be detected.

Generally the in vivo screens are less preferred than in vitro ones because they can be more time-consuming (the Ascaris model can take up to six months to obtain a result) ; more costly; they are all non-human, and; results are frequently unreproducible.

There is therefore a need for a screen which is capable of distinguishing a PTC from a range of putative PTCs. If such a screen were cost-effective, rapid and easy to use then many thousands of possible compounds could be efficiently screened. Such a system should be able to provide reproducible results which correlate well with actual therapeutic activity when administered to humans.

According to the present invention, there is provided a screen which can overcome the above problems and in direct contrast to known screens, the present invention provides a cell-free screen. The present invention provides a screen which utilises a direct biochemical interaction between the moieties to determine whether or not a PTC may be useful as a drug.

The screen will be particularly useful in combinatorial approaches to drug design and testing.

The present invention can provide a means for detecting and quantifying the level of interaction of PTCs with a key three amino acid motif of the C_e4 region of IgE, or similar amino acid motifs.

It is widely accepted that the symptoms of allergies and allergic asthma are caused by the release of mediators, such as histamine, from mast cells and some basophils. These mediators cause inflammation, vasodilation and contraction of smooth muscle. In an allergic individual IgE is synthesised when the individual is first exposed to allergen. This IgE, specific to the individual allergen, becomes bound to specific cells such as mast cells. On re-exposure to allergen two IgE molecules bound to the mast cell are crosslinked by the allergen to trigger the cell to release mediators, including histamine. The precise method of this triggering is not known. Stanworth, et al, WO 90/15878 have proposed that on crosslinking of allergen a key decapeptide region from IgE is exposed. This decapeptide reacts with the cell to trigger the mediator release. Stanworth, (UK Patent Application No. 9320897.3), has also found that the blocking of a key three amino acid motif namely the sequence Lys-Thr-Lys is capable of blocking the triggering. Stanworth has hypothesised that known asthma allergy therapeutics such as sodium cromoglycate and nedocromil function by blocking this three amino acid motif. The screen of the invention allows determination of the binding ability of a PTC with the three amino acid motif of a decapeptide within the C_e4 region of IgE, or a similar amino acid motif. Any such binding should be indicative of a potential therapeutic effect.

The present invention can provide a screen for identifying PTCs comprising:

(i) a binding compound which includes a recognition site and a binding site having the sequence of amino acid residues Xaaj- Xaa₂-Xaa₃; and

(ii) a mono or polyclonal wholly, semi- or non-synthetic antibody or fragment thereof (Al) which is able to bind selectively to the binding site;

wherein Xaa_x and Xaa are the same or different positively charged amino acid residues such as Lys, Arg or His, preferably Lys or Arg, most preferably Lys;

and Xaa₂ is any other amino acid residue, preferably a neutrally charged amino acid residue, more preferably Thr, Pro, Gly or Ala, most preferably Thr.

Preferably the binding compound comprises a recognition site comprising 4 to 7 amino acid residues. The binding compound most preferably has the amino acid residue sequence Lys-Thr- ys- Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:l), in which the first three residues constitute the binding site and the last seven residues constitute the recognition site. In use, the binding compound is mixed with the PTC. This mixture is then incubated with the antibody Al. Competition for binding to the binding site of the binding compound occurs between the PTC and the antibody Al. If the PTC can bind to the binding compound via its binding site it can prevent antibody Al from binding to the binding compound. The binding of antibody Al to the binding compound can be determined for example by chromatography or gel electrophoresis. If binding of antibody Al to the binding compound occurs and binding of the PTC to the binding compound does not occur this is indicative that the PTC is of no value as a drug for treatment of allergies or allergic asthma.

In the screen of the present invention the recognition site on the binding compound is used to determine whether or not the PTC can bind, or to determine how well/badly the PTC is bound and hence how useful the PTC might be.

Preferably the screen of the present invention further comprises:

(iii) a surface onto which antibody Al is immobilised;

(iv) a label for detecting the presence of binding compound bound to antibody Al.

In use, antibody Al is immobilised to the surface. The binding compound is mixed with the PTC. This mixture is then incubated with the antibody Al on the surface. The resulting mixture of antibody Al immobilised to the surface, the PTC and the binding compound is washed to remove any free PTC, any free binding compound and any binding compound-PTC complex. If the binding compound does not form a complex with the PTC then it will be bound to antibody Al.

The label can be attached to the binding compound (e.g. at the recognition site) or the binding compound can comprise the label by having the label already incorporated in the recognition site. Binding of binding compound to antibody Al can be detected by detecting the presence of label.

Alternatively, the screen of the present invention further comprises (v) a mono or polyclonal, wholly, semi or non- synthetic antibody or fragment thereof (A2) which is able to selectively bind to the recognition site of the binding compound. The binding of binding compound to antibody Al can be detected by adding antibody A2 which can bind selectively to the recognition site of the binding compound, washing to remove excess antibody A2 and detecting the presence of label which can be attached to or incorporated in the antibody A2.

Alternatively, the binding of binding compound to antibody Al can be detected by adding antibody A2 and (vi) a mono or polyclonal, wholly, semi or non-synthetic antibody or fragment thereof (A3) which is able to selectively bind to the antibody A2, washing to remove excess antibody A3 and detecting the presence of label which can be attached to or incorporated in the antibody A3.

Additional antibodies could be successively added each one binding to the previously added antibody. Excess antibody is removed by washing. The final antibody AX added comprises a label or has a label attached which can be used to indicate the binding of antibody Al to binding compound. Preferably antibody Al is a rat or rabbit polyclonal antibody.

Preferably the surface is an ELISA plate, bead, test tube or membrane such as a nitro-cellulose membrane. The surface may be manufactured from e.g. glass, polystyrene, PTFE or nylon and optionally coated with e.g. a protein or a carbohydrate or both e.g. albumin (e.g. bovine serum albumin), casein, gelatin, lactose or mixtures thereof. More preferably the surface is a 96 well polystyrene ELISA plate.

The label can be e.g. horse radish peroxidase (HRP) ; alkaline phosphatase; ligands such as biotin which may be detected by avidin binding, fluorophores, or radionucleotides. Preferably the label is HRP which can be detected by adding peroxidase substrate such as orthophenylenediamine (OPD) and measuring colour intensity.

As an alternative to a label, the binding of binding compound to Al can be directly detected by physical means such as by measuring plasmon resonance or such as by making the solid surface part of a Field Effect Transistor detection system to measure the change in EMF when the binding compound binds to the gate of the transistor. In short, the invention is intended to include any means of detecting the presence/absence and/or amount of binding of binding compound to antibody Al.

Antibody A2 is preferably a mouse monoclonal antibody.

Antibody A3 is preferably a sheep or goat anti-mouse antibody.

In a second aspect the present invention can provide a method for screening for PTCs comprising: (i) mixing a PTC with binding compound (described above) ;

(ii) adding the mixture of binding compound and PTC to antibody Al (described above) and allowing free binding compound to bind to antibody Al.

(iii) detecting binding of binding compound to antibody Al. This may be carried out by for example chromatography or gel- electrophoresis.

No binding of binding compound to antibody Al indicates the PTC is of value; complete binding indicates the PTC is of no value and; some binding indicates the PTC is of some value.

Optionally, the method of the present invention can comprise the further steps of:

(iv) immobilising antibody Al on to a surface;

(v) washing to remove any free binding compound, any free PTC and any PTC-binding compound complex following incubation of the PTC and binding compound mixture with antibody Al; and optionally

(vi) adding an antibody A2 which can bind selectively to the recognition site of the binding compound; and

(vii) washing to remove excess antibody A2; and optionally

(viii) adding an antibody A3 which can bind selectively to antibody A2; and (ix) washing to remove excess antibody A3; and optionally

(x) adding antibodies which can bind selectively to the previously added antibodies, the final antibody added being antibody AX;

(xi) washing to remove excess antibodies;

(xii) after (v) , (vii) , (ix) or (xi) detecting label (described above) which can be attached to or incorporated in any of the binding compound, antibody A2, antibody A3 or antibody AX.

The label can be detected e.g. if the label is HRP by addition of peroxidase substrate and measuring colour intensity.

It will be apparent to those skilled in the art that the conditions used for screening and the relative concentrations of reagents can be varied according to the results to be obtained. For example, it may be desirable in certain circumstances to add the PTC in an equal amount to the binding compound. Alternatively it may be preferable to add the PTC in excess. Furthermore, in certain circumstances it may be desirable to allow the binding compound and PTC to be incubated after mixing in order to allow for as much binding as possible. Alternatively, it is possible to add the binding compound and PTC to the antibody Al without pre-incubation in order to allow for a competitive reaction. In any case, there is always the possibility that the competition for the binding site of the binding compound between the PTC and antibody Al will result in antibody Al displacing some already bound PTC. This effect may possibly be used to give a measure of the strength or stability of the conjugation of the PTC and the binding site and hence some measure of the potential value of the PTC. By carrying out experiments to measure the inhibition effect of the addition of a PTC to the ability of antibody Al to bind the binding compound under different conditions and concentrations, a measure of the likely effectiveness of any PTC can be determined. Clearly, the better the PTC binds to the binding compound the greater will be the inhibition of binding of the binding compound to the antibody Al and the better is the likely value of the PTC as a drug for treatment of allergies or allergic asthma.

A preferred embodiment of the screen of the present invention will now be described with reference to Figure 1 which is a schematic of a screen of the present invention.

Antibody Al (1) is a rat or rabbit polyclonal antibody. It is immobilised to a surface (2) which is a 96 well polystyrene ELISA plate. A binding compound (3) having the sequence Lys- Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-NH₂ (SEQ ID NO:2) can bind selectively to antibody Al via its binding site Lys-Thr-Lys (4) if it is not bound to a PTC (5) . Antibody A2, a mouse monoclonal antibody, (6) can bind selectively to the bound binding compound's recognition site Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:3) (7). Antibody A3, a sheep or goat anti-mouse antibody, (8) can bind selectively to antibody A2. A HRP label(9) is attached to antibody A3 which can be used to detect binding of binding compound to antibody Al.

The invention also provides a kit for carrying out screening of PTCs. The kit comprises at least (i) , (ii) , (iii) , (iv) . Optionally the kit comprises one or both of a PTC positive control (a known inhibitor) and a PTC negative control (a known non-inhibitor) . The present invention will now be illustrated by way of the following examples.

Example 1

Inhibition ELISA

This screen measures inhibition of the binding compound decapeptide F30 (Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-NH₂ (SEQ ID NO:2)) to anti-F30 serum (Antibody Al) by pre-incubation of F30 with either the PTC "F78" or standard anti-asthma preparations such as disodium cromoglycate (Intal) , nedocromil sodium (Tilade) and ketotifen (Zadotifen) .

The PTC F78 is a tripeptide having the sequence Glu-Pro-Glu.

Materials

Buffers

1. Phosphate buffered saline (PBS)/Tween (RTM) , pH 7.2; consisting of

0.44g NaH₂PO₄2H₂0

1.02g Na₂ HP0₄

8.7g Na Cl

Tween (RTM) 20 0.05% in 1L distilled water

2. Coating buffer, 0.05M (pH 9.6); consisting of 0.3975g Na₂ C0₃

0.7325g Na₂ HC03 in 250ml of distilled water ELISA Plate

1 . Incubation plates

96-well polystyrene rigid plates, flat bottomed (Dynatech Immulon)

2. Assay plates

Falcon 3912:microtest III Flexible Assay plates, 96 well, flat bottomed

Drugs

1. PTC F78, a tripeptide believed to be an inhibitor of the release of histamine from mast cells

2. Standard preparations such as sodium cromoglycate, ketotifen and nedocromil sodium.

Uej Qd.

1st Staσe Incubation Plate

Coat with 1% bovine serum albumin (prepared from Fraction V albumin, SIGMA) in coating buffer, pH 9.6 incubate for 30 mins at 37°C wash x 3 with PBS/Tween (ELISA washer, Dynatech MRW) . Add lOOμl PBS/Tween (RTM) to all wells except those in row A. Add 200μl of drugs to each well in row A and double dilute down to row H (1:128) . Add lOOμl of PBS/Tween to columns 2, 4, 6, 8, 10 and 12.

Add lOOμl F30, lOμM in PBS/Tween (RTM) to columns 1, 3, 5, 7, 9 and 11. Incubate for 0-60 min, preferably less than 30 mins at 37 °C .

2nd Staσe Assay Plate

Coat with rabbit anti-F30 (No. 2301, Rheumatology and Allergy Research Unit, Birmingham University) at 1:500 in coating buffer for 1 hour at 37°C wash x 3 in PBS/Tween (RTM) .

Transfer lOOμl from each well in the incubation plate to the corresponding well in the assay plate and incubate for 1 hour at 37°C wash x 3 with PBS/Tween (RTM) .

Add lOOμl of mouse monoclonal anti-F30 Dec IB made up at 1:1000 in PBS/Tween (RTM) to each well in the assay plate and incubate for 1 hour at 37°C. Wash x 3 in PBS/Tween (RTM) . Add lOOμl of sheep-anti-mouse IgG (H + L) peroxidase labelled serum diluted 1:1000 in PBS/Tween (RTM) (Serotec Ltd., Oxford, UK) to every well. Incubate for 1 hour at 37°C. Wash x 3 in PBS/Tween (RTM) . Make up substrate b_y adding OPD (2mg/ml) to sodium perborate buffer (both from Sigma Chemical Co. Ltd. , Fancy Road, Poole, UK) . Add lOOμl to every well.

Allow colour to develop fully, about 2-5 min at room temperature and stop the reaction using 30μl 25% H₂S0₄ per well.

Read using a Titretek Twinreader, filter 5 (wavelength 492nm) .

Results

The results are shown in figures 2-6. These data show that F78 is able to bind to F30 and thereby inhibit its binding to Anti- F30 Antibody Al. F78 is an inhibitor and hence may be expected to be a valuable therapeutic compound. These data indicate that F78 is a better inhibitor than disodium cromoglycate in this assay.

Example 2

The decapeptide F30 is elongated by the addition of a tyrosine at the C-terminal end. This tyrosine is then labelled using radioactive Iodine¹²⁵ by methods known in the art.

This peptide can then be mixed with a potential PTC in the same manner as described in Example 1 and applied to an ELISA plate pre-coated with rabbit anti-F30 at 1:500 in coating buffer for 1 hour at 37°C, and then washed with suitable buffer (PBS/Tween (RTM) ) .

The level of radioactivity in each well of the plate is then measured. A high count indicates that the PTC is not inhibiting binding and is not a good candidate PTC. Conversely a low count indicates that the PTC has potential.

Example 3

A peptide which includes the binding site such as F30 can be coupled directly to biotinamidocaproyl hydrozide via its (unamidated) C-terminal end. Following incubation and washing (as in the above examples) the plate can then be incubated with a streptavidin/Peroxidase solution. Following incubation for 1 hour at 37°C the plate is then washed to remove unbound material. Substrate (OPD/H₂0₂) is added to each well and the colour allowed to develop. After 2-15 minutes at room temperature the reaction is stopped using 25% H₂S0₄ added to each well.

The colour is then read using a microtitre plate reader at a wavelength of 492nm. A high colour indicates that the PTC is not inhibiting and is not a good candidate PTC. Conversely a low colour indicates that the PTC has potential.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: PEPTIDE THERAPEUTICS LIMITED

(B) STREET: 321 CAMBRIDGE SCIENCE PARK

(C) CITY: CAMBRIDGE

(D) STATE: CAMBRIDGE

(E) COUNTRY: ENGLAND

(F) POSTAL CODE (ZIP) : CB4 4WG

(G) TELEPHONE: 01223 423333 (H) TELEFAX: 01223 423111

(ii) TITLE OF INVENTION: Screen For Potential Therapeutic Compounds

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9417086.7

(B) FILING DATE: 24-AUG-1994

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 aπ no acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION:10

(D) OTHER INFORMATION:/product= "Phe 10" /note_* "amidated"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Lys Thr Lys Gly Ser Gly Phe Phe Val Phe I S 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gly Ser Gly Phe Phe Val Phe 1 5

Claims

1. A screen for identifying PTCs comprising:

(i) a binding compound which includes a recognition site and a binding site having the sequence of amino acid residues Xaa_!- Xaa₂-Xaa₃; and

wherein Xaaj and Xaa are the same or different positively charged amino acid residues such as Lys, Arg or His, preferably Lys or Arg, most preferably Lys;

2. A screen according to claim 1 wherein the binding compound comprises a recognition site comprising 4 to 7 amino acid residues, preferably it has the amino acid residue sequence Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:l), in which the first three residues constitute the binding site and the last seven residues constitute the recognition site.

3. A screen according to claim 1 or 2 which further comprises:

(iii) a surface onto which antibody Al is immobilised; (iv) a label for detecting the presence of binding compound bound to antibody Al.

4. A method of screening for a PTC using a screen according to any preceding claim in which antibody Al is immobilised to the surface, the binding compound is mixed with the PTC, this mixture is then incubated with the antibody Al on the surface, the resulting mixture of antibody Al immobilised to the surface, the PTC and the binding compound is washed to remove any free PTC, any free binding compound and any binding compound-PTC complex, and if the binding compound does not form a complex with the PTC then it will be bound to antibody Al.

5. A method of screening for PTCs comprising:

(i) mixing a PTC with binding compound according to claim 1;

(ii) adding the mixture of binding compound and PTC to antibody Al according to claim 1 and allowing free binding compound to bind to antibody Al.

(iii) detecting binding of binding compound to antibody Al.

6. The method of claim 5 comprising the further steps of:

(iv) immobilising antibody Al on to a surface;

(v) washing to remove any free binding compound, any free PTC and any PTC-binding compound complex following incubation of the PTC and binding compound mixture with antibody Al; and optionally (vi) adding an antibody A2 which can bind selectively to the recognition site of the binding compound; and

(vii) washing to remove excess antibody A2; and optionally

(viii) adding an antibody A3 which can bind selectively to antibody A2; and

(ix) washing to remove excess antibody A3; and optionally

(xi) washing to remove excess antibodies;

7. A kit for carrying out screening of PTCs according to claim 3 which comprises at least (i) , (ii) , (iii) , (iv) .

8. The kit of claim 7 which also comprises one or both of a PTC positive control (a known inhibitor) and a PTC negative control (a known non-inhibitor) .