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WO1996006357A1 - Criblage pour des composes a potentiel therapeutique - Google Patents

Criblage pour des composes a potentiel therapeutique Download PDF

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Publication number
WO1996006357A1
WO1996006357A1 PCT/GB1995/001912 GB9501912W WO9606357A1 WO 1996006357 A1 WO1996006357 A1 WO 1996006357A1 GB 9501912 W GB9501912 W GB 9501912W WO 9606357 A1 WO9606357 A1 WO 9606357A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
ptc
binding compound
binding
amino acid
Prior art date
Application number
PCT/GB1995/001912
Other languages
English (en)
Inventor
Denis Raymond Stanworth
Ian Victor Lewin
Sarita Maman
Valerie Jones
Original Assignee
Peptide Therapeutics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peptide Therapeutics Limited filed Critical Peptide Therapeutics Limited
Priority to AU32274/95A priority Critical patent/AU3227495A/en
Publication of WO1996006357A1 publication Critical patent/WO1996006357A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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.
  • PTC 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.
  • 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.
  • 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.
  • PCA rat Passive Cutaneous Anaphylaxis
  • TMA Trimellitic Anhydride
  • 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.
  • 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.
  • 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 e 4 region of IgE, or similar amino acid motifs.
  • IgE 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.
  • the present invention can provide a screen for identifying PTCs comprising:
  • a binding compound which includes a recognition site and a binding site having the sequence of amino acid residues Xaaj- Xaa 2 -Xaa 3 ;
  • 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;
  • Xaa 2 is any other amino acid residue, preferably a neutrally charged amino acid residue, more preferably Thr, Pro, Gly or Ala, most preferably Thr.
  • 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.
  • 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.
  • 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.
  • the screen of the present invention further comprises:
  • 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.
  • 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.
  • A2 a mono or polyclonal, wholly, semi or non- synthetic antibody or fragment thereof
  • 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.
  • 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.
  • A3 a mono or polyclonal, wholly, semi or non-synthetic antibody or fragment thereof
  • antibody Al is a rat or rabbit polyclonal antibody.
  • 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.
  • HRP horse radish peroxidase
  • OPD orthophenylenediamine
  • 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.
  • 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.
  • the present invention can provide a method for screening for PTCs comprising: (i) mixing a PTC with binding compound (described above) ;
  • the method of the present invention can comprise the further steps of:
  • the label can be detected e.g. if the label is HRP by addition of peroxidase substrate and measuring colour intensity.
  • 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.
  • a measure of the likely effectiveness of any PTC can be determined.
  • 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.
  • FIG. 1 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-NH 2 (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) .
  • the kit comprises one or both of a PTC positive control (a known inhibitor) and a PTC negative control (a known non-inhibitor) .
  • a PTC positive control a known inhibitor
  • a PTC negative control a known non-inhibitor
  • This screen measures inhibition of the binding compound decapeptide F30 (Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-NH 2 (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.
  • PBS Phosphate buffered saline
  • RTM Tween
  • PTC F78 a tripeptide believed to be an inhibitor of the release of histamine from mast cells
  • Standard preparations such as sodium cromoglycate, ketotifen and nedocromil sodium.
  • the decapeptide F30 is elongated by the addition of a tyrosine at the C-terminal end. This tyrosine is then labelled using radioactive Iodine 125 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) ) .
  • 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.
  • 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/H 2 0 2 ) is added to each well and the colour allowed to develop. After 2-15 minutes at room temperature the reaction is stopped using 25% H 2 S0 4 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.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

La présente invention concerne un criblage faisant appel à une interaction biochimique directe existant entre des fragments de molécules pour déterminer si un composé à potentiel thérapeutique (PTC) a ou non une utilité médicamenteuse. Ce criblage peut se montrer des plus utile s'agissant d'approches combinatoires aux fins du développement d'un médicament et des essais auxquels celui-ci est soumis. Le criblage dont il est ici question permet de déterminer le pouvoir de liaison d'un PTC avec le motif structural de trois acides aminés d'un décapeptide dans la région Cε4 de l'IgE, ou un motif structural d'un acide aminé similaire. Une telle liaison, quelle qu'elle soit, pourrait indiquer un éventuel effet thérapeutique. Le criblage suppose: (i) un composé liant qui inclut un site de reconnaissance et un site de liaison ayant la séquence Xaa1-Xaa2-Xaa3 de résidus aminoacides; (ii) un anticorps monoclonal ou polyclonal, totalement, semi- ou non synthétique ou un fragment de celui-ci (Al) à même de se lier de manière sélective au site de liaison où Xaa1 et Xaa3 désignent les résidus aminoacides chargés positivement, identiques ou différents, comme Lys, Arg ou His, de préférence Lys ou Arg ou mieux, Lys; et Xaa2 désigne tout autre résidu aminoacide, de préférence un résidu aminoacide neutre ou mieux Thr, Pro, Gly ou bien Ala ou idéalement Thr.
PCT/GB1995/001912 1994-08-24 1995-08-14 Criblage pour des composes a potentiel therapeutique WO1996006357A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU32274/95A AU3227495A (en) 1994-08-24 1995-08-14 Screen for potential therapeutic compounds

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9417086.7 1994-08-24
GB9417086A GB9417086D0 (en) 1994-08-24 1994-08-24 Screen for potential therapeutic compounds

Publications (1)

Publication Number Publication Date
WO1996006357A1 true WO1996006357A1 (fr) 1996-02-29

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PCT/GB1995/001912 WO1996006357A1 (fr) 1994-08-24 1995-08-14 Criblage pour des composes a potentiel therapeutique

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AU (1) AU3227495A (fr)
GB (1) GB9417086D0 (fr)
WO (1) WO1996006357A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0811016A4 (fr) * 1994-03-28 1997-12-10
KR101620093B1 (ko) 2010-08-23 2016-05-13 주식회사 녹십자메디스 신규한 쿠마린 유도체 및 이의 제조방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0403312A1 (fr) * 1989-06-15 1990-12-19 Btg International Limited Peptides immunoactifs et anticorps et leurs utilisations dans le traitement antiallergique
EP0592230A1 (fr) * 1992-10-07 1994-04-13 Snow Brand Milk Products Co., Ltd. Anticorps monoclonal anti-peptide et ADN codant pour le même
WO1995010532A1 (fr) * 1993-10-11 1995-04-20 Peptide Therapeutics Limited Composes utilises dans les traitements anti-allergies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0403312A1 (fr) * 1989-06-15 1990-12-19 Btg International Limited Peptides immunoactifs et anticorps et leurs utilisations dans le traitement antiallergique
WO1990015878A1 (fr) * 1989-06-15 1990-12-27 National Research Development Corporation Peptides et anticorps immunoactifs et leur utilisation dans les traitements anti-allergiques
EP0592230A1 (fr) * 1992-10-07 1994-04-13 Snow Brand Milk Products Co., Ltd. Anticorps monoclonal anti-peptide et ADN codant pour le même
WO1995010532A1 (fr) * 1993-10-11 1995-04-20 Peptide Therapeutics Limited Composes utilises dans les traitements anti-allergies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
D. R STANWORTH ET AL.: "Essential structural requirements for triggering of mast cells by a synthetic peptide comprising a sequence in the Ce4 domain of human Ig E.", MOLECULAR IMMUNOLOGY, vol. 21, no. 3, pages 243 - 247 *
D. R. STANWORTH ET AL.: "Anti-e-chain antibodies as probes in the study of mast cell triggering.", MOLECULAR IMMUNOLOGY, vol. 23, no. 11, pages 1231 - 1235 *
R. STANWORTH ET AL.: "Synthetic peptides comprising sequences of the human immunoglobulin E heavy chain capable of releasing histamine.", THE BIOCHEMICAL JOURNAL, vol. 180, no. 3, 15 June 1979 (1979-06-15), pages 665 - 668 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0811016A4 (fr) * 1994-03-28 1997-12-10
KR101620093B1 (ko) 2010-08-23 2016-05-13 주식회사 녹십자메디스 신규한 쿠마린 유도체 및 이의 제조방법

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Publication number Publication date
GB9417086D0 (en) 1994-10-12
AU3227495A (en) 1996-03-14

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