WO2004010145A2 - Procede de criblage - Google Patents

Procede de criblage Download PDF

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Publication number
WO2004010145A2
WO2004010145A2 PCT/GB2003/003231 GB0303231W WO2004010145A2 WO 2004010145 A2 WO2004010145 A2 WO 2004010145A2 GB 0303231 W GB0303231 W GB 0303231W WO 2004010145 A2 WO2004010145 A2 WO 2004010145A2
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WO
WIPO (PCT)
Prior art keywords
activation
gnrh
test compound
receptor
event
Prior art date
Application number
PCT/GB2003/003231
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English (en)
Other versions
WO2004010145A3 (fr
Inventor
Ulrich Conrad Dyer
Stuart Russel Maudsley
Original Assignee
Ardana Bioscience 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
Priority claimed from GB0216960A external-priority patent/GB0216960D0/en
Priority claimed from GB0308890A external-priority patent/GB0308890D0/en
Application filed by Ardana Bioscience Limited filed Critical Ardana Bioscience Limited
Priority to AU2003248955A priority Critical patent/AU2003248955A1/en
Publication of WO2004010145A2 publication Critical patent/WO2004010145A2/fr
Publication of WO2004010145A3 publication Critical patent/WO2004010145A3/fr

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Classifications

    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the invention relates to a method of selective screening of G-proteins- coupled receptor ligands, and in particular GnRH receptor ligands, in order to provide active compounds which can selectively activate or inhibit specific transduction events.
  • GPCR G protein-coupled receptor
  • GPCR agonists do not all stabilise the same active receptor conformations.
  • different serotoninergic compounds can activate distinct active states of the 5-HT 2 c receptor.
  • the agonists DOI (+-l-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane) and quipazine can select between activation of two responses in Chinese hamster ovary (CHO) cells.
  • DOI has a minimal capacity to induce IP n accumulation compared to quipazine.
  • AA arachidonic acid
  • release quipazine has a minimal capacity compared to DOI.
  • Differential activation of G-proteins by GPCRs cannot be accommodated by a mechanism by which one single receptor active state produced by all agonists interacts with G-proteins.
  • differential stimulus pathway activation can occur through the strength of the signal (i.e. highly efficacious agonist activating two pathways whereas a weaker agonist activating only the more sensitive one) the reversal of agonist relative activity (e.g. 5-HT 2 c receptor) cannot be explained in this manner. Therefore it is believed that different ligands stabilise different active conformations of the receptor resulting in the presentation of a different ultrastructural interface of the intracellular face of the receptor to G- proteins.
  • the method comprises the steps of: a) selecting at least one test compound; b) exposing a cell culture, preferably a mammalian cell culture, to the test compound and incubating the culture under suitable conditions; c) selecting at least two distinct intracellular events which are controlled, at least partially, by the G-protein-coupled receptor; d) testing for the ability of said test compound to modulate one of the selected intracellular events; and e) selecting the test compound which selectively modulates at least one of the pre-selected intracellular events and does not modulate at least another pre-selected intracellular event.
  • GPCR G-protein-coupled receptor
  • This method allows the distinction at an early stage and in vitro between test compounds, and in particular known G-protein-coupled receptor ligands, which appear to have potential as a G-protein-coupled receptor modulators, but which activate unwanted transduction pathways and, as such, should be disregarded, and it allows the identification of test compounds which are signal-specific and have a greater potential as a therapeutic agents.
  • the method can be used to distinguish between already known G- protein coupled receptor agonists/antagonist, it can also be used to test other compounds.
  • the method of the invention can advantageously comprise a preliminary step wherein the test compound is tested for its capacity to bind to the G-protein-coupled receptor (GPCR).
  • GPCR G-protein-coupled receptor
  • the compound has a K D for binding the GPCR in question of between 0.1 and lOnM, such as about InM. Whether or not a test compound binds to the GPCR in question can be determined using methods well known in the art, such as a competitive radioligand binding assay.
  • the test compound may be any compound, but typically it is an organic compound of between 100 and 10000 Daltons, preferably between 500 and 5000 Daltons.
  • the test compound is a compound in a library of test compounds, such as those made using combinatorial chemistry techniques.
  • the selected test compound may be a drug-like compound or lead compound for the development of a drug-like compound.
  • drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example, as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 15000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of bioavailability.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • the cell cultures used in the method of the invention are, of course, selected for their particular ability to react to activation of the targeted G-protein receptor.
  • the cells may be unmodified cells which are known to express one or more particular GPCRs, or they may be cells which have been specifically transfected with a polynucleotide which encodes a particular GPCR of interest. Cells which naturally express GPCRs are well known in the art, such as L ⁇ T2 gonadotrope cells as decribed in the examples.
  • COS-7 African green monkey fibroblast cells express the ⁇ 2- adrenoceptor
  • Rat- la rat fibroblast cells express the thrombin receptor
  • PC 12 phaeocromacytoma adrenal tumour
  • NG108-15 rat-mouse sympathetic neurone hybridoma cells express the tacykinin NK1 receptor
  • Ishikawa human endometrial tumour cells express the EP prostanoid receptor.
  • HEK human embryonic kidney
  • SCL60 Type I GnRH receptor
  • Yamaguchi et al (2003) Eur. J. Biochem 270, 1816-1827 describes HEK 293 (human embryonic kidney) cell lines stably expressing the endothelin type B GPCR (Genbank Accession No. JQ1042); and Audinot et al (2001) J. Biol. Chem. 276, 13554-13562 describes HEK 293 cell lines stably expressing the melanin-concentrating hormone (MCH) receptor (Genbank Accession No. NP_005288)
  • Suitable cell culture conditions are one in which the cells are viable and able to express their GPCR.
  • the culture conditions are such that it can be determined whether a test compound can stimulate or inhibit signalling via the GPCR.
  • the intracellular events to be tested may be either the presence, increase, decrease or absence of a specific substance characteristic of said event.
  • GPCRs are associated with intracellular signalling events, and any particular GPCR may be associated with more than one such signalling event or more than one signalling pathway.
  • the intracellular event may be the activation and/or deactivation of Ca 2+ channels, production of inositol phosphate, gonadotropin transcription and/or release, activation of cellular tyrosine kinases such as activation of c-Src, induction of a pro-apopotic phenotype in tumour cells etc. These events can all be measured using methods well known in the art.
  • production of inositol phosphate can be measured in inositol- free growth media supplemented with H-myoinositol; and gonadotropin transcription can be measured by northern blots or by reverse-transcript polymerase chain reaction (RT-PCR).
  • RT-PCR reverse-transcript polymerase chain reaction
  • PS phospholipid
  • Extracellular expression of PS can be readily determined making use of labelled (eg fluorescently labelled) Annexin V which selectively binds PS.
  • Measurement of caspase or procaspase may be useful in determining whether or not a test compound is proapoptotic. Such measurments can be carried out in a high throughput screening format with appropriate enzyme substrates.
  • the assays can be advantageously included in a kit which permits screening of a large number of compounds.
  • results provided by an assay of the invention can be used to select selective inhibitors/activators of specific transduction pathways. It is particularly preferred if compounds are selected that are able to inhibit substantially a specific pre-selected GPCR signalling pathway, but not able to inhibit substantially another specific pre-selected GPCR signalling pathway.
  • test compound substantially modulates said event. It will be appreciated that a test compound may have an effect on the said event but that the effect is not substantial in the context of the invention. For example, if the selected intracellular event is production of inositol phosphate, the test compound that is selected for further study is one which does not substantially activate this event (ie cause production of inositol phosphate).
  • not modulating at least said pre-selected intracellular we include the meaning that the test compound does not substantially modulate said event. It will be appreciated that a test compound may have an effect on the said event but that the effect is not substantial in the context of the invention. For example, if the selected intracellular event is induction of a pro- apopotic phenotype in tumour cells, the test compound that is selected for further study is one which does not substantially activate this event (ie cause induction of a pro-apoptotic phenotype).
  • test compounds selected are ones which exhibit a useful differential between their ability to modulate substantially a first pre-selected intracellular event and their ability to not modulate substantially a second intracellular event.
  • the method or assay of the invention may also be used as a research tool to comprehend and define the different activation states of a specific G- protein-coupled receptor and pinpoint the biological pathway triggered by a specific state. This in turn has important and obvious medical implications for drug design, especially for selecting drug candidates which do not trigger undesirable side-effects.
  • differential signalling events can be initiated via activation of the same receptor by various ligands.
  • activation of differing receptors within the same cellular environment can result in distinct cellular signalling events.
  • GnRH Gonadotropin-Releasing Hormone
  • GnRH-II GnRH-II
  • GnRH I and II, and GnRH III can also exert important therapeutic actions at many other peripheral tissues involved in reproductive biology.
  • GnRH receptor GnRHR
  • GnRHR GnRH receptor
  • the Src tyrosine kinase has been demonstrated over the past five years to be critically involved in the majority of physiological and pathophysiological mechanisms of cell growth and proliferation.
  • GnRH I and II despite being equipotent (at high doses) at stimulating highly amplified events (ERK1/2), show a discrepancy in their capacity in inducing activation of the c-Src non-receptor tyrosine kinase in gonadotrope cells.
  • a l ⁇ M dose of both GnRH I or II results in a maximal stimulation of ERK1/2.
  • the cells possessing Type I GnRH receptors are more sensitive to GnRH I than GnRH II as shown by the greater ERKl/2 activation response to lOnM doses of GnRH I than GnRH II.
  • saturating doses of both ligands (l ⁇ M) induced a similar maximum ERKl/2 stimulation.
  • phosphotyrosine-containing proteins were isolated from the crude cell extracts of L ⁇ T2 cells by immunoprecipitation with an agarose-conjugated anti-phosphotyrosine antibody (PY20, Santa Cruz). The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane and immunoblotted with an antisera that recognises the autophosphorylated (Y418) form of c-Src.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • GnRHR ligands were tested: The two naturally occurring GnRH I and II and two truncated forms of GnRH I: GnRH I 1-5 and GnRH 1-6.
  • GnRH 1-5 is also called “hemi-form" as it comprises the first five amino acid residues of GnRH and is naturally occurring in specific subset of central nervous system neurones.
  • GnRH 1-6 is an artificial ligands and comprises the first six amino acid residues.
  • L ⁇ T2 gonadotrope cells were stimulated with the specified ligands for the indicated time periods.
  • Whole-cell lysate protein extracts were made and resolved by SDS-PAGE and transferred to PVDF membranes.
  • both GnRH I and II (lOOnM) induce a profound activation of ERKl/2
  • GnRH I 1-5 (1 M) yields a much lower degree of ERK 1/2 stimulation with lOOnM GnRH I 1-6 (artificial ligand) having no effect upon ERK 1/2 activation.
  • the anti-apoptotic kinase Aktl becomes activated by phosphorylation of residues near its activation loop, one such residue is Ser .
  • GnRH I 1-5 appears considerably more potent in stimulating c-Src ictivation (measured by c-Src Y418 auto-tyrosine phosphorylation) than ERKl/2.
  • human embryonic kidney (HEK) cells stably expressing the Type I GnRH receptor (designated SCL60) show a robust GnRH ligand induced activation of c-Src.
  • GnRH 1 1-5 shows negligible capacity in activating ERKl/2, a much more distal and a more biochemically amplified event, compared with GnRH I ligand.
  • GnRH 1 1-5 may stabilise the Type I GnRHR in a different conformation, compared to that induced by GnRH I, that specifically has a high affinity for the c-Src activation pathway compared to the ERKl/2 activation pathway.
  • GnRH 1-5 The specific function of GnRH 1-5 has yet to be adequately identified. However, as shown in Figure 2, GnRH I 1-5 appears to be able to preferentially activate the anti-apoptotic serine/threonine kinase Aktl (also known as protein kinase B). Compared to the endogenous GnRH I and II ligands the truncated GnRH 1 1-5 ligand shows minimal capacity to activate the highly amplified biochemical pathway to the activation of ERKl/2 mitogen-activated protein kinase. The activation of the mitogen-activated protein kinase via GnRH 1 1-5 may serve in a neuroprotective role during neurotransmission.
  • Aktl also known as protein kinase B
  • Transient expression of either the Type I human GnRHR or marmoset Type II GnRHR in HEK293 cells allowed the application of either GnRH I or II to stimulate the activation of ERKl/2 as shown in the top panel of Figure 4. Similar amounts of total ERK protein was present in all the experimental conditions (middle panel). However, unlike GnRH I stimulation of Type I GnRHR expressing cells, stimulation of HEK293 cells expressing the Type II GnRHR with GnRH II failed to induce any activation of c-Src. The activation of c-Src was estimated by detecting the amount of auto-tyrosine phosphorylated (Y418) c-Src in the lowest of the three panels.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé d'identification de substances d'essai qui inhibent ou activent sélectivement uniquement un nombre limité de voies de transduction d'un récepteur couplé aux protéines G (GPCR), le procédé comprenant les étapes consistant: a) à sélectionner au moins un composé d'essai; b) à exposer une culture cellulaire, de préférence une culture de cellules mammifères, au composé d'essai et à faire incuber la culture dans des conditions appropriées; c) à sélectionner au moins deux événements intracellulaires distincts lesquels sont régulés, au moins partiellement, par le récepteur couplé aux protéines G; d) à tester la capacité dudit composé d'essai à moduler un des événements intracellulaires sélectionnés; et à sélectionner le composé d'essai qui module sélectivement au moins un des événements intracellulaires présélectionnés et ne module pas au moins un autre événement intracellulaire présélectionné.
PCT/GB2003/003231 2002-07-22 2003-07-22 Procede de criblage WO2004010145A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003248955A AU2003248955A1 (en) 2002-07-22 2003-07-22 Screening method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0216960.5 2002-07-22
GB0216960A GB0216960D0 (en) 2002-07-22 2002-07-22 Screening method
GB0308890.3 2003-04-17
GB0308890A GB0308890D0 (en) 2003-04-17 2003-04-17 Screening method

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040337A1 (fr) * 2004-10-13 2006-04-20 Evotec Technologies Gmbh Procede pour l'identification de composes qui affectent le transport d'une proteine a travers la voie de trafic membranaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854004A (en) * 1991-11-25 1998-12-29 Boehringer Ingleheim Gmbh Process for screening substances capable of modulating a receptor-dependent cellular signal transmission path
WO2001078796A1 (fr) * 2000-04-15 2001-10-25 Medical Res Council Recepteur de la gonadoliberine de type ii et polynucleotides codant pour ce recepteur

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1512016T3 (da) * 2002-07-22 2006-07-03 Ardana Bioscience Ltd Screeningsmetode og antitumor-medikamentkandidat opnået derved

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Publication number Priority date Publication date Assignee Title
US5854004A (en) * 1991-11-25 1998-12-29 Boehringer Ingleheim Gmbh Process for screening substances capable of modulating a receptor-dependent cellular signal transmission path
WO2001078796A1 (fr) * 2000-04-15 2001-10-25 Medical Res Council Recepteur de la gonadoliberine de type ii et polynucleotides codant pour ce recepteur

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BENARD O ET AL: "Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone." THE JOURNAL OF BIOLOGICAL CHEMISTRY. UNITED STATES 16 FEB 2001, vol. 276, no. 7, 16 February 2001 (2001-02-16), pages 4554-4563, XP002269312 ISSN: 0021-9258 *
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; MAUDSLEY, STUART RUSSELL: "Screening method and anti-tumor drug candidate obtained therefrom" retrieved from STN Database accession no. 2004:80979 XP002269313 & WO 2004 010146 A (ARDANA BIOSCIENCE LIMITED, UK) 29 January 2004 (2004-01-29) *
KENAKIN T: "Agonist-receptor efficacy II: agonist trafficking of receptor signals" TRENDS IN PHARMACOLOGICAL SCIENCES, ELSEVIER TRENDS JOURNAL, CAMBRIDGE, GB, vol. 16, no. 7, July 1995 (1995-07), pages 232-238, XP004207507 ISSN: 0165-6147 *
KRAUS S ET AL: "Intracellular signaling pathways mediated by the gonadotropin-releasing hormone (GnRH) receptor." ARCHIVES OF MEDICAL RESEARCH. UNITED STATES 2001 NOV-DEC, vol. 32, no. 6, November 2001 (2001-11), pages 499-509, XP002269311 ISSN: 0188-4409 *
LEIBOVITZ D ET AL: "Sequential degradation of the neuropeptide gonadotropin-releasing hormone by the 20 S granulosa cell proteasomes." FEBS LETTERS. NETHERLANDS 13 JUN 1994, vol. 346, no. 2-3, 13 June 1994 (1994-06-13), pages 203-206, XP002269395 ISSN: 0014-5793 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040337A1 (fr) * 2004-10-13 2006-04-20 Evotec Technologies Gmbh Procede pour l'identification de composes qui affectent le transport d'une proteine a travers la voie de trafic membranaire

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WO2004010145A3 (fr) 2004-04-29
AU2003248955A1 (en) 2004-02-09
AU2003248955A8 (en) 2004-02-09

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