WO2003011901A1 - Techniques d'identification de substances interagissant avec des domaines d'homologie avec la pleckstrine (ph) et proteine contenant des domaines d'homologie avec la pleckstrine mutes - Google Patents

Techniques d'identification de substances interagissant avec des domaines d'homologie avec la pleckstrine (ph) et proteine contenant des domaines d'homologie avec la pleckstrine mutes Download PDF

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WO2003011901A1
WO2003011901A1 PCT/GB2002/003262 GB0203262W WO03011901A1 WO 2003011901 A1 WO2003011901 A1 WO 2003011901A1 GB 0203262 W GB0203262 W GB 0203262W WO 03011901 A1 WO03011901 A1 WO 03011901A1
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compound
domain
ptdins
tapp
mutated
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PCT/GB2002/003262
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English (en)
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Dario Alessi
Daan Van Aalten
Christine Thomas
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University Of Dundee
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Priority to GB0402001A priority Critical patent/GB2393725B/en
Publication of WO2003011901A1 publication Critical patent/WO2003011901A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the 3-phosphoinositides phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P 3 ) and PtdIns(3,4)P 2 function as cellular second messengers. Their concentrations 5 are increased following stimulation of cells with extracellular agonists and they are thought to trigger the activation of signal transduction networks that regulate a plethora of processes including physiological responses to insulin and controlling cell survival [1, 2 .
  • PtdIns(3,4,5)P3 is generated following growth factor/insulin- induced activation of members of the phosphoinositide 3 -kinase (PI 3 -kinase) 0 family, which phosphorylate PtdIns(4,5)P 2 at the D-3 position of the inositol ring [2].
  • PtdIns(3,4,5)P 3 can then be converted either back to PtdIns(4,5)P 2 through the action of a 3-phosphatase termed PTEN [3] or through the action of 5- phosphatases termed SH2-containing inositol phosphatase- 1 (SHIP 1) or SHIP2 to PtdIns(3,4)P 2 [4].
  • PtdIns(3,4)P 2 can also be generated 5 via a PI3 -kinase independent pathway in response to agonists such as hydrogen peroxide [5] and crosslinking of platelet integrin receptors [6].
  • PH pleckstrin homology
  • Proteins that possess PH domains that can interact with PtdIns(3,4,5)P 3 include the PH domains that are found on the serine/threonine-specific protein kinases, Protein Kinase B (PKB) and the 3 -Phosphoinositide Dependent Kinase- 1 (PDK1) [8], the Bruton Tyrosine Kinase (BTK) family of tyrosine kinases [9], certain adaptor proteins 5 such as Dual Adaptor of Phosphotyrosine and 3Phosphoinositides (DAPPl) [10, 11, 12, 13] and the Grb2-Associated Binder-I (Gabl) [14], as well as the ADP Ribosylation Factor (ARF), the GTPase activating protein (GAP) centaurin- ⁇ [15] and the ARF guanine nucleotide exchange factor, GRPl [16].
  • PPKB Protein Kinase B
  • PDK1 3
  • DAPPl PH domain unlike those of GRPl and BTK, forms high affinity interactions with the 3 and 4 phosphate groups of the inositol ring and not with the 5-phosphate group that is accomodated in a largely empty space in the ligand binding pocket [18].
  • TAPP 1 and TAPP2 TAndem PH domain containing Protein- I (TAPP 1) and TAPP2 because they possessed 2 sequential PH domains [21].
  • the N-terminal PH domain of TAPP1 and TAPP2 did not interact with any phosphoinositide tested, whilst the C-terminal PH domain bound PtdIns(3,4)P 2 with high affinity, but did not bind PtdIns(3,4,5)P 3 or any other phosphoinositide tested.
  • TAPP1 and TAPP2 are the first proteins reported to interact specifically with PtdIns(3,4)P 2 and not with PtdIns(3,4,5)P 3 and may therefore be mediators of cellular responses that are triggered bv PtdIns(3,4)P 2 .
  • a first aspect of the invention provides a method for selecting or designing a compound for modulating the activity of Tandem PH domain containing Protein (TAPP), the method comprising the step of using molecular modelling means to select or design a compound that is predicted to interact with the phosphoinositide binding domain of TAPP, wherein a three-dimensional structure of at least a part of the phosphoinoisitide binding domain of the TAPP is compared with a three- dimensional structure of a compound, and a compound that is predicted to interact with the said phosphoinositide binding domain is selected. It is preferred that the compound is for modulating the phosphoinositide binding activity of TAPP. Other activities of TAPP that may be modulated include interactions with other polypeptides, which may in turn be modulated by binding of phosphoinositides to TAPP.
  • the compound may be capable of affecting the intracellular location of the polypeptide; for example, it may inhibit or promote the translocation of the polypeptide to a membrane, for example the plasma membrane or golgi, vacuole, lysosome or endosome membrane.
  • a membrane for example the plasma membrane or golgi, vacuole, lysosome or endosome membrane.
  • Possible association with cellular membranes of polypeptides comprising a PH domain with the required phosphoinositide binding properties are discussed further in Example 1.
  • the compound may modulate any interaction of the polypeptide with further identical polypeptide molecules (ie self-association, for example dimerisation).
  • a compound that is capable of modulating the ability of the polypeptide to bind to a phosphoinositide may thereby modulate the intracellular location of the polypeptide molecule and/or modulate any post-translational modification, for example phosphorylation, of the polypeptide.
  • the three-dimensional structure of at least a part of the phosphoinositide binding domain of the TAPP is a three-dimensional structure of at least a part of the phosphoinositide binding site of the TAPP and a compound that is predicted to interact with the said phosphoinositide binding site is selected.
  • the compound may bind to a portion of said TAPP polypeptide that is not the binding site so as to interfere with the binding of the TAPP polypeptide to the phosphoinositide.
  • the compound may bind to a portion of said polypeptide so as to decrease said polypeptide's binding activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide' s activity.
  • the three-dimensional structure of at least a part of the phosphoinositide binding site of the TAPP is a three-dimensional structure of the part of the phosphoinositide binding site of TAPP that is defined by loops ⁇ l- ⁇ 2, ⁇ -3- ⁇ 4, ⁇ 6- ⁇ 7 of full-length TAPPl and a compound that is predicted to interact with the said part of the phosphoinositide binding site is selected.
  • Tandem Pleckstrin Homolgy domain containing protein is included the polypeptides termed mammalian (for example human or mouse) TAPPl and TAPP2 identified in Dowler et al (2000) Biochem J 351, 19-31 and in GB patent applications No 0018908.4 (filed on 3 August 2000) and 0021685.3 (filed on 5 September 2000).
  • the term also includes fragments and fusions thereof that comprise the C-terminal PH domain, as discussed further below and in Example 1.
  • a particularly preferred TAPP polypeptide is the C-terminal PH domain of TAPPl, optionally with an N-terminal GST tag, as described in Example 1.
  • able to bind or “capable of binding” is meant that binding of the said polypeptide to the said phosphoinositide can be detected using a surface plasmon resonance or protein lipid overlay technique as described in Example 1 and discussed further below.
  • substantially unable to bind is meant that binding of the said polypeptide to the said phosphoinositide is not detected, or is only weakly detected using a surface plasmon resonance or protein lipid overlay technique as described in Example 1. It is preferred that the TAPP polypeptide binds to PtdIns(3,4)P 2 with at least two, preferably 3, 5, 10, 15, 20, 30 or 50-fold higher affinity than to other phosphoinositides, in particular PtdIns(3,4,5)P 3 .
  • the binding of the said TAPP polypeptide to PtdIns(3,4)P 2 has an apparent K D of less than about 2000 nM, 1000 nM or 500 nM, preferably less than about 400 or 350 nM, for example between about 350 nM and 10 nM, when measured using the method described in Example 1. It is preferred that the binding of the said polypeptide to other phosphoinositides, particularly PtdIns(3,4,5)P 3 has an apparent K D of more than about 2000 nM, 1000 nM or 500 nM when measured using the method described in Example 1.
  • TAPP polypeptide for example a fragment, variant, derivative or fusion of full length TAPP, or fusion of a variant, fragment or derivative
  • PtdIns(3,4)P 2 phospholipids
  • Example 1 Dowler et al (supra) and GB application Nos 0018908.4 and 0021685.3 (supra) and include a protein-lipid overlay assay in which the lipid is spotted onto a support, for example Hybond-C extra membrane, and protein bound to the support by virtue of interaction with the lipid is detected, for example using an antibody-based method, as well know to those skilled in the art.
  • a surface plasmon resonance assay for example as described in GB application Nos 0018908.4 and 0021685.3 (supra) or in Plant et al (1995) Analyt Biochem 226(2), 342-348, may alternatively be used.
  • Methods may make use of a said polypeptide, for example comprising a PH domain, or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative that is labelled, for example with a radioactive or fluorescent label.
  • Suitable methods may also be ⁇ described in, for example, Shirai et al (1998) Biochim Biophys Ada 1402(3), 292- 302 (use of an affinity column prepared using phosphatidylinositol analogues) and Rao et al (1999) J Biol Chem 274, 37893-37900 (use of avidin-coated beads bound to biotinylated phosphatidylinositol analogues).
  • the variant or fragment or derivative or fusion of the TAPP, or the fusion of the variant or fragment or derivative has at least 30% of the PtdIns(3,4)P 2 binding activity of full-length human TAPPl. It is more preferred if the variant or fragment or derivative or fusion of the said TAPP, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the PtdIns(3,4)P 2 binding activity of full-length human TAPPl .
  • a variant, fragment, derivative or fusion of TAPP comprises the N-terminal of the two PH domains of TAPP.
  • This PH domain may be capable of interacting with polypeptides, as discussed in GB application Nos 0018908.4 and 0021685.3 (supra).
  • a variant, fragment, derivative or fusion of TAPP comprises (preferably as the C-terminal three residues) the last three residues of TAPP (for example TAPPl or TAPP2), which conform to the minimal sequence motif (Ser/Thr-Xaa-Val/Ile) required for binding to a PDZ domain (as discussed in Example 1); and or one or more proline rich regions found towards the C-terminus of TAPP2 (as discussed in GB application Nos 0018908.4 and 0021685.3 (supra), which may form a binding site for an SH3 domain).
  • reagents including any fragment, derivative, variant or fusion of the polypeptide or fusion of a variant, fragment or derivative
  • conditions used in screening methods as discussed further below may be chosen such that the interactions between the said polypeptide and a phosphoinositide, for example PtdIns(3,4)P 2 , or an interacting polypeptide are substantially the same as between the wild-type, preferably human polypeptide (for example TAPPl or TAPP2) and the phosphoinositide or interacting polypeptide in vivo.
  • variants of a polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of TAPP, as described above.
  • the TAPP variant has an amino acid sequence which has at least 65% identity with the amino acid sequence of TAPPl or TAPP2 referred to above, more preferably at least 70%, 71%, 72%, 73% or 74%, still more preferably at least 75%, yet still more preferably at least 80%, in further preference at least 85%, in still further preference at least 90% and most preferably at least 95% or 97% identity with the amino acid sequence defined above.
  • the TAPP variant has an amino acid sequence which has at least 65% identity with the amino acid sequence of the C-terminal PH domain, particularly the residues forming the phosphoinositide binding site, of TAPP in the appropriate sequence referred to above, more preferably at least 70%, 71%, 72%, 73% or 74%, still more preferably at least 75%, yet still more preferably at least 80%, in further preference at least 83 or 85%, in still further preference at least 90% and most preferably at least 95% or 97% identity with the amino acid sequence defined above.
  • PH domain may be readily identified by a person skilled in the art, for example using sequence comparisons, for example as described in Dowler et al (2000) supra and GB Application Nos 0018908.4 and 0021685.3 (supra).
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson et al (1994) Nucl Acid Res 22, 4673-4680). The parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
  • the TAPP is a polypeptide which consists of the amino acid sequence of the TAPPl or TAPP2 (preferably TAPPl) sequence referred to above or naturally occurring allelic variants thereof. It is preferred that the naturally occurring allelic variants are mammalian, preferably human, but may alternatively be homologues from parasitic or pathogenic or potentially pathogenic organisms.
  • the TAPP may be Myc epitope-tagged or His-tagged or GST-tagged, as described in Example 1. It may be a GFP (green fluorescent protein) fusion, as known to those skilled in the art.
  • GFP green fluorescent protein
  • the method of the first aspect of the invention may further comprise the step of comparing a three-dimensional structure of at least a part of a Ptdlns (3,4,5)P 3 binding domain with a three-dimensional structure of a compound, and a compound that is predicted to interact with the said phosphoinositide binding domain (or site or part thereof) of TAPP with a higher affinity than it is predicted to interact with the said Ptdlns (3,4,5)P 3 binding domain is selected.
  • the three-dimensional structure of at least a part of the Ptdlns (3,4,5)P 3 binding domain is a three-dimensional structure of at least a part of the Ptdlns (3,4,5)P 3 binding site of the Ptdlns (3,4,5)P 3 binding domain and a compound that is predicted to interact with the said phosphoinositide binding domain (or site or part thereof) of TAPP with a higher affinity than it is predicted to interact with the said Ptdlns (3,4,5)P 3 binding site is selected.
  • the three- dimensional structure of at least a part of the Ptdlns (3,4,5)P 3 binding site is a three-dimensional structure of the part of the Ptdlns (3,4,5)P 3 binding site that is defined by loops ⁇ l- ⁇ 2, ⁇ -3- ⁇ 4, ⁇ 6- ⁇ 7 of a Ptdlns (3,4,5)P 3 binding domain and a compound that is predicted to interact with the said phosphoinositide binding domain (or site or part thereof) of TAPP with a higher affinity than it is predicted to interact with the said part of the Ptdlns (3,4,5)P 3 binding site is selected.
  • the structures of the portion of the TAPP phosphoinositide binding site that is responsible for its specificity for PtdIns(3,4)P 2 , and the portion of the PtdIns(3,4,5)P 3 binding site that is responsible for its specificity for PtdIns(3,4,5)P 3 are compared with the structure of the test compound.
  • a compound is selected that matches this portion of the PtdIns(3,4)P 2 binding site more closely than it matches this portion of the PtdIns(3,4,5)P 3 binding site.
  • the Ptdlns (3,4,5)P 3 binding domain is the Ptdlns (3,4,5)P 3 binding domain of Dual Adaptor for Phosphotyrosine and Phosphoinositide (DAPP).
  • the PtdIns(3,4,5)P 3 binding domain may be a mutated phosphoinositide binding domain of TAPP which is capable of binding to PtdIns(3,4,5)P 3 , for example in which the residue equivalent to Ala203 is mutated, for example to a small residue, for example a gly cine residue, as described in Example 1, or in which both the residue equivalent to Ala203 and the residue equivalent to Val204 are mutated, for example to a small residue, for example a glycine residue, as described in Example 1. It is preferred that the residue equivalent to Met205 is not additionally mutated, for example to a glycine, as discussed in Example 1.
  • DAPP is included the polypeptides termed mammalian (for example human or mouse) DAPPl identified in Dowler et al (1999) Biochem J 342, 7-12 (and also references 11 to 13 of Example 1) and in WO01/11042.
  • the term also includes fragments and fusions thereof that comprise the PH domain, as discussed in Example 1, Dowler et al (1999; supra) and WO01/11042.
  • a particularly preferred DAPP polypeptide is the PH domain of DAPPl, optionally with an N- terminal GST tag, as described in Example 1.
  • DAPP polypeptide for example a fragment, variant, derivative or fusion of full length DAPP, or fusion of a variant, fragment or derivative
  • PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 are described in Example 1, Dowler et al (1999) and WO01//11042 and include techniques mentioned above in relation to TAPP.
  • the variant or fragment or derivative or fusion of the DAPP, or the fusion of the variant or fragment or derivative has at least 30% of the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding activity of full-length human DAPPl. It is more preferred if the variant or fragment or derivative or fusion of the said DAPP, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding activity of full-length human DAPP 1.
  • a further aspect of the invention provides a method for selecting or designing a compound for modulating signalling via PtdIns(3,4)P 2 , the method comprising the step of using molecular modelling means to select or design a compound that is predicted to interact with a PtdIns(3,4)P 2 -specific binding domain, wherein a three-dimensional structure of at least a part of the PtdIns(3,4)P 2 binding domain is compared with a three-dimensional structure of a compound, and a compound that is predicted to interact with the said PtdIns(3,4)P 2 binding domain is selected.
  • the three-dimensional structures that are compared may be predicted three-dimensional structures or may be three-dimensional structures that have been determined, for example by techniques such as X-ray crystallography, as well known to those skilled in the art and as described in Example 1.
  • the three-dimensional structures may be displayed by a computer in a two-dimensional form, for example on a computer screen. The comparison may be performed using such two-dimensional displays.
  • the structure is of the PtdIns(3,4)P 2 binding domain of TAPP, still more preferably TAPPl . Most preferably, it is the structure reported in Example 1, for which the coordinates are shown in Figure 9.
  • the structure may be a structure of another PtdIns(3,4)P 2 -specific binding domain obtained or obtainable by modelling based on the structure of the PtdIns(3,4)P 2 binding domain of TAPP and any other relevant structural information.
  • the structure may be a structure of the PH domain of DAPP mutated so that it binds PtdIns(3,4)P 2 but does not detectably bind PtdIns(3,4,5)P 3 , for example mutated to replace the residue equivalent to Gly 176 of full length DAPPl with a larger residue, for example an alanine residue or other residue of similar bulk, for example serine, valine, tln-eonine or cysteine, as described in Example 1.
  • the structure is not a structure of a DAPP polypeptide that has the same (ie substantially the same) binding specificity as wild-type DAPPl, ie that is capable of binding to both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 .
  • a further aspect of the invention provides method for selecting or designing a compound for modulating signalling via PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 to different extents, the method comprising the step of using molecular modelling means to select or design a compound that is predicted to interact with a PtdIns(3,4)P 2 binding domain and a PtdIns(3,4,5)P 3 binding domain with different affinities, comprising the steps of (1) comparing a three-dimensional structure of at least a part of the PtdIns(3,4)P 2 binding domain with a three-dimensional structure of a compound, and (2) comparing a three-dimensional structure of at least a part of a Ptdlns (3,4,5)P 3 binding domain with a three-dimensional structure of the compound, and a compound that is predicted to interact with the said PtdIns(3,4)P 2 binding domain and the said Ptdlns (3,4,5)P 3 binding domain with different affinities is selected.
  • the PtdIns(3,4)P 2 binding domain is the PtdIns(3,4)P 2 binding domain of TAPP. It is further preferred that the Ptdlns (3,4,5)P 3 binding domain is the Ptdlns (3 ,4,5)P 3 binding domain of DAPP. Alternatively, the Ptdlns (3,4,5)P 3 binding domain may be a mutated phosphoinositide binding domain of TAPP which is capable of binding to PtdIns(3,4,5)P 3 , as discussed above.
  • a further aspect of the invention provides a method of selecting or designing a compound that modulates the activity of a PH domain-containing polypeptide, the method comprising the step of using molecular modelling means to select or design a compound that is predicted to interact with a PH domain, wherein a three- dimensional structure of a polycarboxylate, for example citrate, is compared with a three-dimensional structure of a compound, and a compound that is predicted on the basis of the structure comparison to interact with the PH domain is selected.
  • a polycarboxylate molecule is able to bind to a PH domain, at least with low affinity.
  • a polycarboxylate molecule is able to bind to a PH domain, at least with low affinity.
  • a compound that can interact with the PH domain preferably with the phosphoinositide binding site, in particular the residues considered to confer PtdIns(3,4)P 2 specificity, in a similar manner (for example similar separation and/or type of interaction ie hydrophobic or ionic, and/or similar cumulative energy of interaction) to the interacting polycarboxylate may be selected.
  • Methods of assessing the interaction are well known to those skilled in the art.
  • the PH domain is capable of binding to a phosphoinositide and a compound that is predicted to interact with the phosphoinositide binding site of the PH domain is selected. It is further preferred that the PH domain is the phosphoinositide binding PH domain of TAPP, for example TAPPl or TAPP2.
  • design we include the meaning that the agent is made or modified so that it contains one or more functional groups which it is expected or known will bind all or part of the desired phosphoinositide binding domain, particularly phosphoinositide binding site, still more particularly part of a phosphoinositide binding site that is important in determining what type(s) of phosphoinositide are bound.
  • Exemplary methods include computer aided design.
  • the aim of using a computer for drug design is to analyse the interactions between the drug and its receptor site and to "design" molecules that give an optimal fit.
  • the central assumption is that a good fit results from structural and chemical complementarity to the target receptor.
  • the techniques provided by computational methods include computer graphics for visualisation and the methodology of theoretical chemistry. By means of quantum mechanics the structure of small molecules can be predicted to experimental accuracy. Statistical mechanics permit molecular motion and solvent effects to be incorporated.
  • US Patents Nos. 5,891,643; 5,804,390; 5,698,401; and 5,989,827 relate to methods for designing compounds which bind to a specific target molecule, involving two- dimensional 15 N-1H NMR correlation spectroscopy techniques. Such methods may be used in accordance with the present invention and the disclosure of those patents incorporated herein by reference.
  • GRID Goodford (1985) J Med Chem 28, 849-857; available from Oxford University, Oxford, UK
  • MCSS Miranker et al (1991) Proteins: Structure, Function and Genetics 11, 29-34; available from Molecular Simulations, Burlington, MA
  • AUTODOCK Goodsell et al (1990) Proteins: Structure, Function and Genetics 8, 195-202; available from Scripps Research Institute, La Jolla, CA
  • DOCK Kuntz et al (1982) JMol Biol 161, 269- 288; available from the University of California, San Francisco, CA
  • LUDI Bohm (1992) J Comp Aid Molec Design 6, 61-78; available from Biosym Technologies, San Diego, CA
  • LEGEND Neishibata et al (1991) Tetrahedron 47, 8985; available from Molecular Simulations, Burlington, MA
  • LeapF LeapF
  • selection we include affimty purification techniques for identifying agents which are capable of binding to all or part of the PtdIns(3,4)P 2 binding site. Examples include affinity chromatography and phage display technologies.
  • the agents identified or obtained according to the above aspects of the invention may be a drug-like compound or lead compound for the development of a drug-like compound.
  • the methods may be methods for identifying a drag-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 5000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.
  • 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 drag (for example because it is only weakly potent against its intended target, non- selective in its action, unstable, poorly soluble, difficult to synthesise, too toxic or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • the compounds identified in the methods of the invention may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds.
  • the selected or designed compound may be synthesised (if not already synthesised) and tested for its effect on a phosphoinositide binding domain containing polypeptide, for example TAPP or DAPP, for example its effect on the phosphoinositide binding activity.
  • the compound may be tested in a screening method of the invention, as discussed further below.
  • a further aspect of the invention provides a method for identifying a compound that modulates the phosphoinositide binding activity of a PH domain containing polypeptide, comprising the step of detennining the effect of the compound on the phosphoinositide binding activity of, or ability of the compound to bind to, the PH domain containing polypeptide, wherein the compound is a poly-carboxylate or variant or derivative thereof.
  • the PH domain is capable of interacting with PtdIns(3,4)P 2 .
  • the PH domam-containing polypeptide is or comprises TAPP (for example TAPPl or TAPP2) or the C-terminal pH domain of full length TAPP.
  • the compound may be a variant or derivative of citrate.
  • derivatives of -C0 2 H groups include groups which are commonly derived from a carboxylic acid and/or groups that contain a central carbon atom that is at the same oxidation state as -C(0)OH. Derivatives of -C0 2 H groups therefore includes groups such as:
  • esters e.g. those formed with an alcohol of formula R ⁇ H, wherein R 1 represents aryl or alkyl
  • thioesters e.g. those formed with a thiol of formula R ⁇ H, wherein R 1 is as hereinbefore defined
  • salts e.g. those formed with a nitrogen-containing base such as ammonia, an alkylamine, a dialkylamine, a Ixialkylamine and pyridine or alkali or alkaline earth metal salts (e.g. Na, K, Cs, Mg or Ca salts).
  • Preferred derivatives of -C0 2 H groups include those that are pharmaceutically acceptable.
  • the compound is capable of adopting a conformation that has a similar charge distribution to a citrate molecule or a PtdIns(3,4)P 2 molecule.
  • the compound may be a phosphoinositide derivative in which one or more phosphate groups is replaced by a carboxyl group. Such a compound may be kinase- resistant.
  • a further aspect of the invention provides a method for identifying a compound for modulating the activity of TAPP and/or modulating signalling via PtdIns(3,4)P 2 or PtdIns(3,4,5)P 3 , the method comprising the step of determining the effect of the compound on the activity of, or ability of the compound to bind to, (1) a mutated TAPP or mutated phosphoinositide binding domain of TAPP, mutated at a residue of loop ⁇ l- ⁇ 2, ⁇ -3- ⁇ 4 or ⁇ 6- ⁇ 7 defining at least part of the phosphoinositide binding site of TAPP, and optionally also (2) TAPP or the phosphoinositide binding domain of TAPP which is not mutated at a said residue, and selecting a compound which affects to different extents the activity of, or binds with different affinities to, the unmutated TAPP/domain and the mutated TAPP/domain.
  • the effect of the compound on the phosphoinositide binding activity of the mutated TAPP/domain and optionally unmutated TAPP/domain is determined.
  • the effect of the compound on an activity that is modulated by phosphoinositide binding to TAPP may be measured. Examples of such activities are noted above and may include changes in conformation, cellular location and/or interaction with other polypeptide(s).
  • the screening method may identify compounds which inhibit binding of phosphoinositides to TAPP, and compounds which mimic binding of phosphoinositides to TAPP, as will be apparent to those skilled in the art. It will be appreciated that screens in which binding of compounds to TAPP is measured may be used in conjunction with screens in which the effect of compounds (for example compounds identified in the binding screen(s)) on one or more activities of TAPP (for example phosphoinositide binding activity) is measured.
  • mics the effect of the interaction of a phosphoinositide for example PtdIns(3,4)P 2 with the TAPP is meant that the compound has a quantitative or qualitative effect on the TAPP, for example on its conformation, cellular location, or ability to interact with other polypeptide(s), that is the same as an effect of the phosphoinositide on the TAPP, for example on its confonnation, cellular location, or ability to interact with other polypeptide(s).
  • the mutated TAPP/domain is capable of binding to PtdIns(3,4,5)P 3 .
  • Examples of such mutated TAPP are discussed in Example 1. It is particularly preferred that the TAPP/domain is mutated at a residue of loop ⁇ l- ⁇ 2, for example at the residue equivalent to Arg212, Ala203 and/or Val204 of full length TAPPl. It is most preferred that the TAPP is mutated at the residue equivalent to Ala203 of full length TAPPl, or at the residues equivalent to Ala203 and Val204 of full length TAPP 1 , as discussed in Example 1.
  • a further aspect of the invention provides method for identifying a compound for modulating the activity of Dual Adaptor for phosphotyrosine and phosphoinositide (DAPP), and/or modulating signalling via PtdIns(3,4)P 2 or PtdIns(3,4,5)P 3 , the method comprising the step of determining the effect of the compound on the activity of, or ability of the compound to bind to, (1) a mutated DAPP or mutated phosphoinositide binding domain of DAPP, which is capable of binding PtdIns(3,4)P 2 and is less capable of binding to PtdIns(3,4,5)P 3 than unmutated DAPP, and (2) DAPP or the phosphoinositide binding domain of DAPP which is not so mutated, and selecting a compound which affects to different extents the activity of, or binds with different affinities to, the unmutated DAPP/domain and the mutated DAPP/domain.
  • DAPP Dual Adaptor for phosphotyrosine and
  • a further aspect of the invention provides a method for identifying a compound for modulating signalling via PtdIns(3,4)P 2 , the method comprising the step of deterrnuding the effect of the compound on the activity of, or ability of the compound to bind to, mutated DAPP, or mutated phosphoinositide binding domain of DAPP, which is capable of binding PtdIns(3,4)P 2 and is less capable of binding to PtdIns(3,4,5)P 3 than unmutated DAPP; and selecting a compound which affects the activity of, or is capable of binding to, the said mutated DAPP/domain.
  • the DAPP/domain is mutated at the residue equivalent to Gly 176 of full-length DAPPl, to a larger residue such as alanine, valine, serine, cysteine or fhreonine as discussed in Example 1.
  • any convenient method may be used to measure the interaction of the DAPP/TAPP domain with the compound or the effect of such binding.
  • the appropriate methods make use of the methods described in Example 1 for detecting and/or quantifying the interaction between a polypeptide and a phospholipid, for example a protein-lipid overlay or surface plasmon resonance method, as discussed above. It is preferred that a GST-tagged fusion polypeptide is used. Methods in which radioactively or fluorescently labelled lipids are used may also be useful.
  • Suitable methods include yeast two-hybrid interactions, co-purification, ELISA, co-immunoprecipitation methods and cellular response assays.
  • Cellular response assays may be carried out in a variety of cell types, for example in adipocytes or adipocyte cell lines, in a skeletal muscle cell line (such as the L6 myotubule cell line), liver cells or liver cell lines or cancer cells or cancer cell lines.
  • Platelets may be preferred when the polypeptide is TAPP.
  • NIH Swiss mouse embryo cells NIH/3T3 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (ATCC) as CRL 1658
  • human embryonic kidney 293 cells also available from the ATCC
  • preferred cell types include haematopoietic cells or cell lines (for example RAW macrophages, BT cells, primary differentiated T cells).
  • Other cell types for example 293 cells or NIH3T3 cells may also be used.
  • the method may be perfonned in vitro, either in intact cells or tissues, with broken cell or tissue preparations or at least partially purified components. Alternatively, they may be performed in vivo.
  • the cells, tissues or organisms in/on which the method is performed are preferably transgenic.
  • they may be transgenic for TAPP and/or DAPP, for example may express the said mutated TAPP or DAPP in place of endogenous TAPP or DAPP.
  • They may express human TAPP or DAPP in place of endogenous TAPP or DAPP.
  • They may express mutated DAPP in place of TAPP.
  • the interaction of the DAPP/TAPP/domain with the compound is measured using fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the interaction of the DAPP/TAPP/domain with the compound is measured using surface plasmon resonance.
  • screening assays which are capable of high throughput operation will be particularly preferred.
  • Examples may include cell based assays, and protein-protein binding assays.
  • An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used, as well know to those skilled in the art.
  • SPA-based Scintillation Proximity Assay; Amersham International
  • either the polypeptide or a phosphoinositide may be immobilised on the SPA beads and the ability of the test compound to disrupt the interaction between the polypeptide and phosphoinositide may be measured.
  • Phosphoinositides or analogues thereof may be immobilised on SPA beads, for example using methods as described in Shirai et al (1998) Biochim Biophys Ada 1402(3), 292-302 or in Rao et al (1999) JBiol Chem 274, 37893-37900.
  • the ability of the compound to bind to the said polypeptide may be measured by measuring the ability of the compound to disrupt or prevent the interaction between the polypeptide (or variant, fragment, derivative or fusion) and the interacting phosphoinositide.
  • the binding constant for the binding of the compound to the relevant polypeptide may be determined.
  • Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example using a method capable of high throughput operation, for example a chip-based method in which the compounds to be tested are immobilised in a microarray on a solid support, as known to those skilled in the art.
  • screening assays of the invention are useful for identifying compounds which may be useful in the treatment of diabetes, defects of glycogen metabolism, cancer (including melanoma), inflammatory conditions, ischaemic conditions, for example stroke, thrombosis or tendency to thrombosis (for example useful as an antithrombotic agent).
  • the following techniques may be useful in assessing compound or phosphoinositide binding: calorimetric measurements, measurements using rritiated or other labelled phosphoinositides or headgroups, gel filtration methods (to separate bound and unbound compound or phosphoinositide), protein- lipid overlay (for example in a 384-well assay format).
  • drag-like compounds” and “lead compounds” identified in the screening assays of the invention are suitably screened in further screens to determine their potential usefulness in treating diabetes, defects of glycogen metabolism, cancer (including melanoma), inflammatory conditions, ischaemic conditions, for example stroke, or thrombosis or tendency to thrombosis. Additional screens which may be carried out include deterrnuding the effect of the compounds on blood glucose levels, tumour growth or blood clotting tendency/time, as appropriate. This may typically be done in rodents.
  • a further aspect of the invention provides a mutated TAPP wherein one or more residues defining the PtdIns(3,4)P 2 binding site of TAPP is mutated and the mutated TAPP is capable of binding PtdIns(3,4,5)P 3 .
  • a further aspect of the invention provides a mutated TAPP wherein one or more of residues equivalent to residue Ala203, Val204, Met205, Thr207 of full-length human TAPPl is mutated.
  • residue at the position equivalent to residue Ala203 is mutated to a small residue, for example Gly. It is further preferred that the residue at the position equivalent to residue Ala203 and the residue at the position equivalent to residue Val204 are both mutated to a small residue, for example Gly.
  • residue at the position equivalent to residue Val204 is mutated to a Leu
  • residue at the position equivalent to residue Mef205 is mutated to a Val
  • residue at the position equivalent to residue Thr207 is mutated to Asn.
  • Mutation of the residue equivalent to Ala203 may be necessary for the mutated TAPP to bind to PtdIns(3,4,5)P 3 . It is preferred that the residues at the positions equivalent to Ala203, Val204 and Met205 are not all mutated to Gly, as discussed in Example 1.
  • a further aspect of the invention provides a mutated DAPP wherein one or more residues defining the PtdIns(3,4,5)P 3 binding site of DAPP is mutated and the mutated DAPP is less capable of binding PtdIns(3,4,5)P 3 than the unmutated DAPP.
  • the mutated residue is the residue equivalent to residue Gly 176 of full length human DAPPl, which is preferably mutated to a larger residue, for example an alanine or another small residue, for example serine, cysteine, valine or threonine.
  • residue equivalent to a particular residue, for example the residue equivalent to Ala203 of human TAPPl, is included the meaning that the amino acid residue occupies a position in the native two or three dimensional structure of a polypeptide corresponding to the position occupied by the said particular residue, for example Ala203, in the native two or three dimensional structure of full-length human TAPPl.
  • the residue equivalent to a particular residue may be identified by alignment of the sequence of the polypeptide with that of full-length human TAPPl in such a way as to maximise the match between the sequences.
  • the alignment may be carried out by visual inspection and/or by the use of suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, which will also allow the percent identity of the polypeptides to be calculated, or using the Align program (Pearson (1994) in: Methods in Molecular Biology, Computer Analysis of Sequence Data, Part II (Griffin, AM and Griffin, HG eds) pp 365-389, Humana Press, Clifton).
  • residues identified in this manner are also "equivalent residues”.
  • a further aspect of the invention provides a recombinant polynucleotide suitable for expressing a mutated polypeptide of the invention.
  • a still further aspect of the invention provides a host cell comprising a polynucleotide of the invention.
  • the host cell is capable of expressing a mutated polypeptide of the invention in place of the unmutated polypeptide (ie in place of endogenous TAPP or DAPP).
  • a further aspect of the invention provides a transgenic, non-human animal comprising a host cell as defined above, in particular comprising cells which are capable of expressing a mutated polypeptide of the invention in place of the unmutated polypeptide.
  • a further aspect of the invention provides a method of making a mutated polypeptide of the invention, the method comprising culturing a host cell of the invention (preferably in cell or tissue culture) which expresses said polypeptide and isolating said polypeptide.
  • a further aspect of the invention provides a polypeptide obtainable by this method.
  • a further aspect of the invention provides an antibody which binds to a mutated polypeptide of the invention but does not bind to the corresponding unmutated polypeptide.
  • the antibody may interact with an epitope comprising the amino acid sequence of TAPP flanking the residue equivalent to Ala203 or Val204 of full length TAPPl, or with an epitope comprising the amino acid sequence of DAPP flanking the residue equivalent to Gly 176 of full length DAPPl.
  • Antibodies reactive towards the said polypeptides may be made by methods well known in the art.
  • the antibodies may be polyclonal or monoclonal.
  • Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982).
  • Such antibodies may mimic binding of a phosphoinositide to the polypeptide or may alternatively or in addition act as inhibitors of phosphoinositide binding to the polyppeptide.
  • a further aspect of the invention provides a kit of parts useful in carrying out a screening method of the invention relating to polycarboxylates, comprising a PH domain containing polypeptide, preferably a PtdIns(3,4)P 2 binding PH domain, still more preferably TAPP or the C-terminal PH domain of full length TAPP, and at least one polycarboxylate or polycarboxylate variant or derivative compound.
  • a further aspect of the invention provides a kit of parts useful in carrying out other screening methods of the invention comprising (1) TAPP which is not a mutated TAPP of the invention, or DAPP which is not a mutated DAPP of the invention and (2) a mutated TAPP of the invention, or a mutated DAPP of the invention.
  • a further aspect of the invention provides a compound identified or identifiable by a screening or selection/design method of the invention, wherein the compound is not citrate, PtdIns(3,4)P 2 , Ptdlns(3,4,5), or an inositol phosphate.
  • the compound may be an antibody of the invention, for example an antibody capable of binding to the part of the TAPP phosphoinositide binding site that is responsible for its specificity for PtdIns(3,4)P 2 , or may be a polycarboxylate molecule.
  • the compound may be capable of modulating the phosphoinositide binding activity of a PtdIns(3,4)P binding polypeptide and a PtdIns(3,4,5)P 3 binding polypeptide and/or the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding to a phosphoinositide binding polypeptide to different extents.
  • the compound may be capable of modulating the phosphoinositide binding activities of DAPP and TAPP to different extents, and/or may be capable of modulating the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding activities of DAPP to different extents.
  • the compound is not a phosphoinositide.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound identified or identifiable by a screening or selection/design method of the invention, wherein the compound is not citrate, PtdIns(3,4)P 2 , Ptdlns(3,4,5), or an inositol phosphate, and a pharmaceutically acceptable excipient.
  • a further aspect of the invention provides a said compound or composition for use in medicine.
  • Compounds, identifiable in the screening method, which mimic the effect of a particular phosphoinositide on a polypeptide, for example TAPP or DAPP are believed to be useful in treating diabetes and/or other conditions, as indicated above.
  • Compounds identifiable in the screening methods of the invention that inhibit binding of a phosphoinositide to the said polypeptide are believed to be useful in treating cancer.
  • Compounds may be used, for example, for treatment of diabetes by switching on insulin-stimulated signal transduction pathways or for the treatment of cancer by inhibiting cell proliferation or promoting apoptosis.
  • Compounds may also be useful in the modulation or resolution of inflammation or platelet activation, as discussed above.
  • certain compounds found in the screening methods may be able to enhance cell proliferation in a beneficial way and may be useful, for example in the regeneration of nerves or in wound healing.
  • a further aspect of the invention provides the use of a said compound or composition in the manufacture of a medicament for the treatment of a patient in need of modulation of phosphoinositide binding or signalling, or a patient with an inflammatory or an ischaemic disease, cancer, diabetes, thrombosis or a defect in glycogen metabolism (or at risk of such a condition).
  • inflammatory disease is included immune system disorders, for example autoimmune diseases, as will be apparent to those skilled in the art.
  • a further aspect of the invention provides a method of treating a patient in need of modulation of phosphoinositide binding or signalling, or a patient with an inflammatory or an ischaemic disease, cancer, diabetes, thrombosis or a defect in glycogen metabolism (or at risk of such a condition), wherein the patient is administered an effective amount of a said compound or composition.
  • a further aspect of the invention provides a method of modulating the activity, for example phosphoinositide binding activity of a PH domain containing polypeptide (preferably TAPP or DAPP) the method comprising the step of exposing the PH domain containing polypeptide to a said compound or composition.
  • the method may be performed in vitro or in vivo. It may be performed in cells or in cell free systems, for example using purified components.
  • a further aspect of the invention provides a mutated TAPP or DAPP of the invention or polynucleotide encoding said mutated TAPP or DAPP for use in medicine.
  • a further aspect of the invention accordingly provides a pharmaceutical composition
  • a pharmaceutical composition comprising a mutated TAPP or DAPP of the invention or polynucleotide encoding said mutated TAPP or DAPP and a pharmaceutically acceptable excipient.
  • a further aspect of the invention provides the use of a said polypeptide or polynucleotide or composition in the manufacture of a medicament for the treatment of a patient in need of modulation of phosphoinositide binding or signalling, or a patient with an inflammatory or an ischaemic disease, cancer, diabetes, thrombosis or a defect in glycogen metabolism (or at risk of such a condition).
  • a further aspect of the invention provides a method of treating a patient in need of modulation of phosphoinositide binding or signalling, or a patient with an inflammatory or an ischaemic disease, cancer, diabetes, thrombosis or a defect in glycogen metabolism (or at risk of such a condition), wherein the patient is administered an effective amount of a said polypeptide or polynucleotide or composition.
  • a further aspect of the invention provides the use of a mutated TAPP or DAPP of the invention, or polynucleotide encoding said mutated TAPP or DAPP in a compound screening method, for example a screening method as described above.
  • the method may be performed in vitro or in vivo. It may be performed in cells or in cell free systems, for example using purified components.
  • the compound (or polypeptide or polynucleotide) may be administered to a patient (or test subject) in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intravesically, in standard sterile, non-pyrogenic formulations of diluents and carriers.
  • the compound (or polypeptide or polynucleotide) may also be administered topically.
  • the compound (or polypeptide or polynucleotide) may also be administered in a localised manner, for example by injection.
  • the patient is in need of inhibition of PtdIns(3,4)P 2 signalling but not in need of inhibition of PtdIns(3,4,5)P 3 signalling.
  • the patient may be in need of inhibition of PtdIns(3,4,5)P 3 signalling but not in need of inhibition of PtdIns(3,4)P 2 signalling.
  • TAPPl-PH cr is shown as a grey ribbon drawing, with residues lining the ligand binding pocket drawn in a sticks representation with grey carbons.
  • the PtdIns(3,4,5)P 3 headgroup observed in the DAPPl structure is shown with the ring in dark grey and the phosphate groups in dark and light grey.
  • DAPPl VL1 is shown as a string, with residues contacting the inositol head group in black.
  • the ordered citrate molecule observed in the TAPP1-PH CT structure is shown as a sticks model.
  • Residue Ala203 in TAPP 1 which is mutated to a glycine in DAPP 1 , is shown.
  • FIG. 3 Phosphoinositide binding properties of wild type and VL1 mutant DAPPl /TAPPl PH domains.
  • a to K The ability of the indicated GST fusion proteins to bind a variety of phosphoinositides was analysed using a protein-lipid overlay assay. Serial dilutions of the indicated phosphoinositides (1000 pmol, 250 pmol, 125 pmol, 63 pmol, 31 pmol, 16 pmol, 8 pmol, 4 pmol, 2 pmol and 1 pmol) were spotted onto a nitrocellulose membranes which were then incubated with the indicated wild type and mutant purified GST fusions of the isolated PH domains of the DAPPl and TAPPl proteins.
  • the membranes were washed and the GST- fusion proteins bound to the membrane by virtue of their interaction with lipid were detected using a GST antibody.
  • a representative of at least 3 separate experiments carried out is shown.
  • An alignment of the sequences that encompass the VL1 loop in TAPPl and DAPPl is shown and the residues mutated are indicated.
  • - represents an identical residue to the wild type sequence.
  • Figure 4 Inhibition of TAPPl and DAPPl binding to 3 -phosphoinositides by citrate.
  • Figure 9 Crystal structure coordinates for TAPPl.
  • Example 1 Crystal structure of the phosphatidylinositol (3,4)-bisphosphate binding PH domain of TAPPl- molecular basis of lipid specificity.
  • Phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) and its immediate breakdown product PtdIns(3,4)P 2 function as second messengers in growth factor and insulin induced signalling pathways.
  • PtdIns(3,4,5)P 3 Phosphatidylinositol-(3,4,5)P 3
  • PtdIns(3,4)P 2 function as second messengers in growth factor and insulin induced signalling pathways.
  • PtdIns(3,4,5)P 3 Phosphatidylinositol-(3,4,5)P 3
  • PtdIns(3,4)P 2 function as second messengers in growth factor and insulin induced signalling pathways.
  • PtdIns(3,4)P 2 proteins
  • PtdIns(3,4)P 2 proteins
  • PtdIns(3,4)P 2 proteins
  • PtdIns(3,4)P 2 proteins
  • TAPPl Tandem Pleckstrin Homology domain containing protein- 1
  • the structure was solved in complex with an ordered citrate molecule, which provides a scaffold for possible Ptdlns-mimetics that could have a chemotherapeutical potential.
  • TAPPl -PH CT -Citrate complex is the first structure of a PH domain bound to a non-natural ligand.
  • an ordered citrate molecule has also been observed in the structure of a FYVE domain, where it also occupies the Ptdlns bmding site [23].
  • Our TAPPl -PH CT - citrate complex may provide a scaffold for the design of Ptdlns-mimetics which could selectively block the recruitment of proteins carrying PH domains, thus interfering with the signal transduction cascade.
  • DAPP 1 can bind to both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 because the 5-phosphate can be accommodated in the ligand binding pocket.
  • the 5-phosphate in the DAPP1 PH- PtdIns(3,4,5)P 3 complex has relatively weak interactions with the protein, through two hydrogen bonds with the backbone nitrogens of residues Leul77 and Vall78 (Fig. 2B).
  • TAPP! -PH CT structure there is no room for the 5-phosphate to bind.
  • the backbone conformation at residue 176 in DAPPl is near the edge of an "additional allowed" region in the Ramachandran plot, whereas residue 203 in TAPPl is near the center of a highly populated allowed region (right-handed ⁇ helical).
  • residue 203 in TAPPl is near the center of a highly populated allowed region (right-handed ⁇ helical).
  • TAPPl [V204L] only interacted with PtdIns(3,4)P 2 but with reduced affinity compared to that of type TAPPl (Fig 3E).
  • the TAPP1[M205V] mutant also exclusively bound to PtdIns(3,4)P 2 with similar apparent affinity as wild type TAPPl.
  • a mutant of TAPPl in which the 3 residues in the VL1 loop of TAPPl (e.g. AVM) were altered to those found in DAPPl (e.g. GLV) was able to interact with PtdIns(3,4,5)P 3 to a similar extent as the TAPPl [A203G] mutant (compare Figs 3D and 3G).
  • TAPP1[V204G] interacted only with PtdIns(3,4)P 2 like wild type TAPPl (Fig 3H).
  • TAPPl [A203G, V204G] mutant interacted with PtdIns(3,4,5)P 3 with significantly higher affinity than the TAPP1[A203G] mutant and remarkably even possessed higher affinity for PtdIns(3,4,5)P 3 than PtdIns(3,4)P 2 (Fig 31).
  • TAPPl -PH CT structure obtained had an ordered citrate molecule bound in the lipid binding pocket (Figs. IA, 2B) and we were unable to obtain crystals of TAPPl bound to inositol(l,3,4)P 3 , we verified whether citrate could inhibit the binding of TAPP1-PH CT and the isolated PH domain of DAPPl to 3- phosphoinositides.
  • Buffer A 50 mM Tris-HCI pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium- ⁇ - glycerophosphate, 50 mM sodium fluoride, 5 mM dithiothreitol and "complete" proteinase inhibitor cocktail (one tablet per 25 ml).
  • Buffer B 50mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.2 M NaCI and 5 mM dithiothreitol.
  • Buffer C 50 mM MES/NaOH pH6.0, 150 mM NaCI and 0.1 % (by mass) Tween-20.
  • TAPPl-PHcx The PtdIns(3,41)P 2 binding PH domain of TAPPl (TAPPl-PHcx) was amplified by PCR using the Hi-fidelity PCR system with the full length TAPPl cDNA [21] as the template, and the 5' primer
  • E. coli BL21 cells transformed with the pGEX4T-l vector encoding the expression of GST-TAPPl-PHc T were grown at 37 °C in 2 litres of Luria Broth with 50 mg/ml carbenicillin until OD 60 o reached 0.7.
  • the expression of GST-TAPP1-PH CT was induced by the addition of 250 mM isopropyl- ⁇ -D-thiogalactopyranoside, and the bacteria were then grown for a further 18 hours at 27 °C.
  • the cells were harvested by centrifugation at 3500 x g for 15 min, then lysed by resuspension in 100 ml of Buffer A containing DNAse and lysozyme and passing through a French Press.
  • the resulting solution was centrifuged at 13000 g for 30 min to remove residual debris, briefly sonicated and passed through a 0.45 ⁇ M filter. The supernatant was incubated for one hour at 4 °C in 4 ml of Glutathione-Sepharose previously equilibrated in Buffer A, and then the beads washed 6 times with 5 column volumes of Buffer B. The TAPPl PH domain was then separated from the GST-tag by incubating the Glutathione Sepharose beads conjugated to GST- TAPPI-PH CT in a 1 :1 volume of resin to Buffer B with thrombin 100 U/ml at 4°C overnight.
  • TAPP1-PH CT was analysed by SDS-polyacrylamide gel electrophoresis followed by Coomasie Blue staining of the gel and found to be essentially homogenous. It was analysed by electrospray mass spectroscopy, revealing a major single species of molecular mass of 14138.39, close to the predicted mass of 14162.24 for the TAPP1-PH CT fragment.
  • the TAPP1-PH CT protein was concentrated to a final concentration of 14.5 mg/ml (as determined by a Bradford assay) using a VivaSpin concentrator.
  • Hanging drops were formed by mixing 1 ⁇ l of protein solution with I ⁇ l of a mother liquor solution containing 0.085 M sodium citrate, 25.5% PEG 4000, 15% glycerol, and 0.17 M ammonium acetate. The drop was then equilibrated by vapour diffusion against a reservoir containing mother liquor. Crystals were grown at 20°C and appeared after 1 day, growing to 0.3mm x 0.15mm x 0.1mm over 6 days. Crystals were frozen straight from the drop without additional cryo-protection.
  • the structure of the TAPPl PH domain was solved by molecular replacement with AMoRe [28], using the DAPPl structure [18] as a search model.
  • a single solution was obtained with an R- factor of 0.501 and a conelation coefficient of 0.392.
  • the resulting model phases were used as input for warpNrrace [29] which was able to build 92 out of the 123 possible residues.
  • the wild type and mutant PH domains of TAPPl and DAPPl used for the lipid binding experiments were expressed in E. coli and purified as described before [21]. Lipid binding studies were carried out using the protein-lipid overlay assay described previously [10, 21]. Briefly, lyophilised lipids were reconstituted in 1:1 mixture of chloroform:methanol to a concentration of 1 mM. The stock solution was serially diluted by 2-fold in a mixture chloroform:methanol:water (1:2:0.8) and 1ml of this solution, corresponding to between 1000 and 1 pmole of phosphoinositide, was spotted onto HybondC-extra nitrocellulose membrane, which was allowed to dry at room temperature for 1 hr.
  • Membranes were blocked for 1 hr at room temperature in Buffer C containing 2% (by mass) bovine serum albumin (BSA) and then were incubated in Buffer C containing 2% (by mass) BSA and 10 nM of the indicated purified wild type or mutant GST-fusion PH domains of DAPPl and TAPPl. After incubation overnight at 4°C with gentle rocking, the membranes were washed in 5 times over 40 min in Buffer C, then incubated with 1 :2000 dilution of a monoclonal anti-GST antibody in Buffer C containing 2% by mass BSA for 1 h at room temperature.
  • BSA bovine serum albumin
  • Membranes were washed a further 5 times over 40 min in Buffer C and then incubated with 1 :5000 dilution of goat anti-mouse secondary antibody conjugated to horse Radish Peroxidase In Buffer C containing 2% (by mass) BSA for 1 hr. The membranes were washed 8 times over 1 hour and detection of GST fusion proteins bound to the membrane was achieved by enhanced chemiluminescence. Table 1

Abstract

La présente invention concerne une technique de sélection ou de conception de composé destiné à réguler l'activité de la protéine contenant le domaine PH tandem (TAPP). Cette technique consiste à utiliser un moyen de modélisation moléculaire pour sélectionner ou concevoir un composé dont on prévoit qu'il interagit avec le domaine de liaison à la phosphoinositide de la protéine TAPP. On compare une structure tridimensionnelle d'au moins une partie du domaine de liaison à la phosphoinositide de la protéine TAPP avec une structure tridimensionnelle d'un composé et on sélectionne un composé dont on prévoit qu'il interagit avec ce domaine. Cette invention concerne aussi une technique de sélection ou de conception de composé qui régule l'activité d'un polypeptide contenant le domaine PH. Cette technique consiste à utiliser un moyen de modélisation moléculaire pour sélectionner ou concevoir un composé dont on prévoit qu'il interagit avec un domaine PH. On compare une structure tridimensionnelle d'un polycarboxylate, par exemple un citrate, avec une structure tridimensionnelle d'un composé et on sélectionne un composé dont on prévoit qu'il interagit avec le domaine PH. On identifie des résidus du site de liaison à la phosphoinositide de la protéine TAPP et de la protéine DAPP entrant en jeu dans la détermination de leur spécificité de liaison à la phosphoinositide. Des polypeptides mutés au niveau de ces résidus conviennent, par exemple, pour des composés de dosages d'analyse.
PCT/GB2002/003262 2001-07-31 2002-07-16 Techniques d'identification de substances interagissant avec des domaines d'homologie avec la pleckstrine (ph) et proteine contenant des domaines d'homologie avec la pleckstrine mutes WO2003011901A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2402743A (en) * 2003-06-11 2004-12-15 Amersham Biosciences Uk Ltd Inositol 1, 4, 5 trisphosphate assays
WO2005031347A1 (fr) * 2003-09-27 2005-04-07 Medical Research Council Procedes d'identification de composes chimiques impliques dans la signalisation mediee par phosphoinositide et leur utilisation dans la fabrication de medicaments

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DOWLER S ET AL: "IDENTIFICATION OF PLECKSTRIN-HOMOLOGY-DOMAIN-CONTAINING PROTEINS WITH NOVEL PHOSPHOINOSITIDE-BINDING SPECIFICITIES", BIOCHEMICAL JOURNAL, THE BIOCHEMICAL SOCIETY, LONDON, GB, vol. 351, 2000, pages 19 - 31, XP001096514, ISSN: 0264-6021 *
FERGUSON KATHRYN M ET AL: "Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains.", MOLECULAR CELL, vol. 6, no. 2, August 2000 (2000-08-01), pages 373 - 384, XP002226095, ISSN: 1097-2765 *
LEEVERS SALLY J ET AL: "Signalling through phosphoinositide 3-kinases: The lipids take centre stage.", CURRENT OPINION IN CELL BIOLOGY, vol. 11, no. 2, April 1999 (1999-04-01), pages 219 - 225, XP002226099, ISSN: 0955-0674 *
LIETZKE SUSAN E ET AL: "Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains.", MOLECULAR CELL, vol. 6, no. 2, August 2000 (2000-08-01), pages 385 - 394, XP002226098, ISSN: 1097-2765 *
THOMAS C C ET AL: "CRYSTAL STRUCTURE OF THE PHOSPHATIDYLINOSITOL 3,4-BISPHOPHATE-BINDING PLECKSTRIN HOMOLOGY (PH) DOMAIN OF TANDEM PH-DOMAIN-CONTAINING PROTEIN 1 (TAPP1): MOLECULAR BASIS OF LIQUID SPECIFICITY", BIOCHEMICAL JOURNAL, THE BIOCHEMICAL SOCIETY, LONDON, GB, vol. 358, no. PART 2, 1 September 2001 (2001-09-01), pages 287 - 294, XP001057248, ISSN: 0264-6021 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2402743A (en) * 2003-06-11 2004-12-15 Amersham Biosciences Uk Ltd Inositol 1, 4, 5 trisphosphate assays
WO2005031347A1 (fr) * 2003-09-27 2005-04-07 Medical Research Council Procedes d'identification de composes chimiques impliques dans la signalisation mediee par phosphoinositide et leur utilisation dans la fabrication de medicaments

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GB0402001D0 (en) 2004-03-03
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GB2393725B (en) 2005-08-31

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