WO2003083097A1 - Structure de domaine d'homologie avec la pleckstrine - Google Patents

Structure de domaine d'homologie avec la pleckstrine Download PDF

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WO2003083097A1
WO2003083097A1 PCT/GB2003/001455 GB0301455W WO03083097A1 WO 2003083097 A1 WO2003083097 A1 WO 2003083097A1 GB 0301455 W GB0301455 W GB 0301455W WO 03083097 A1 WO03083097 A1 WO 03083097A1
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atom
anisou
hoh
pkb
glu
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Dario Alessi
Daan Van Aalten
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University Of Dundee
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to the structure of protein kinases, and particularly to the structure of protein kinase B.
  • PtdIns(4,5)P 2 phosphatidylinositol 4,5-bisphosphate
  • PtdIns(3,4,5)P 3 As well as its immediate breakdown product PtdIns(3,4)P 2 , trigger physiological processes indicated that these lipids exert their effects by interacting with proteins possessing pleckstrin homology (PH) domains [4, 5].
  • PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 effector proteins is the serine/threonine protein kinase termed Protein Kinase B (PKB) also known as Akt.
  • PKB is activated downstream of PI 3-kinase and phosphorylates numerous regulatory proteins to enhance insulin-induced metabolic responses such as stimulating glucose uptake and glycogen synthesis in addition to promoting cell proliferation and inhibiting apoptosis [6, 7, 8]. Indeed, in a significant number of cancer cell types, PKB activity is elevated and this has been demonstrated to contribute to the enhanced ability of these cells to survive and proliferate (reviewed in [8]).
  • PKB possesses a pleckstrin homology domain located at its N-terminus, which binds PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 with similar affinity, but does not interact with PtdIns(4,5)P 2 , PtdIns3P or PtdIns4P [9, 10].
  • PKB following stimulation of cells with agonists that activate PI 3-kinase, PKB by virtue of its interaction with PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 , is recruited from the cytosol to the plasma membrane where these lipids are located [11, 12, 13].
  • PKB1 3- Phosphoinositide Dependent Protein Kinase- 1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 binding PH domain [14, 15].
  • PDK1 3- Phosphoinositide Dependent Protein Kinase- 1
  • PKB-PH PKB PH domain
  • Previous predicted structures of the PKB PH domain were obtained using homology modelling methods based upon structural information available from the PH domain of Bruton's tyrosine kinase (BTK) [27], or from a hybrid of the PH domains of PLC ⁇ and pleckstrin N [12], together with the PKB-PH domain sequence. These predictions of the PKB PH domain structure were thus biased towards the PH domain from which the structural information was obtained.
  • BTK Bruton's tyrosine kinase
  • a first aspect of the present invention provides a method of identifying a compound which modulates the interaction between PKB and PtdIns(3,4,5)P or PtdIns(3,4)P 2 , the method comprising determining whether, and optionally to what extent, a conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , is enhanced or disrupted in the presence of the compound.
  • a second aspect of the present invention provides a method of identifying a compound which mimics the effect of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 on PKB, the method comprising determining whether, and optionally to what extent, a conformational change in a PH domain of the PKB upon contact with the compound, matches a conformational change in the PH domain of the PKB upon contact with the PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 .
  • the PKB ⁇ PH structure reveals a standard PH domain fold with seven ⁇ - strands forming two orthogonal anti-parallel ⁇ -sheets closed at one end by a C-terminal ⁇ -helix.
  • At the other end of the ⁇ -barrel lie three loops (NL1-3). These loops form a bowl lined with basic residues into which the Ins(l,3,4,5)P head group can bind.
  • the ligand binding pocket of the PH domain was found to have a closed conformation, occupied by a hydrogen bonding network involving several basic residues.
  • the hydrogen bonding network centres around an ionic interaction between Arg86/Lysl4 and Glu 17, and also involving Asn53 and several water molecules.
  • the numbering of residues is based upon the numbering of full- length human PKB ⁇ .
  • Ins(l,3,4,5)P Upon binding of Ins(l,3,4,5)P to the PH domain, the hydrogen bonding network is disrupted and Ins(l,3,4,5)P forms specific interactions with several basic amino acid residues of the PH domain, as well as with other side chains and with the protein backbone.
  • the DI -phosphate interacts with Arg23 and the backbone nitrogen of Ilel9; the D3-phosphate interacts with the side chains of Lysl4, Arg23, Arg25, and Asn53; and the D4-phosphate interacts with Lys 14, Asn53 and Arg86.
  • the D5-phosphate does not interact with any residue in the PH domain — it is oriented towards the solvent and interacts only with five ordered water molecules.
  • Binding of Ins(l,3,4,5)P 4 to the PH domain was shown to result in a surprising and unexpected conformational change in the variable loops of the PH domain, a phenomenon not previously observed in any other PH domain structure. Displacements of up to 7.6 A in the PKB PH backbone were shown to occur upon ligand binding. Glu 17 is pushed outwards, resulting in a relatively minor conformational change of VL1 with backbone shifts up to 2.5 A, with Arg86 following the movement of Glu 17. Arg86 lies at the base of NL3 and pulls the entire loop closer to the phosphoinositide binding pocket, leading to backbone shifts of up to 7.4 A at Trp80 which is located at the tip of VL3.
  • VL2 the entire loop is rearranged, with backbone shifts up to 7.6 A on Glu49, and forms an ordered ⁇ -helix with 3-4 negative charges (Asp44, Asp46, Glu49 and to a lesser extent Glu40) clustered together facing the solvent.
  • the structure of the PH domain as provided herein differs from the previously published predicted structures [12, 27] as the predicted molecular models were based upon structures obtained from different PH domains, and using a ligand having a lipid chain (-OP03CH2CH(OCOC2H5)CH20COC2H4), while the structure provided herein is of the PKB-PH domain complexed with the 3-phosphoinositide headgroup Ins(l,3,4,5)P 4 and no lipid chains.
  • Glu40 is described as having a role in the binding affinity of the ligand PtdIns3,4,5P 3 . Our structure, however, shows this is not the case but that Glu40 could have an effect on binding to the kinase domain.
  • VL2 region could be the region which interacts with the kinase domain.
  • the method of the first and second aspects of the invention may also comprise the step of determining a baseline or normal conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in the absence of the compound. Additionally or alternatively, a previously determined baseline or normal conformational change can be used.
  • Disrupting a conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in the presence of the compound may comprise reducing the rate at which or extent to which the baseline or normal conformational change occurs.
  • disrupting a conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in the presence of the compound may comprise abolishing a baseline or normal conformational change.
  • disrupting a conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in the presence of the compound may comprise causing a different conformational change to occur.
  • Enhancing a conformational change in the PH domain of the PKB upon contact with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in the presence of the compound may comprise increasing the rate at which or extent to which the baseline or normal conformational change occurs.
  • PtdIns(3,4)P 2 on PKB is meant that the compound has a quantitative or qualitative effect on at least one activity or function or property of PKB, for example on its ability to be phosphorylated by PDK1, or its protein kinase activity, that is the same as the effect of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 on PKB.
  • PKB as used herein includes a polypeptide comprising the amino acid sequence identified as human PKB ⁇ (NCBI Accession Number P31749); or human PKB p (NCBI Accession Number P31751); or human PKB y (NCBI Accession Number Q9Y243) or a variant, fragment, fusion or derivative thereof, or a fusion of a variant or fragment or derivative.
  • the PKB retains the protein kinase activity of full length human PKB ⁇ . It is further preferred that the PKB is capable of binding PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 and being activated by PDK1.
  • the variant or fragment or derivative or fusion of the PKB, or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of full- length human PKB ⁇ . It is more preferred if the variant or fragment or derivative or fusion of the PKB, 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 enzyme activity of human PKB ⁇ .
  • the variant or fragment or derivative or fusion of the PKB, or the fusion of the variant or fragment or derivative binds PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 at at least 30% of the rate or extent of full-length human PKB ⁇ . It is more preferred if the variant or fragment or derivative or fusion of the PKB, or the fusion of the variant or fragment or derivative, binds PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 at at least 50%, preferably at at least 70% and more preferably at at least 90% of the rate or extent of human PKB ⁇ .
  • the variant or fragment or derivative or fusion of the PKB, or the fusion of the variant or fragment or derivative can be activated by PDK1 at at least 30% of the rate or extent of full-length human PKB ⁇ . It is more preferred if the variant or fragment or derivative or fusion of the PKB, or the fusion of the variant or fragment or derivative, can be activated by PDK1 at at least 50%, preferably at at least 70% and more preferably at at least 90% of the rate or extent of human PKB ⁇ .
  • PDK1 is a ubiquitously expressed enzyme belonging to the serine/threonine family of protein kinases, which, like PKB, possesses a PH domain. It activates not only PKB, but also PKA, PKC-zeta, p70S6K and p90S6K/RSK, and is believed to play a general role in cellular signalling processes.
  • PDK1 as used herein includes a polypeptide comprising the amino acid sequence identified as human PDK1 (NCBI Accession Number 015530), or a variant, fragment, fusion or derivative thereof, or a fusion of a variant or fragment or derivative, and having at least 30% of the ability of full-length human PDK1 to phosphorylate PKB.
  • PDK1 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 ability of full-length human PDK1 to phosphorylate PKB.
  • PKB PH domain as used herein includes a polypeptide comprising the amino acid sequence identified as PKB in Figure 2 as SEQ ID NO: 1, or the equivalent region of PKB ⁇ or PKB ⁇ , or a variant, fragment, fusion or derivative thereof, or a fusion of a variant or fragment or derivative.
  • the PKB PH domain has a similar structure to that of PKB ⁇ PH as defined herein, more preferably, the same structure as that of PKB ⁇ PH.
  • the PKB PH domain undergoes a similar conformational change upon binding of 3-phosphoinositides as does PKB ⁇ PH, and more preferably, undergoes the same conformational change as that of PKB ⁇ PH.
  • the PKB PH domain is capable of binding 3-phosphoinositides at at least 30% of the rate or extent of full-length human PKB ⁇ . It is more preferred if the PKB PH binds 3-phosphoinositides at at least 50%, preferably at at least 70% and more preferably at at least 90% of the rate or extent of human PKB ⁇ .
  • 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 PKB, or the PKB PH.
  • variants of a polypeptide we also include polypeptides comprising the native sequence of homologues of the polypeptide from other organisms.
  • substitutions is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the PKB or PKB-PH variant has an amino acid sequence which has at least 65% identity with the respective amino acid sequences of PKB or PKB-PH 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 respective amino acid sequences defined above. It is appreciated that percent sequence identity between two polypeptides may be readily identified by a person skilled in the art, for example using sequence comparisons as described below.
  • 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 PKB or PKB PH domain are polypeptides which comprise the amino acid sequence of the respective PKB or PKB PH domain sequences 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 other organisms.
  • compounds that modulate the binding of 3- phosphoinositides to PKB-PH may themselves bind to PKB-PH at the 3- phosphoinositide binding site. Such compounds may inhibit the normal or baseline conformational change in the PH domain by preventing 3- phosphoinositide binding to PKB-PH, thus reducing or abolishing the normal or baseline conformational change in the PH domain.
  • such compounds may bind to the 3-phosphoinositide binding site of the PKB-PH and cause a different conformational change from the normal or baseline conformational change to occur in the PH domain.
  • compounds which bind to the 3-phosphoinositide binding site of the PKB-PH may mimic the normal or baseline conformational change that occurs upon 3-phosphoinositide binding.
  • the mimicked conformational change may occur at a different rate or to a different extent to that of the normal or baseline conformational change that occurs upon 3-phosphoinositide binding to PKB-PH.
  • compounds that modulate the binding of 3- phosphoinositides to PKB-PH may themselves bind to other regions of the PKB PH domain other than the 3-phosphoinositide binding site. Such compounds would typically either inhibit or enhance the normal or baseline conformational change that occurs upon 3-phosphoinositide binding to PKB-PH.
  • measuring the conformational change may include measuring any of the changes to the PKB PH domain structure that occur upon binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , as described herein. More preferably, the measured conformational change is selected from the following:
  • determining the conformational change comprises using the 3D structural co-ordinates of the PH domain bound to a 3-phosphoinositide or headgroup thereof, and/or the 3D structural co-ordinates of the PH domain alone, or a subset thereof, and a molecular modelling technique, preferably, one of the molecular modelling techniques described herein.
  • using the 3D structural co-ordinates of the PH domain bound to a 3-phosphoinositide or headgroup thereof, or subset thereof comprises using the 3D structural co-ordinates of PKB ⁇ PH-Ins(l,3,4,5)P 4 as set forth in Table 2, or a subset thereof.
  • using the 3D structural co-ordinates of the PH domain alone, or a subset thereof comprises using the 3D structural co-ordinates of apo PKB ⁇ PH as set forth in Table 3, or a subset thereof.
  • determining the conformational change in the PH domain of PKB comprises directly measuring the conformational change.
  • this comprises use of one or more of ⁇ MR; X-ray crystallography; circular dichroism, (CD) which may be useful in detecting, for example, the degree of presence or absence of the VL2 loop ⁇ -helix; or FRET.
  • determining the conformational change comprises indirectly measuring the conformational change.
  • At least one amino acid residue of the PKB PH domain is substituted with a detectably-labelled cysteine residue, and indirectly measuring the conformational change in the PH domain of PKB comprises detecting the label.
  • the detectably-labelled cysteine residue is fluorescently-labelled, and detecting the label comprises detecting a change in fluorescence intensity or frequency.
  • Any thiol-reactive fluorophore for example BAD AN (see, for example, Wadum et al Fluorescently labeled bovine acyl- CoA binding protein - an acyl-CoA sensor.
  • the substituted amino acid residue of the PH domain is not involved in binding to PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 .
  • the substituted amino acid residue is selected from residues 1-123 of PKB ⁇ PH as listed in SEQ ID No 1, with the exception of Lysl4, Ilel9, Arg23, Arg25, Asn53 and Arg86.
  • the substituted amino acid residue of the PH domain is not involved in binding to a negatively charged lipid membrane.
  • the substituted amino acid residue is selected from residues 1-123 of PKB ⁇ PH as listed in SEQ ID No 1, with the exception of Argl5, Lys20, Arg67 and Arg69.
  • the substituted amino acid residue of the PH domain is exposed to the solvent, and is located close to the binding pocket such that when a ligand (such as a 3,4 phosphoinositide) is not bound to the PH domain in the binding pocket, the fluorophore is located in the binding pocket.
  • a ligand such as a 3,4 phosphoinositide
  • the substituted amino acid residue is selected from a residue located in VL1.
  • the substituted amino acid residue is selected from Glu 17, Glul ⁇ , and Tyrl9 of SEQ ID NO: 1.
  • the interaction or conformational change can be confirmed using a technique such as isothermal titration calorimetry (ITC) or a protein-lipid overlay binding assay.
  • ITC isothermal titration calorimetry
  • the present invention provides a method of selecting or designing a compound to be assessed in the method of identifying compounds that modulate or mimic the effect of 3-phosphoinositides on PKB as described herein.
  • the method comprises the step of using the 3D structural co-ordinates of a PKB PH domain bound to a 3-phosphoinositide or headgroup thereof, and/or the 3D structural co-ordinates of a PKB PH domain alone, or a subset thereof, and a molecular modelling technique, to select the compound from a plurality of test compounds.
  • the compound may be based on or superimposed on the 3-phosphoinositide or headgroup structure.
  • the method also comprises the steps of identifying, in at least one database of chemical structures, a set of test compounds containing a specific structural feature, identifying a subset of test compounds by discarding from the set of test compounds those compounds that are obviously unable to bind to the PKB PH domain based upon their size, charge and/or steric hindrance, and using the 3D structural co-ordinates of a PKB PH domain bound to a 3-phosphoinositide or headgroup thereof, and/or the 3D structural co-ordinates of a PH domain alone, or a subset thereof, and a molecular modelling technique, to identify from the subset of test compounds a compound likely to be able to bind to the PH domain.
  • the method also comprises varying, typically computationally, the possible atoms at each site on the 3-phosphoinositide or headgroup thereof (or test compound, for example based on or superimposed on the 3- phosphoinositide or headgroup structure) that is identified in the 3D structural co-ordinates of the PH domain bound to the 3-phosphoinositide or headgroup thereof as being in contact with the PH domain, and screening for resultant compounds that minimise repulsion or hindrance while keeping the remaining protein co-ordinates and ligand co-ordinates of the PH domain bound to the 3-phosphoinositide or headgroup thereof fixed.
  • using the 3D structural co-ordinates of a PH domain bound to a 3-phosphoinositide or headgroup thereof, and/or the 3D structural co-ordinates of a PH domain alone, or a subset thereof comprises using a molecular model of a PH domain derived from the 3D structural coordinates of the PH domain bound to a 3-phosphoinsotide or headgroup thereof, and/or the 3D structural co-ordinates of the PH domain alone, or a subset thereof.
  • using the 3D structural co-ordinates of a PH domain bound to a 3-phosphoinositide or headgroup thereof, or subset thereof comprises using the 3D structural co-ordinates of PKB ⁇ PH-Ins(l,3,4,5)P as set forth in Table 2, or a subset thereof.
  • using the 3D structural co-ordinates of a PH domain alone, or a subset thereof comprises using the 3D structural co-ordinates of apo PKB ⁇ PH as set forth in Table 3, or a subset thereof.
  • the 3D structure of a compound to be tested may be compared with the 3D structure of a PKB PH domain such as a 3D structure modelled from the data contained in Table 2 and/or Table 3.
  • the compound structure may be a predicted 3D structure or a previously determined 3D structure.
  • the 3D structures may be displayed by a computer in a 2D form, for example on a computer screen. The comparison may be performed using such 2D displays.
  • a compound predicted to interact with the PKB PH domain and to cause a conformational change in a similar manner to PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 may be selected.
  • Methods of assessing a conformational change are well known to those skilled in the art, and include methods such as CD, NMR, X-ray crystallography and FRET.
  • Known compounds that bind the PH domain of PKB are obtained or synthesised, co-crystallised together with the PKB PH domain, and the structure of the PH domain with the compound determined.
  • the compound is modified, typically by adding, subtracting, or changing atoms in the compound, with the aim of improving the binding of the compound to the PH domain.
  • the modified compound is obtained or synthesised, co-crystallised together with the PKB PH domain, and the structure of the PH domain with the modified compound determined.
  • the steps of modification, co-crystallisation and structure determination can be iterated until compounds with the desired effect on the PKB PH domain are obtained.
  • 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) J Mol 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
  • LeapFrog available
  • the selected or designed compound may be synthesised (if not already synthesised) and tested further for its effect on PKB, for example its effect on the protein kinase activity or lipid binding activity of PKB.
  • the compound may be tested in one of a variety of screening methods which are well known in the art.
  • a further aspect of the invention provides a method of modulating an activity, for example the protein kinase activity, of PKB wherein the PKB is exposed to a compound identified by any of the methods disclosed herein.
  • a yet further aspect of the invention is a compound identified or identifiable by the above selection/design method of the invention.
  • the ability of the compound to disrupt or enhance normal conformational change upon interaction of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 with PKB-PH may be measured by detecting/measuring the conformational change using any suitable method and comparing the conformational change detected/measured in the presence of different concentrations of the test compound, for example in the absence and in the presence of the test compound, for example at a concentration of about lOO ⁇ M, 30 ⁇ M, lO ⁇ M, 3 ⁇ M, l ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M and/or 0.001 ⁇ M.
  • Suitable methods include methods analogous to those discussed herein.
  • the present invention provides a PKB, or a fragment, variant, derivative or fusion thereof, having at least one amino acid residue of the PH domain substituted by a detectably-labelled cysteine residue.
  • the present invention provides a PKB, or a fragment, variant, derivative or fusion thereof, having at least one amino acid residue of the PH domain substituted by a cysteine residue that can be detectably labelled.
  • the substituted amino acid residue is as defined above.
  • the invention provides for the use of the PKB having at least one amino acid residue of the PH domain substituted by a detectably- labelled cysteine residue, in an assay for detecting conformational change of the PH domain.
  • the invention provides for a kit of parts comprising the PKB having at least one amino acid residue of the PH domain substituted by a detectably-labelled cysteine residue, or PKB having at least one amino acid residue of the PH domain substituted by a cysteine residue that can be detectably-labelled, and means for carrying out an assay for detecting conformational change of the PH domain.
  • Means for carrying out an assay may include one or more of phosphoinositides, or headgroups thereof, particularly PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 which can be used as normal or baseline positive controls, and Ins(l,3,4,5)P which can be used as a negative control; PKB mutants, for example Lysl4->Ala, Arg25->Ala or Arg86->Ala as negative controls (ie unable to bind to phosphoinositides); assay buffers; assay instructions; and a fluorophore for labelling the substituted cysteine residue, and reaction buffers for the labelling reaction.
  • PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 which can be used as normal or baseline positive controls
  • Ins(l,3,4,5)P which can be used as a negative control
  • PKB mutants for example Lysl4->Ala, Arg25->Ala or Arg86->Ala as negative controls (i
  • the present invention provides for a nucleic acid encoding the PKB, or a fragment, variant, derivative or fusion thereof, having at least one amino acid residue of the PH domain substituted by a cysteine residue that can be detectably-labelled.
  • the invention provides for a vector comprising such a nucleic acid, and in still yet a further aspect, the invention provides for a host cell comprising such a vector.
  • a compound identified by a method of the invention may, by modulating the conformational change of the PH domain, modulate the ability of the PKB to phosphorylate different substrates to different extents.
  • the compound may inhibit the protein kinase activity in relation to one substrate but may increase or not affect the protein kinase activity in relation to a second substrate.
  • a compound at a given concentration may inhibit phosphorylation of one substrate to a greater extent than another substrate.
  • a compound may have different IC 50 S in relation to phosphorylation of different substrates.
  • the term IC 50 is well known to those skilled in the art and indicates the concentration of compound necessary to inhibit the observed parameter (i.e. phosphorylation of a particular substrate under particular conditions) to 50% of the value in the absence of the compound.
  • IC 50 the more potent the compound.
  • Methods of calculating IC 50 values are well known to those skilled in the art.
  • a similar measure of the effect of the compound may be calculated for compounds that increase phosphorylation of a given substrate (for example the concentration necessary to increase phosphorylation by, for example 20% or 50%).
  • the invention provides screening assays for drugs which may be useful in modulating, for example either enhancing or inhibiting, the protein kinase activity of PKB.
  • the compounds identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds.
  • the compound may be a drug-like compound or lead compound for the development of a drug-like compound for each of the above methods of identifying a compound. It will be appreciated that the methods may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art.
  • 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.
  • 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 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 drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • screening assays which are capable of high throughput operation are 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.
  • beads comprising scintillant and a substrate polypeptide or interacting polypeptide may be prepared.
  • the beads may be mixed with a sample comprising P- or P- ⁇ -labelled PKB (as defined above) and with the test compound. Conveniently this is done in a 96-well or 384- well format.
  • the plate is then counted using a suitable scintillation counter,
  • T? 3 1 ? 33 using known parameters for P or P SPA assays. Only P or P that is in proximity to the scintillant, i.e. only that bound to the substrate or interacting polypeptide that is bound to the beads, is detected. Variants of such an assay, for example in which the substrate or interacting polypeptide is immobilised on the scintillant beads via binding to an antibody or antibody fragment, may also be used.
  • High throughput crystallography techniques may be utilised as particularly preferred screening assays.
  • High throughput crystallography techniques may include those described in the following publications, each of which is incorporated herein by reference: J. Crystal Growth 90:318-324 (1988); J. Crystal Growth 90:325-339 (1988); PCT Patent Publication WO 00/78445; US Patent No. 5,221,410 and European Patent No. EP 0 553 539.
  • reagents and conditions used in the method may be chosen such that the interactions between, for example, the PKB and the compound, are substantially the same as between human PKB and PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 in vivo.
  • the compounds tested in screening methods or in other assays in which the ability of a compound to modulate the conformational change in the PKB PH domain is measured may have been selected and/or designed (including modified) using molecular modelling techniques, for example using computer techniques, as indicated above.
  • a further aspect of the invention provides a compound identified by any of the methods described herein that modulates or mimics a conformational change in the PH domain of the PKB upon contact with the PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , for use in medicine.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound identified by any of the methods described herein that modulates or mimics a conformational change in the PH domain of the PKB upon contact with the PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2) and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may be administered 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 pharmaceutical composition may also be administered in a localised manner, for example by injection.
  • the compound identified by the methods described herein may be an inhibitor of PKB.
  • PKB-PH binding of PKB-PH to 3-phosphoinositides is a prerequisite for activation of PKB by PDKl
  • compounds, typically small molecules, that disrupt the normal conformational change of the PH domain upon the interaction of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 with PKB would decrease or abolish the activity of PKB.
  • Inhibitors of PKB may block binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to PKB by themselves binding to the 3-phosphoinositide binding site in the PH domain of PKB, without causing the conformational change.
  • PKB inhibition would be expected to impair growth and promote apoptosis of cancer cells in which PKB activity is elevated.
  • Phosphoinositide derivatives containing a hydroxymethyl group at the D3 position, and/or a carbonate rather than a phosphate group at DI were previously shown to inhibit PDGF-induced PKB phosphorylation and inhibited the growth of four different cancer cell lines [25, 26, 27].
  • the present invention provides the use of a compound identified by any of the methods described herein, that disrupts the conformational change in the PKB-PH domain on interaction with PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , in the preparation of a medicament for the treatment of cancer. More preferably, the medicament impairs the growth or promotes apoptosis of cancer cells.
  • Such a compound could disrupt the conformational change in the PKB-PH domain by binding directly to the 3-phosphoinositide ligand binding site of PKB-PH, or to another region of the PKB, or directly to PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , for example so that the 3-phosphoinositides may bind to PKB but without inducing the conformational change.
  • the identified compound may be an activator of PKB.
  • compounds that bind to the 3-phosphoinositide ligand binding site of PKB- PH and induce a similar or enhanced conformational change on the PH domain as does the binding of Ins(l,3,4,5)P 4 could promote the PtdIns(3,4,5)P 3 -independent activation of PKB by PDKl.
  • Such compounds could be useful in treating any disorder characterised in having a PKB deficiency.
  • such compounds could be used to stimulate insulin dependent responses in diabetic patients, to promote cellular survival following an ischaemic event, for example the survival of neuronal cells following a stroke, or to reduce apoptosis.
  • a compound identified by any of the methods described herein in the preparation of a medicament for stimulating insulin dependent responses in diabetic patients, or for promoting cellular survival following an ischaemic event, for example for promoting the survival of neuronal cells following a stroke, or for reducing apoptosis.
  • the present invention provides the use of a compound identified by any of the methods described herein, that binds to the 3-phosphoinositide ligand binding site of PKB ⁇ PH and induces a conformational change which promotes the PtdIns(3,4,5)P 3 -independent activation of PKB, in the preparation of a medicament for stimulating insulin dependent responses in diabetic patients or for promoting cellular survival following an ischaemic event, for example for promoting the survival of neuronal cells following a stroke, or for reducing apoptosis.
  • the present invention provides the use of a compound identified by any of the methods described herein, that enhances the normal conformational change upon binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to the PKB PH domain, in the preparation of a medicament for stimulating insulin dependent responses in diabetic patients or for promoting cellular survival following an ischaemic event, for example for promoting the survival of neuronal cells following a stroke, or for reducing apoptosis.
  • enhancing the normal conformational change upon binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to the PKB PH domain promotes activation of PKB by PDKl.
  • Figure 1 consists of three pairs of stereo figures illustrating the structure of PKB ⁇ PH complexed to Ins(l,3,4,5)P , in accordance with a preferred embodiment of the present invention.
  • panel A the experimental electron density map from SOLVE after density modification is shown in grey (contoured at 1.25 ⁇ ).
  • the final PKB ⁇ PH structure is shown as a sticks model.
  • Ins( 1, 3, 4,5)P is represented by a sticks model.
  • Panel B is a ribbon drawing of the PKB ⁇ PH-Ins(l,3,4,5)P 4 complex, with the seven ⁇ -strands (labelled ⁇ -l) and the ⁇ -helices (labelled ⁇ l-2) shown.
  • Ins(l,3,4,5)P 4 is also shown.
  • the side chains of residues interacting with this molecule are shown with grey carbons.
  • the basic residues thought to interact with the membrane also have their side chains shown as sticks.
  • the negatively charge residues on VL2 that are hypothesised to interact with the kinase domain are also shown.
  • Panel C is a ribbon diagram of the Ins(l,3,4,5)P 4 binding site, with the ligand shown.
  • , ⁇ ca ⁇ c map is shown, contoured at 2.25 ⁇ . Residues making hydrogen bonds to the ligand are shown as sticks with grey carbons, hydrogen bonds are shown as black dotted lines.
  • Figure 2 shows a structure-based sequence alignment of four PtdIns(3,4,5)P 3 binding PH domains, constructed by superposition of their structures using the MOTIF option in WHAT IF [37]. conserveed residues are shown in black. Homologous residues are highlighted in grey. Secondary structure is shown as black arrows for ⁇ -strands and as bars for the ⁇ -helices. Helix ⁇ l one is shown as a shaded bar. Arrows indicate residues in PKB ⁇ PH that interact with the lipid head group. Variable loops 1,2 and 3 are labelled as VL1, VL2 and VL3 respectively. The serine residue immediately prior to the methionine residue at position 1 is the protease cleavage site.
  • Figure 3 shows the lipid binding properties of various PKB ⁇ mutants.
  • the ability of the wild type and mutant GST-PKB ⁇ fusion proteins to bind to the indicated amounts of PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 was analysed using a protein lipid overlay assay.
  • Figure 4 is a structural comparison of apo-PKB ⁇ PH and PKB ⁇ PH- Ins(l,3,4,5)P .
  • Panel A is an illustration of electrostatic surface potential calculated with GRASP. Dark grey areas (+6kT) represent highly positively charged residues, mid-grey areas (-6kT) indicated highly negatively charged residues.
  • the left image shows the structure of the complex, with Ins(l,3,4,5)P 4 shown as a stick model.
  • the right image shows the apo structure.
  • Panel B is a ribbon diagram highlighting the conformational changes between the a ⁇ o-PKB ⁇ PH and PKB ⁇ PH-Ins(l,3,4,5)P 4 structures.
  • the apo- PKB ⁇ PH structure is shown with ?-strands and the C-terminal helix, with the loop regions in grey, and side chains shown as stick models.
  • the hydrogen bonding network between residues Arg86, Asn53, Glul7, Lys 14 and two water molecules (spheres) is also shown.
  • the PKB PH- Ins(l,3,4,5)P 4 complex protein backbone is shown, only showing loops or strands that undergo a conformational change upon ligand binding.
  • the Ins(l,3,4,5)P molecule is shown.
  • Panel C is a ribbon diagram of the PKB ⁇ PH structure with a stick model of an Ins(l,3,4,5)P 4 derivative described previously [26] that has a hydroxymethyl group at D3 and a carbonate group at DI . Side chains capable of interacting with the inhibitor are shown. Predicted hydrogen bonds are shown in black, including the hydrogen bond between the axial hydroxymethyl group and Arg25, which is mentioned in [26] and the discussion.
  • PKB ⁇ PH phosphatidylinositide binding PH domain of protein kinase B
  • E. coli BL21 cells transformed with the pGEX4T-l vector encoding the expression of GST-PKB ⁇ PH were grown at 37°C in 4 1 of Luria-Bertani broth with 50 g/ml ampicillin, until OD 600 reached 0.7.
  • the expression of GST-PKB ⁇ PH was induced by the addition of 250 M isopropyl- ?-D- thiogalactopyranoside (IPTG), and the bacteria were then grown for a further 16 h at 27°C.
  • the cells were harvested by centrifugation at 3500 g for 15 min, then lysed by resuspension in 200 ml of buffer A (50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM NaV0 4 , 10 mM sodium-glycerophosphate, 50 mM NaF, 5 mM dithiothreitol and 'complete' proteinase inhibitor cocktail (one tablet per 25 ml; Roche) containing additional DNAse (1.5-2 mg/ml) and 1 mg/ml lysozyme. The cells were then incubated on ice for 30 min before a final sonication step.
  • buffer A 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM NaV0 4 , 10 mM sodium-glycerophosphate, 50 mM NaF, 5 mM dithioth
  • the resulting solution was centrifuged at 14500 g for 30 min to remove residual debris and passed through a 0.45 ⁇ m filter. The supernatant was incubated for 1 h at 4°C with 4 ml of glutathione-Sepharose beads equilibrated against buffer A. The beads were then washed 4 times with 5 column volumes of buffer A, and subsequently washed 6 times with 5 column volumes of buffer B (50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.3 M NaCI and 5 mM dithiothreitol).
  • the PKB ⁇ PH domain was then separated from the GST-tag by incubating the glutathione-Sepharose beads conjugated to GST-PKB ⁇ PH in a ratio of 2 units of thrombin to 10 / ⁇ 1 of resin at 4°C overnight.
  • the resin was centrifuged, washed four times with 10 ml of buffer B and the combined supernatants containing PKB ⁇ PH were applied to a 0.2 ml benzamidine-agarose column to remove the thrombin.
  • the eluate was subsequently applied to a 1 ml glutathione-Sepharose column equilibrated in buffer B to remove trace contamination of GST.
  • Selenomethionine-substituted protein was prepared by expressing protein in the methionine auxotrophic strain B834. Cells were grown overnight in Luria Bertani media and then further amplified in M9 minimal media supplemented with 1 g/1 of amino acid mix [29] lacking methionine. Adenine, guanosine, thymine and uracil were added to 5 g/1, FeS0 4 to 4 mg/1 and ZnCl 2 to 40 mg/ml. The media was then filtered through a 0.22 ⁇ m filter and L-selenomethionine was added to a final concentration of 125 mg/1.
  • the complex was crystallised using a mother liquor containing 0.25 M ammonium acetate, 30% PEG 4000, 0.1 M sodium acetate (pH 4.6). Monoclinic crystals appeared after 2 days, growing to approximately 0.2x0.2x0.3 mm after four days. Crystals were frozen in a nitrogen gas stream after being soaked in 10% 2-methyl-2,4- pentanediol for 30 seconds.
  • Table 1 lists details of data collection & structure refinement for a MAD data set collected on PKB ⁇ PH-Ins(l,3,4,5)P 4 crystals, and a native apo PKB ⁇ PH data set. Values between brackets are for the highest resolution shell. All measured data were included in structure refinement.
  • Table 2 lists the data set of structural co-ordinates collected on PKB ⁇ PH- Ins(l ,3,4,5)P 4 crystals.

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Abstract

L'invention concerne un procédé d'identification d'un composé qui module l'interaction entre la protéine kinase B (PKB) et le phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) ou le phosphatidylinositol (3,4)-diphosphate (PtdIns(3,4)P2), ce procédé consistant à déterminer si, et éventuellement à quelle proportion, un changement conformationnel dans un domaine d'homologie avec la pleckstrine (PH) de la PKB après mise en contact avec le PtdIns(3,4,5)P3 ou le PtdIns(3,4)P2, est augmenté ou interrompu en présence dudit composé. L'invention concerne également un procédé d'identification d'un composé qui reproduit l'effet du PtdIns(3,4,5)P3 ou du PtdIns(3,4)P2 sur la PKB, ce procédé consistant à déterminer si, et éventuellement à quelle proportion, un changement conformationnel dans un domaine de PH de la PKB après mise en contact avec ledit composé, correspond à un changement conformationnel dans le domaine de PH après mise en contact avec le PtdIns(3,4,5)P3 ou le PtdIns(3,4)P2.
PCT/GB2003/001455 2002-04-03 2003-04-02 Structure de domaine d'homologie avec la pleckstrine WO2003083097A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN109030447A (zh) * 2018-09-29 2018-12-18 西安交通大学 一种酵母中有机硒的测定方法

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* Cited by examiner, † Cited by third party
Title
THOMAS CC ET AL: "High-resolution structure of the pleckstrin homology domain of protein kinase B/Akt bound to phosphatidylinositol (3,4,5)-trisphosphate", CURRENT BIOLOGY, vol. 12, - 23 July 2002 (2002-07-23), pages 1256 - 1262, XP002249971 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109030447A (zh) * 2018-09-29 2018-12-18 西安交通大学 一种酵母中有机硒的测定方法
CN109030447B (zh) * 2018-09-29 2020-05-22 西安交通大学 一种酵母中有机硒的测定方法

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