EP2205756A2 - Procédés - Google Patents

Procédés

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Publication number
EP2205756A2
EP2205756A2 EP08806500A EP08806500A EP2205756A2 EP 2205756 A2 EP2205756 A2 EP 2205756A2 EP 08806500 A EP08806500 A EP 08806500A EP 08806500 A EP08806500 A EP 08806500A EP 2205756 A2 EP2205756 A2 EP 2205756A2
Authority
EP
European Patent Office
Prior art keywords
atom
ehd
polypeptide
leu
family polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08806500A
Other languages
German (de)
English (en)
Inventor
Harvey Mcmahon
Gary Doherty
Richard Lundmark
Oli Daumke
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Medical Research Council
Original Assignee
Medical Research Council
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Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of EP2205756A2 publication Critical patent/EP2205756A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • C12N9/14—Hydrolases (3)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the invention relates to the field of EHD polypeptides and their structure and/or biological function(s).
  • the dynamin superfamily of large GTPases are multi-domain proteins that include the classical dynamins (Dynl, Dyn2, Dyn3), dynamin-related proteins (Mx i proteins, Dip, OPA and mitofusins) and the GBP/atlastin family .
  • the proteins have an amino-(N-)terminal guanine nucleotide binding domain (G-domain) with a low affinity for nucleotides which is followed by a helical (or middle) domain. Additional domains are involved in membrane-binding and recruitment to sites of activation.
  • Dynamin is the best characterised member where oligomerisation- stimulated GTP hydrolysis has been proposed to lead to scission of clathrin-coated
  • EHDs comprise a highly conserved eukaryotic protein family with four members (EHD 1-4) in mammals and a single member in C. elegans, D. melanogaster and many eukaryotic parasites such as Plasmodium, Leishmania and Entamboeba.
  • the proteins have a molecular mass of approximately 6OkD and contain an N-terminal G-domain, followed by a helical domain and a C-terminal EH-domain (Fig. Ia), although in plant homologues the EH-domain is N-terminal.
  • the EH-domain is known to interact with asparagine-proline-phenylalanine (NPF) motifs in proteins involved in endocytosis.
  • NPF asparagine-proline-phenylalanine
  • EHD polypeptide structure is poorly understood in the art.
  • EHD polypeptides are regarded as GTPases in the art.
  • EHD biological functions are largely unknown in the art.
  • the macromolecular behaviour of EHD polypeptides is incompletely understood in the art.
  • the design or selection/screening for inhibitors or activators of EHD is not possible based on the inadequate information regarding the structure/function of EHD in the art.
  • the present invention seeks to overcome problem(s) associated with the prior art.
  • EHD proteins are known by sequence analysis to contain a guanine nucleotide binding domain (G-domain). It has been suggested that EHD might bind to adenine nucleotides, with the attendant possibility that such nucleotides might be hydrolysed by the protein. However, there has been no accurate scientific study to date which reliably attributes ATP hydrolysis to a G domain. In the prior art, cross-nucleotide activities of that nature are typically attributed to contamination effects. The prior art view is very clearly established that G-domains have guanine nucleotide binding and/or guanine nucleotide hydrolysis activities.
  • EHD family polypeptides are in fact ATP binding polypeptides. Furthermore, we disclose how ATP binds to those polypeptides. In addition, the structural insights allowed a modelling of the likely mechanism of ATP hydrolysis by EHD. family polypeptides, which hypothesis has been demonstrated to be accurate by mutational studies of the ATP ase activity.
  • the invention is based on these surprising findings.
  • the invention relates to a method of identifying a modulator of an EHD family polypeptide, said method comprising
  • the candidate modulator is identified as an enhancer of EHD family polypeptide activity.
  • the candidate modulator is identified as an inhibitor of EHD family polypeptide activity.
  • ATP hydrolysis is monitored in the presence of lipid.
  • lipid is in the form of liposomes.
  • lipid is in the form of phosphatidylserine (PS) at a final concentration of about 10%.
  • PS phosphatidylserine
  • the invention relates to a method as described above further comprising the step of providing a further sample of EHD family polypeptide, said further sample comprising an EHD family polypeptide bearing a T94A mutation, said further sample being used to determine the reference or background level of spontaneous ATP hydrolysis.
  • the invention relates to a method as described above further comprising the step of providing a further sample of EHD family polypeptide, said further sample comprising an EHD family polypeptide bearing an I157Q mutation, said further sample being used to determine the reference level of ATP hydrolysis in the absence of lipids.
  • the invention relates to a method as described above further comprising the step of providing a further reference sample, said further reference sample comprising a dynamin polypeptide together with GTP nucleotide and candidate modulator, said further sample being used to determine whether the candidate modulator has an EHD-specif ⁇ c effect, or whether it is also capable of affecting dynamin GTPase activity.
  • the invention relates to a crystalline EHD family polypeptide, said polypeptide being bound to an adenosine nucleotide or an analogue thereof.
  • the invention relates to a EHD polypeptide having the structure defined by the structural coordinates as shown in Table A.
  • the invention relates to a method for identifying a candidate modulator of EHD family polypeptide activity, said method comprising
  • a molecular modelling apparatus is suitably a computer programmed with the appropriate tools for molecular modelling. Suitable programs/tools are noted in the examples section. .
  • the structural coordinates of at least the EH-domain are selected.
  • the first crystal structure of an EH domain is presented herein - in the prior art only low resolution NMR structural information has been available.
  • the crystal structure of the EH domain enables it to be effectively targeted, for example to find or test inhibitors of the interaction with the NPF motifs of the target cargo.
  • the structural coordinates of at least the G-domain are selected.
  • the structural coordinates of at least the dimerisation interface are selected.
  • the structural coordinates of the oligomerisation interface are selected.
  • oligomerisation E91Q, R167E, K193D, D198R.
  • the structural coordinates of at least the membrane binding site are selected.
  • each of the structural coordinates of Table A are selected.
  • the invention in another aspect, relates to a method for identifying a candidate therapeutic agent, said method comprising application of rational drug design to the crystal structure of EHD2.
  • Rational design of candidate agents likely to be able to interact with the target i protein may be based upon structural studies of the molecular shapes of the target protein as disclosed herein. These will provide guidance as to which amino acid residues form molecular contact regions.
  • the invention relates to a method of manufacturing a modulator of an EHD family polypeptide, said method comprising identifying a candidate modulator as described above, and synthesising a quantity of said modulator. ⁇ . • ⁇
  • the candidate therapeutic agent (or candidate modulator or molecular entity of interest (interchangeably referred to as 'agent' below)) may be an organic compound or other chemical.
  • the agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.
  • the agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.
  • the agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule.
  • the agent may be an antibody.
  • the agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.
  • the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised agent, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesiser or.
  • the agent will be an organic compound.
  • the organic compounds will comprise two or more hydrocarbyl groups.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group.
  • the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.
  • at least two of the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the agent comprises at least one cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the agent may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt — or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt - such as an acid addition salt or a base salt — or a solvate thereof, including a hydrate thereof.
  • the invention relates to use of a candidate modulator of EHD family polypeptide activity identified as described above, wherein said EHD family polypeptide is EHD2, in the manufacture of a medicament for diabetes. . .
  • the invention relates to use of the atomic coordinates as shown in Table A in the modelling of an EHD family polypeptide.
  • the invention relates to a method for the design of one or more ligands of an EHD family polypeptide, said method comprising the use of coordinates as shown in Table A.
  • the invention relates to use of an EHD family polypeptide in the tabulation of a biological membrane.
  • the invention in another aspect, relates to a method of tubulating a biological membrane comprising contacting said membrane with an EHD family polypeptide.
  • the invention relates to a method as described above or a use as described above wherein said membrane is comprised by a liposome.
  • said membrane comprises phosphatidylserine (PS).
  • PS phosphatidylserine
  • the invention relates to use of an EHD family polypeptide in membrane scission.
  • the invention in another aspect, relates to a method of inducing membrane scission, said method comprising contacting said membrane with an EHD family polypeptide.
  • said method further comprises contacting said membrane- EHD family polypeptide complex with nucleotide in conditions permissive of nucleotide hydrolysis.
  • nucleotide is adenosine triphosphate (ATP).
  • Mutating has it normal meaning in the art and may refer to the substitution or truncation or deletion of the residue, motif or domain referred to. Mutation may be effected at the polypeptide level e.g. by synthesis of a polypeptide having the mutated sequence, or may be effected at the nucleotide level e.g. by making a nucleic acid encoding the mutated sequence, which nucleic acid may be - subsequently translated to produce the mutated polypeptide. Where no amino acid is specified as the replacement amino acid for a given mutation site, suitably alanine (A) is used.
  • A alanine
  • the invention relates to a method of inhibiting dimerisation of an EHD family polypeptide, said method comprising mutating amino acid W238 of said polypeptide.
  • the invention relates to a method of inhibiting dimerisation of an EHD family polypeptide, said method comprising mutating amino acids neighbouring W238 of said polypeptide, such as amino acids within
  • the invention relates to a method of modifying an EHD family polypeptide to permit guanine nucleotide binding thereto, said method comprising mutating said EHD polypeptide at one or more amino acid residues within the region Hl 92 to M223, wherein said mutation alleviates steric exclusion of an amino group at carbon 2 of said guanine nucleotide.
  • the invention relates to a method of modifying an EHD family polypeptide to reduce or prevent membrane binding, said method comprising mutating any of K324, K327, K328, K329, K334, K341, V321 or F322.
  • amino acid V321 and/or F322 is mutated — these are considered to have similar effects.
  • the invention in another aspect, relates to a method of modifying an EHD family polypeptide to reduce or prevent membrane binding, said method comprising mutating any of K324, K327, K328, K329 or F322.
  • amino acid F322 is mutated.
  • the invention relates to a method of modifying an EHD family polypeptide to reduce or prevent ATP hydrolysis by said polypeptide, said method comprising mutating amino acid T72A or T94 of said polypeptide, suitably T94.
  • the invention relates to a method of modifying an EHD family polypeptide to reduce or prevent breakdown of membrane structures by said polypeptide, said method comprising mutating amino acid T72 A or T94 of said polypeptide, suitably T94.
  • the invention relates to a method of modifying an EHD family polypeptide to increase ATP hydrolysis, such as to increase intrinsic ATP hydrolysis, by said polypeptide, said method comprising mutating amino acid 1157 of said polypeptide.
  • the invention relates to a method of modifying an EHD family polypeptide to enhance breakdown of membrane structures by said polypeptide, said method comprising mutating amino acid 1157 of said polypeptide.
  • the invention relates to a method of inducing membrane fission, said method comprising contacting said membrane with an EHD family polypeptide comprising an Il 57Q mutation.
  • the invention in another aspect, relates to a method of modifying an EHD family polypeptide to reduce or abolish assembly stimulated ATP hydrolysis, said method comprising mutating said EHD polypeptide at E91, R167, K193, D198, F122, F 128, or by deletion of the EH domain.
  • said mutation(s) comprise one or more of the mutations set out in Figure 4b.
  • the invention relates to an EHD family polypeptide or fragment thereof comprising one or more of the following mutations: (i) T94A; (ii) I157Q; and (iii) F322A.
  • a fragment is suitably at least 10 amino acids in length, suitably at least 25 amino acids, suitably at least 50 amino acids, suitably at least 100 amino acids, suitably at least 200 amino acids, suitably the majority of the EHD polypeptide of interest.
  • a fragment comprises a whole motif or a whole domain of the EHD polypeptide of interest.
  • a fragment comprises at least 10 amino acids either side of the given mutation of interest.
  • a fragment comprises at least 10 amino acids each side of the two or more mutations, and suitably further comprises the intemvening amino acid sequence too.
  • the fragment comprises the amino acids between said mutation and said end (e.g. the N- or C- terminus).
  • the invention relates to a method of destabilising a membrane, said method comprising contacting said membrane with a EHD family polypeptide and a nucleotide under conditions permissive of nucleotide hydrolysis.
  • the invention relates to an EHD family polypeptide comprising one or more of the mutations described herein.
  • said EHD family polypeptide or fragment thereof is or is derived from mammalian EHD2.
  • the invention provides architectural and mechanistic insights into an EHD ATPase involved in membrane remodeling.
  • EH epsin homology domain-containing proteins
  • dynamin superfamily such as low affinity to nucleotides, the ability to tubulate liposomes in vitro, to oligomerise around lipid tubules in ring-like structures and to stimulate nucleotide hydrolysis in response to lipid binding.
  • EHD2 epsin homology domain-containing proteins
  • the nucleotide-binding domain is involved in dimerisation which creates a highly curved membrane-binding region. Oligomerisation of dimers occurs on another nucleotide-binding domain interface, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for an understanding of membrane deformation.
  • Dynamins are distinguished from classical signalling GTPases by their large size, their low affinity for nucleotide and assembly-stimulated nucleotide hydrolysis. From studies of GBPl, dynamin, bacterial dynamin-like protein, and particularly the insights into EHD disclosed herein, it appears that the mechanism of assembly- stimulated nucleotide hydrolysis is also conserved. In cases where data are available assembly involves the same conserved interface in the G-domain, with the same orientation of G-domains, a phosphate cap and an activation mechanism which is dependent on a catalytic serine or- threonine from switch I.
  • This mechanism is different for the signal-recognition particle and its receptor where the nucleotides are found anti-parallel in the dimer and GTP hydrolysis involves catalysis by the 2'-hydroxyl group of opposing nucleotides. This assembly- stimulated GTP hydrolysis mechanism has likely been maintained across the dynamin superfamily.
  • nucleotide hydrolysis is most likely leading to membrane scission in vivo, and thus conformational changes induced by nucleotide hydrolysis are leading to further membrane destabilisation. .
  • the invention may be useful in the attachment of entities to biological membranes.
  • the invention may relate to a method of attaching an entity to a biological membrane, the method comprising attaching said entity to a membrane binding element of an EHD family protein, and contacting the resulting complex with a biological membrane.
  • the invention may involve use of an EHD family polypeptide in the hydrolysis of ATP.
  • a biological membrane is typically a lipid bilayer membrane.
  • An example of a biological membrane is a plasma membrane.
  • the biological membranes of the invention are often intracellular membranes, for example those involved in vesicle trafficking, or those forming a part of the endocytic recycling compartment.
  • the term liposome has its normal meaning in the art, namely a single or multi laminar vesicle. Liposomes may be made from lecithins or other lipids. Preferably liposomes are made from brain derived lipids. Preferably liposomes are made from Folch extract.
  • liposomes may be made from 100% anionic phosphatidyl serine (PS) liposomes (it is to be noted that this 100% refers to the composition of the liposomes in this particular embodiment and should not infer the proportion of lipid present overall which is discussed elsewhere herein).
  • PS anionic phosphatidyl serine
  • liposomes may contain phosphatidyl inositol 4,5 bisphosphate (PEP2).
  • EHD FAMILY POLYPEPTIDES comprise a highly conserved eukaryotic protein family. EHDs have a molecular mass of approximately 60KD. EHD proteins contain a G domain, a helical domain, and a EH domain. Typically these domains occur in the order N terminus - G domain - helical domain - EH domain - C terminus. However, it should be noted that in plant EHDs, the EH domain may be at the N terminus.
  • an EHD family polypeptide For a polypeptide to be considered as an EHD family polypeptide, it should possess one or more of the above characteristics. More suitably, it should possess sufficient sequence identity to EHD1/2/3/4 to be classified in the same molecular family. Most suitably, it should be a mammalian EHD polypeptide. Most suitably, it should comprise mammalian EHD2 amino acid sequence. In particular, an exemplary EHD family polypeptide has the sequence shown as mmEHD2 in figure 8.
  • mouse EHD2 mouse EHD2
  • mmEHD2 mouse EHD2 amino acid sequence
  • This is to be used as is well understood in the art to locate the residue of interest. This is not always a strict counting exercise — attention must be paid to the context.
  • the protein of interest such as human EHD2.is of a slightly different length
  • location of the correct residue in the human sequence correseponding to (for example) T94 may require the sequences to be aligned and the equivalent or corresponding residue picked, rather than simply taking the 94 th residue of the sequence of interest. This is well within the ambit of the skilled reader.
  • figure 8 presents a comprehensive alignment of sequences of interest with the reference sequence mrnEHD2 at the top which both illustrates the principle and provides a robust reference chart for ease of location of the correct residues.
  • EHD2 exhibits high sequence homology with other EHD family polypeptides.
  • the invention relates to the use of EHD2 in the development of therapeutics for application to other EHD family proteins, hi particular, the crystal structure of the ATPase domain of EHD2 complexed with a bound ligand is applicable across the EHD family polypeptides since those polypeptides are also regarded as ATPases (having previously been thought to be GTPases).
  • EHD family polypeptides can rescue a C.elegans RME knockout.
  • a particular polypeptide is indeed to be considered an EHD family polypeptide, it may be tested whether or not that polypeptide can rescue a C.elegans RME knockout. If the knockout is rescued, the polypeptide may be regarded as an EHD family polypeptide.
  • EHD4 / Pincher is predominantly localised to the plasma membrane and is involved in the uptake of the TrkA
  • TrkA in the presence of NGF .
  • Other members of the family also mediate trafficking of various ligands and overexpression of these EHDs leads to their
  • EHDl is involved in MHC class I recycling at the membrane (Caplan et al. (2002) A tubular EHDl -containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane. EMBO J. 21 p2557-67). Overexpression of EHDl increases recycling. By inhibiting EHDl activity, for example in a transplanted organ, the transplant may be protected from effects of autorejection. Conversely, activation of EHDl activity may promote an immunological response.
  • the invention relates to use of a candidate modulator of EHD family polypeptide activity identified as described above, wherein said EHD family polypeptide is EHDl, in the manufacture of a medicament for ameliorating or enhancing immune responses.
  • Amelioration relates to inhibitors of EHDl, augmentation applies to activators of EHDl.
  • EHDl may have an involvement in cystic fibrosis. .
  • EHD2 is . known to be involved in insulin mediated GLUT4 transport to the membrane (Park et al. (2004) EHD2 interacts with the insulin-responsive glucose transporter (GLUT4) in rat adipocytes and may participate in insulin-induced GLUT4 recruitment. Biochemistry 43 p7552-62). Thus inhibition of EHD2 provides therapeutic benefit in the treatment of diabetes such as type II diabetes.
  • the invention relates to use of a candidate modulator of EHD family polypeptide activity identified as described above, wherein said EHD family polypeptide is EHD2, in the manufacture of a medicament for diabetes.
  • EHD4 mediates pinocytic endocytosis of functionally specialised nerve growth factor (NGFJ/neurotr ⁇ phic tyrosine kinase receptor type 1 (TrkA) to endosomes and TrkA-erk5 mitogen-activated protein kinase signalling (Shao et al. (2002) Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes. J. Cell Biol. 157 p679-691).
  • NGFJ/neurotr ⁇ phic tyrosine kinase receptor type 1 (TrkA) to endosomes
  • TrkA-erk5 mitogen-activated protein kinase signalling
  • the invention relates to use of a candidate modulator of EHD family polypeptide activity identified as described above, wherein said EHD family polypeptide is EHD4, in the manufacture of a medicament for pain, hi another aspect, the invention relates to use of a candidate modulator of EHD family polypeptide activity identified as described above, wherein said EHD family polypeptide is EHD4, in the manufacture of a medicament for cancer.
  • EHDs are involved in clathrin-independent endocytosis, which is also known to be a mechanism of entry for viruses into cells (Damm et al. (2005) Clathrin- and caveolin-1 -independent endocytosis : entry of simian virus 40 into cells devoid of caveolae. . J. Cell Biol. 168 ⁇ 477 ⁇ 488).
  • broad inhibition of the EHD class finds application in viral infections.
  • the EHD sequence is conserved from parasites to higher mammals such as humans.
  • treatments for infections may usefully be provided.
  • the invention provides use of a candidate modulator of EHD family polypeptide activity identified as described above, in the manufacture of a medicament for viral or parasite infection.
  • EHDl is involved in the recycling of MHC class 1 molecules.
  • modulation of EHDl has application in immune modulation, for example in transplant patients or in graft versus host disease.
  • EHD4 is involved in the internalisation of the TrkA receptor. This receptor responds to nerve growth factor (NGF).
  • NGF nerve growth factor
  • modulation of EHD4 is relevant to the modulation of, or treatment of, pain.
  • NGF nerve growth factor
  • modulation of EHD4 is relevant to the modulation of, or treatment of, pain.
  • NGF is also implicated in the survival and homeostatic maintenance of neurons; thus modulation of EHD4, for example EHD4 response to NGF, finds application in the treatment of neurodegeneration.
  • EHD2 is important in diabetes.
  • modulation of EHD2 is important for the treatment of diabetes.
  • inhibition of EHD2 is important for the treatment of diabetes.
  • the present invention particularly concerns screening for or validation of EHD2 inhibitors.
  • the invention relates to the use of EHD2 inhibitors in the treatment of diabetes.
  • the invention relates to the screening for compounds capable of inhibiting EHD2, and to the use of such inhibitors in the manufacture of a medicament for diabetes.
  • ASSAYS There are numerous biological activities ascribed to EHD family polypeptides for the first time by the present inventors. These include oligomerisation (including dimerisation), lipid binding, nucleotide binding and nucleotide hydrolysis. The invention provides assays for modulators of one or more of these activities.
  • oligomerisation may provide specificity between different EHD family members such as EHD 1/2/3/4. These individual proteins are known to localise to different places within the cell.
  • EHD family members such as EHD 1/2/3/4.
  • these individual proteins are known to localise to different places within the cell.
  • an advantageous specificity can be provided for molecules so identified.
  • the assays of the present invention readout or screen for factors affecting oligomerisation. . .
  • oligomerisation facilitates lipid binding. Therefore, for certain assay readouts it may be possible that one or more underlying biological effects might be detected. For example, if the readout of a particular assay was lipid binding, this would be expected to be adversely affected by anything which inhibited oligomerisation. Such factors should be borne in mind by the skilled operator.
  • the assays of the invention suitably monitor EHD ATP ase activity.
  • Colorimetric ATPase assays are widely available commercially (e.g. http://www.innovabiosciences.com/products/atpase.php).
  • the assay is suitably conducted according to the manufacturer's recommendations for ATPase assay.
  • the assay may be conducted using regaents of InnovaBiosciences' catalogue number 601-0120.
  • the assay is conducted essentially as set out in Innova Biosciences' Technical Bulletin number 654 (release 007; July 2005).
  • EHD polypeptides may exhibit a slow rate of ATP turnover - this may advantageously be enhanced by including a lipid preparation such as a liposome preparation into the assay of the invention.
  • functional assay step(s) may be additionally used in order to better characterise the effect of the modulator(s) or treatment(s) being studied, or to verify the in vivo significance.
  • An example of such an assay is to monitor internalisation of labelled NGF for candidate EHD4 modulators.
  • a direct lipid binding assay might be employed.
  • a FRET based assay might be used, hi this embodiment, a first FRET element would be attached to the EHD family polypeptide of interest, and a second FRET element would be attached to the lipid of interest.
  • the FRET effect fluorescence resonance energy transfer
  • nucleotide binding assay Any nucleotide binding assay known in the art may be employed for this subsequent step or assay, for example the direct binding of a radiolabeled or a fluorescently labelled nucleotide to the EHD family polypeptide of interest under the different treatments being investigated may be assessed.
  • molecular modelling aspects to the design of a nucleotide analogue. For example, it may be possible to design a nucleotide analogue which would selectively bind to an EHD family polypeptide such as EHD2.
  • the invention relates to such design methods, and to a nucleotide analogue so designed.
  • the assays of the invention are likely to identify inhibitors of oligomerisation more often or more reliably than specific inhibitors of membrane binding. The reason is that the oligomerisation interface is much more extensive than the relatively much smaller interface which is believed to mediate membrane binding. Since disruption of oligomerisation leads to disruption of membrane binding, the hits which are detected by assaying for disrupted membrane binding would be expected to contain a greater proportion of hits disrupting oligomerisation and a smaller proportion of hits which directly interfere with the actual mechanism of membrane binding.
  • candidate modulators of EHD family polypeptides may be applied to cells harbouring a fluorescently labelled EHD family polypeptide.
  • the EHD family polypeptide would normally be localised to one or more membrane locations within the cell, dependent on which EHD family member was being studied. If the presence of the candidate modulator changes the expected cellular distribution of the EHD family polypeptide being studied, then this is a dramatic indicator that membrane binding had been affected by that modulator.
  • One example of an altered distribution would be a cytosolic distribution.
  • the invention relates to a three step screening procedure having a. first step of a ATPase screen, a second step of a cell based localisation screen, and an optional third step of a cargo internalisation screen
  • the invention may relate to a two step procedure involving a first step of an ATPase screen and a second step of a cargo internalisation screen
  • the invention may relate to a two step procedure involving a first step of an ATPase screen and a second step of a cell based localisation screen.
  • a dynamin control may be included as a reference sample in the assay of the invention.
  • a dynamin family polypeptide as a control provides numerous advantages. Dynamin is one of the most homologous proteins to EHD in terms of sequence identity. It is expected that dynamin works via a similar mechanism for membrane scission. The activity and affinity profile for dynamin is thought to be similar to EHD. Furthermore, the lipid specificity of dynamin is very close to the specificity of EHD family polypeptides. Thus, by including dynamin as a parallel reference sample in the assays of the invention, false positives having a general effect (rather than an EHD specific effect) may beneficially be excluded from the screen at a very early stage.
  • dynamin is a GTPase. Therefore, the reference sample featuring dynamin should have GTP as a substrate, and should measure the hydrolysis of GTP (rather than ATP which will of course be used in assaying EHD activity).
  • GTP GTPase
  • ATP ATP-phosphate
  • two dynamin controls are used, one with candidate modulator and one without. This advantageously permits internal calibration of the background for dynamin, making the dynamin control more robust.
  • the candidate compounds or treatments of most interest will be those which have ho effect on dynamin action, such as no effect on the GTP hydrolysis activity of dynamin, but which do affect EHD activity, such as ATP hydrolysis by EHD.
  • a control or reference sample which comprises an inactive EHD family polypeptide.
  • an inactive EHD family polypeptide is suitably constructed by mutation of the wild type EHD sequence.
  • the catalytic residue may be substituted, residues important in activation of hydrolysis may be substituted, residues involved in ATP binding may be substituted, or any other suitable alteration to the ATP hydrolytic elements of EHD may be made.
  • the most stable polypeptide is selected as an inactive EHD mutant.
  • the most suitable EHD mutant to select is that which knocks out the ATPase activity.
  • a most suitable mutant is a T94A or T72A mutant, more suitably a T94A mutant, of an EHD family polypeptide (or the equivalent residue).
  • This has the advantage of binding ATP but also has the advantage of being catalytically inactive, in other words the T94A mutant does not catalyse the hydrolysis of ATP.
  • Design of this mutant has been enabled by the structural insights into EHD presented herein.
  • the T94A mutant has the further advantage that it is exceptionally well suited to the search for factors affecting oligomerisation. This is because this mutant actually oligomerises slightly more readily even than the wild type EHD family polypeptide family itself.
  • the assays of the invention suitably comprise a lipid component such as a membrane component.
  • the lipid component may comprise or contain any negatively charged lipid.
  • the reason that the lipids should be negatively charged is due to the lysines present in the lipid binding site of EHD family polypeptides. Without wishing to be bound by theory, it is believed that the initial long range charge mediated interaction is between the negatively charged elements of the lipids and the positively charged lysine residues on the EHD family polypeptide.
  • the negatively charged lipid component may be phosphatidyl serine.
  • the negatively charged lipid component may be any phosphatidyl inositol lipid.
  • the negatively charged lipids may be provided in the form of liposomes such as Folch liposomes.
  • PIP2 phosphatidyl inositol 4,5 bisphosphate
  • PS phosphatidyl serine
  • lipid such as phosphatidyl serine (PS) is used at approximately 10%-20% final concentration.
  • PS phosphatidyl serine
  • the invention provides a super-active EHD family polypeptide mutant.
  • This mutant is suitably the I157Q mutant.
  • EHD family polypeptides bearing this mutation are not well activated by lipids.
  • EHD family polypeptides bearing the I157Q mutation are already very highly active in terms of
  • the present invention relates to an EHD family polypeptide bearing the Il 57Q mutation.
  • such mutants are useful in embodiments of the assay of the invention.
  • the inclusion of lipids in the assay may advantageously be avoided.
  • an I157Q EHD family polypeptide may be used as a reference or control sample. In this way, if a particular compound or treatment was shown to affect ATP hydrolysis of an EHD family polypeptide both with and without the I157Q mutation, in particular when lipids were absent from the I157Q assay sample, then this effectively, provides another level of information about the action of the compound or treatment being studied.
  • an I157Q mutant EHD is affected in a similar manner to a normal EHD polypeptide, then this would indicate that the action of the compound or treatment is via an ATP or an oligomerisation effect, and is far less likely to be via a lipid binding effect.
  • the assays of the invention may employ I157Q mutants in order to distinguish between the effects on different biological aspects of the EHD family polypeptide being studied.
  • EHD3 may be involved in transferrin uptake. Specifically, EHD3 may inhibit transferrin uptake. Transferrin uptake is an essential biological function, and therefore it is not desirable to interfere with this function. For these reasons, preferably inhibition of transferrin uptake by EHD3 is not a target of the present invention. However, the invention may be applied to the identification or development of inhibitors of EHD3 which still allow transferrin uptake, hi another embodiment transferrin uptake may be used as a readout in a cellular screen to ensure EHD modulators do not interfere with this vital function (i.e. a counter screen).
  • Determination of the 3D structure of EHD provides important information about the likely active sites of EHD, particularly when comparisons are made with similar enzymes. This information may then be used for rational design of EHD inhibitors or interactors, e.g. by computational techniques which identify possible binding ligands for the active sites, by enabling linked-fragment approaches to drug design, and by enabling the identification and location of bound ligands using X-ray crystallographic analysis. These techniques are discussed in more detail below.
  • EHD inhibitors may also be designed in the this way. More specifically, a ligand (e.g. a potential inhibitor) of EHD may be designed that complements the functionalities of the EHD active site(s) such as the oligomerisation site or ATP ase site. The ligand can then be synthesised, formed into a complex with EHD, and the complex then analysed by X-ray crystallography to identify the actual position of the bound ligand.
  • a ligand e.g. a potential inhibitor of EHD may be designed that complements the functionalities of the EHD active site(s) such as the oligomerisation site or ATP ase site.
  • the ligand can then be synthesised, formed into a complex with EHD, and the complex then analysed by X-ray crystallography to identify the actual position of the bound ligand.
  • the structure and/or functional groups of the ligand can then be adjusted, if necessary, in view of the results of the X-ray analysis, and the synthesis and analysis sequence repeated until an optimised ligand is obtained.
  • Related approaches to structure-based drug design are also discussed in Bohacek et al., Medicinal Research Reviews, Vol.16, (1996), 3-50.
  • EHD inhibitors or activators
  • automated ligand-receptor docking programs discussed e.g. by Jones et al. in Current Opinion in Biotechnology,
  • Linked-fragment approaches to drug design also require accurate information on the atomic coordinates of target molecules.
  • the idea behind these approaches is to determine (computationally or experimentally) the binding locations of plural ligands to a target molecule, and then construct a molecular scaffold to connect the ligands together in such a way that their relative binding positions are preserved.
  • the connected ligands thus form a potential lead compound that can be further refined using e.g. iterative technique(s).
  • EHD electronic-based drug design
  • these compounds are known e.g. from the research literature.
  • a first stage of the drug design program may involve computer-based in silico screening of compound databases (such as the Cambridge Structural Database) with the aim of identifying compounds which interact with the active site or sites of the target bio-molecule. Screening selection criteria may be based on pharmacokinetic properties such as metabolic stability and toxicity.
  • determination of the EHD structure allows the architecture and chemical nature of each active site to be identified, which in turn allows the geometric and functional constraints of a descriptor for the potential inhibitor to be derived.
  • the descriptor is, therefore, a type of virtual 3-D pharmacophore, which can also be used as selection criteria or filter for database screening.
  • the invention relates to the selection and/or design and/or screening for inhibitors or activators or molecules capable of interfering with or binding to EHD polypeptides.
  • the invention relates to screening for inhibitors of EHD polypeptides.
  • FIG. 2 shows the structure of EHD2: a, Ribbon-type presentation of the EHD2 dimer. Molecule A is coloured according to the secondary structure and molecule B according to the domain structure. Disordered loops are represented by dashed lines.
  • Figure 3 shows membrane binding and the role of ATP hydrolysis: a, Ribbon-type representation of the putative membrane-binding site with residues tested for membrane-binding in ball-and-stick representation, b, Coomassie-stained gels of sedimentation assays in the absence (lower panel) and presence of Folch liposomes using wild-type EHD2 and the indicated mutants, c, Nucleotide hydrolysis of the lipid binding mutants was carried out as described in Fig. 1f. The F322A mutant utilizat intrinsic, ⁇ stimulated reaction) showed a 40% decrease in the stimulated ATPase
  • Mouse EHD2 was expressed in Escherichia coli as a His-fusion protein as described in Materials and Methods.
  • NI Non-induced culture.
  • I Induced culture.
  • SN Soluble extract.
  • FT Soluble extract after application to NiNTA Sepharose.
  • E1- EHD2 after elution from NiNTA-Sepharose.
  • E2 - EHD2 after dialysis and thrombin cleavage.
  • E3 - EHD2 after re-application and elution from the NiNTA column. This protein was further purified by size-exclusion chromatography using a Sephadex S200 column (data not shown).
  • Figure 6 shows ultracentrifugation analysis indicates that EHD2 is a dimeric protein.
  • Sedimentation velocity experiments were performed as described in methods. Selected scans (at equal, ⁇ 15min intervals), and of g(s20,w) (the amount of material sedimenting between s20,w and (s20,w+ ⁇ s)) and also the residuals for fitting the data with DCDT+, were plotted with the program profit v.5.6.7 (Quantum soft, Switzerland). The fitted value is 113 ⁇ 4kDa which corresponds well with the calculated mass of the dimer of 124kDa.
  • FIG. 7 shows EHD2 tubulation of PS- and synthetic liposomes.
  • EHD2 was incubated with the indicated liposomes in the presence and absence of nucleotides and analysed by EM as described in Methods, a, EHD2 deformed PS liposomes into tubular networks, here in the presence of ATP-Y-S.
  • b Enlarged views of the indicated area in a. Note the presence of regularly spaced EHD2 rings (some are indicated with arrows).
  • EHD2 In the presence of ADP and in the absence of nucleotide (f,g), EHD2 also tubulated PS liposomes and formed ring-like structures around the tubules. e,g are enlarged views of the indicated areas in d and f, respectively. We did not observe a noticeable change of size of the tubules with the different nucleotide conditions.
  • Figure 8 shows alignment and secondary structure assignment. Multiple sequence alignment of the EHD family. The following sequences were aligned (expasy accession number in brackets): Mouse musculus EHD2 (Q8BH64), Homo sapiens EHD1 (Q9H4M9), Homo sapiens EHD3 (Q9NZN3), Homo sapiens EHD4 (Q9H223), Danio rerio EHD (Q6P3J7), Xenopus laevis EHD1 (Q7SYA1), Xenopus laevis EHD4 (Q7ZXE8), Drosophila melanogaster PAST-1 (Q8IGN0), Caenorhabditis elegans RME 1 (Q966F0), Schistosoma japonicum EHD (Q5DG45), Dictyostelium discoideum EHD (Q54ST5), Plasmodium falciparum EHD (Q9NLB8), Entamoeba his
  • Figure 9 shows the EHD dimer interface.
  • the alignment in Fig. 8 was used to create a surface conservation plot of the EHD2 G-domain and helical domain, with conserved residues shown in purple and non- conserved residues shown in cyan. Helix ⁇ 6 with its invariant W238 and the contiguous loop of the opposing EHD2 monomer are shown in orange interacting with the conserved surface.
  • Figure 10 shows the Ca binding site of the EH-domain. Residues
  • FIG. 11 shows Top: Electrostatic surface representation of EHD2. Red indicates negative charge and blue positive charge at neutral pH. The orientation of EHD2 is the same as in Fig. 2a. The membrane interaction site is highly curved which is a consequence of EHD2 dimerisation. The diameter of the sphere is approximately 7nm, but this may not be so extreme if the tips of the loops (a phenylalanine and a valine residue) are inserted into the membrane.
  • Figure 13 shows: a, Top and side view of the EHD2 oligomeric ring model in a surface representation. For better clarity, the EH domains are not included.
  • the diameter of the embraced lipid tubule is «18nm and the thickness of the EHD2 ring is «10nm, in agreement to what is observed in the EM assays.
  • Approximately twenty EHD2 dimers constitute one turn in this model, b, Arrangement of the EHD2 dimers in the oligomer.
  • the high curvature of the membrane interaction site of EHD2 (Fig. 11 ) is oriented perpendicular to the curvature of the lipid tubule.
  • Figure 14 shows analysis of peptide binding to the EH-domain.
  • the affinity of hepta-peptides from EHD2 containing the indicated xPF motifs to the EH-domain of EHD2 was measured by ITC at 10°C in 10OmM HEPES (pH 7.5), 5OmM NaCl 5
  • ITC measurements were performed at 10 0 C in 2OmM HEPES (pH 7.5), 30OmM NaCl, 2mM MgC12. Liposome binding assays were performed as described previously (www.endocytosis.org). Multiple turnover ATPase assays were performed in 2OmM HEPES (pH 7.5), 135mM NaCl 5 15mM KCl, ImM MgC12 at 30 0 C with lO ⁇ M EHD2 (or mutants) as enzyme and lOO ⁇ M ATP as substrate, in the absence or presence of lmg/ml Folch liposomes (Sigma- Aldrich). Reactions were started by the addition of the protein to the final reaction mix and
  • Mouse EHD2 full-length protein and all mutants were expressed as N-terminal His-fusions followed by a PreScission cleavage site in Escherichia coli BL21 DE3 Rosetta (Novagen) from a modified pET28 vector.
  • Bacteria cultures in TB medium were induced at an OD of 0.2 with 40 ⁇ M IPTG and grown overnight at 18°C.
  • Bacteria were lysed in lysis buffer containing 5OmM HEPES (pH 7.5), 40OmM NaCl, 25mM Imidazole, 2.5mM ⁇ -Mercaptoethanol ( ⁇ -ME), 500 ⁇ M Pefablock SC (Boehringer Ingelheim) using an Emulsiflex homogeniser (Avestin, Canada).
  • the soluble extract was applied to, a NiNTA-column (Qiagen, Hildesheim) equilibrated with lysis buffer.
  • the column was extensively washed with 2OmM HEPES (pH 7.5), 70OmM NaCl, 3OmM Imidazole, 2.5mM ⁇ -ME, ImM ATP, 1OmM KCl and shortly with 2OmM
  • HEPES pH 7.5
  • 30OmM NaCl 25mM Imidazole, 2.5mM ⁇ -ME.
  • Bound protein was eluted with 2OmM HEPES (pH 7.5), 30OmM NaCl, 10OmM Imidazole, 2.5mM ⁇ -ME and dialysed overnight at 4°C against 2OmM HEPES (pH 7.5), 30OmM NaCl, 2.5mM ⁇ -Mercaptoethanol in the presence of 250 ⁇ g PreScission protease to cleave the His-tag.
  • the protein was re-applied to a NiNTA column to which it bound under these buffer conditions also in the absence of the His-tag.
  • the column was extensively washed with 2OmM HEPES, 30OmM NaCl, 2.5mM ⁇ -ME, and the protein finally eluted with 2OmM HEPES, 30OmM NaCl, 2.5mM ⁇ -Me, 25mM Imidazole, concentrated and further purified using a Sephadex200 size-exclusion column (two consecutive runs for proteins used for the ATP ase assays). Typical yields were 4mg purified EHD2 / 1 bacteria culture. At 30OmM NaCl we could concentrate the protein to 40mg/ml but at lower salt concentration we observed some precipitation at this protein concentration. The protein was partially stabilised by ImM MgC12.
  • 30 selenomethioninesubstituted point mutant Q41 OA was prepared as described .
  • This mutant showed identical biochemical properties as the wild-type protein but displayed less degradation in the linker region when incubated over longer periods of time.
  • the protein was concentrated to 40mg/ml and supplemented with 4mM MgC12, 2mM AMP-PNP (Sigma-Aldrich, both final concentrations).
  • the hanging- drop vapour-diffusion method was used for crystallisation. 2 ⁇ l protein solution were mixed with an equal volume of reservoir solution containing 3% PEG2000 MME, 5OmM MES (pH 6.4), 4mM MgC12. Crystals appeared after one week at
  • 3 5 model was refined using Refmac5 with 3 TLS groups ( Figure 15 ("Supplementary Table 1"))-
  • the asymmetric unit contains 477 amino acids, one AMPPNP, one magnesium, one calcium and five water molecules and has an excellent geometry with all residues in the favoured and most favoured region of
  • Electron potential maps were generated using ccp4 molecular graphics.
  • EHD2 Mouse full-length EHD2 was expressed in bacteria and purified to homogeneity (Fig. 5). The purified protein was nucleotide-free as judged by HPLC analysis. In 30OmM NaCl, EHD2 was highly soluble, eluted as a dimeric protein by size- exclusion chromatography, and was found to be a dimer in dynamic light scattering experiments and in analytical velocity centrifugation (Fig. 6). At 50 ⁇ M protein concentration, the hydrodynamic radius did not change in the presence or absence of nucleotides (or in 15OmM versus 30OmM NaCl), as judged by dynamic light scattering experiments.
  • nucleptide-free T72A mutant bound to Folch liposomes in vitro (Fig. 1C), it showed a cytoplasmic distribution when over- expressed in vivo (Fig. 1 e), indicating that nucleotide binding is required for
  • the nucleotide binding domain of EHD2 possesses a typical G-domain fold with a central ⁇ -sheet surrounded by ⁇ -helices (Fig. 2a, 2b and Fig. 8).
  • AMP-PNP molecule occupies the canonical nucleotide-binding site.
  • EHD2 contains an insertion of two additional ⁇ -strands
  • the helical domain is composed of helix ⁇ l and ⁇ 2 from the N-terminal region (residues 18-55, which follow disordered residues 1-18) and helices ⁇ 8, oc9, ⁇ lO, ⁇ l 1 and ⁇ l2 (residues 285-400) following the G-domain (Fig. 2a).
  • Helix ⁇ 8 of EHD is the organising scaffold against which most of the other helices fold. It has also extensive contacts with the G-domain.
  • the dimeric G-domain together with the helical region adopts a scissor shape, where the membrane is proposed to bind between blades (see later).
  • EH- domain of EHD2 is similar to the previously determined second EH-domain of
  • Epsl 5 solved by NMR studies with a root-mean square deviation of 1.5 A for the main-chain atoms (Fig. 2c). It is built of two closely packed perpendicular EF
  • the peptide binding sites of both EH-domains are occupied by a GPF motif (residues 420-422) from the linker region (Fig. 2a and c).
  • the GPF motif adopts a similar conformation as an NPF-containing peptide bound
  • G4 a highly conserved NKxD motif
  • Asparagine side-chain forms a hydrogen bond to the carbonyl group at carbon ⁇ of the guanosine base and the aspartate side-chain forms a double hydrogen bond to nitrogenl of the guanosine base and the amine group at carbon2 (Fig. 2d, left).
  • Aspartate has been shown to be crucial since mutating it to asparagine in Ras reduces nucleotide affinity by more than 1000- 23 fold .
  • a large hydrophobic residue (leucine in Ras and dynamin, M223 in EHD2) lines the nucleotide binding pocket.
  • the NKxD motif of EHD2 (starting at residue 219) is also highly conserved in EHD family members (Fig. 8).
  • the carboxyamide group of N219 forms a hydrogen bond to the C6 amino group of the adenine base (Fig. 2d, right).
  • M223, whose side-chain is buttressed by the side chain of H 192, is closer to the purine base than the corresponding leucine residue in Ras and sterically excludes an amino group at C2, thus explaining the inability of EHDs to bind to guanine nucleotides.
  • EHD2 membrane-binding properties of EHD2 are pronounced of a subset of the small GTPase family which have recently been shown to require polybasic stretches for their PIP2- and PI(3,4,5)P3-dependent targeting to the plasma
  • 25 cap is also present in GBPl whose mechanism of GTP hydrolysis has been shown to involve dimerisation-dependent positioning of the attacking nucleophilic
  • T94A mutant bound to ATP- ⁇ -S with nearly wild-type affinity (Fig. 3e) and oligomerised around PS liposomes (Fig. 4e), but did not show any membrane-stimulated ATPase activity, consistent with a catalytic function for T94.
  • the T94A mutant labelled extensive tubular structures with essentially no punctate staining (compare wild-type and T94A in Fig. 3d, 3f and 3g), suggesting that ATP hydrolysis is involved in the break-down of tubular structures in vivo.
  • the EHD2 dimer may further oligomerise into the observed rings using this second G-domain interface.
  • the oligomer could also form along the length of a tubule (parallel to its long axis) and thus maximise the use of the highly curved membrane interface of the dimer (Fig. 13 c).
  • a tubule parallel to its long axis
  • Fig. 13 c highly curved membrane interface of the dimer
  • EH-domains are low affinity protein-protein interactors normally found in endocytic multi-subunit assemblies. A likely purpose of the EH-domain is in recruitment of the protein to sites of action. In the oligomer it could further function to concentrate NPF-containing binding partners around membrane-bound EHD, but we did not observe any co-localisation of described EHD binding
  • EHD2 wild-type was over-expressed in HeLa cells for 24h and imaged by EPI- fluorescence for approximately 30min. Some of the tubules and puncta are dynamic.
  • EHD2 T94A was over-expressed in HeLa cells for 24h and imaged by EPI- fluorescence for approximately 30min. There a only tubules, and these are mostly static.
  • EHD2 I157Q was over-expressed in HeLa cells for 24h and imaged by EPI- fiuoresce ⁇ ce for approximately 30min. No tubules can be found and the puncta are mostly motile.
  • Four EHD2 dimers (in the absence of the EH domain) were aligned as described in Methods. All lipid interaction sites point towards the putative membrane interface. Molecules B and C which have been used for the initial alignment with GBPl are related via a 2-fold axis, and the nucleotides of these molecules are oriented in a head-to-head fashion.
  • This example relates to a method of identifying a modulator of an EHD family polypeptide.
  • a first and second sample of an EHD polypeptide are provided.
  • the EHD polypeptide is EHD2.
  • the first EHD sample is contacted with a candidate modulator.
  • the candidate modulator is added to the medium containing the EHD polypeptide.
  • ATP reagent is added to the first and second samples.
  • the ATP reagent is as per InnovaBiosciences' catalogue number 601-0120, permitting colorimetric readout of ATP hydrolysis.
  • ATP hydrolysis in said first and second samples is monitored in accordance with the manufacturer's instructions. .
  • a difference between the ATP hydrolysis in said first and second samples identifies said candidate modulator as a modulator of an EHD family polypeptide.
  • the candidate modulator is identified as an enhancer of EHD family polypeptide activity. Conversely, if hydrolysis of ATP is lower in said, first sample than in said second sample then the candidate modulator is identified as an inhibitor of EHD family polypeptide activity.
  • the ATP hydrolysis is optionally monitored in the presence of lipid, in which case liposomes and/or phosphatidylserine (PS) at a final concentration of about 10% are added to both the samples, suitably after addition of candidate modulator but before addition of ATP reagent.
  • PS phosphatidylserine
  • ATOM 749 CA LEUA 133 23.870 -40.539 6.181 1.0066.14 C ATOM 750 CB LEU A 133 25.374 -40.769 6.373 1.00 66.19 C ATOM 751 CG LEU A 133 26.382 -40.435 5.265 1.00 66.22 C ATOM 752 CDl LEU A 133 26.089 -39.104 4.584 1.00 66.32 C ATOM 753 CD2 LEU A 133 27.804 -40.457 5.821 1.00 66.13 C ATOM 754 C LEU A 133 23.101 -41.546 7.032 1.00 65.93 C ATOM 755 O LEU A 133 23.329 -42.751 6.937 1.00 65.82 O ' ATOM 756 N ASN A 134 22.205 -41.051 7.882 1.00 65.76 N ' ATOM 757 CA ASN A 134 21.409 -41.930 8.732 1.00 65.55 C • ATOM 758 CB
  • ATOM 831 CB ASN A 143 30.167-62.745 -1.756 1.0057.34 C 5- ATOM 832 CG ASN A 143 29.403 -63.022 -3.037 1.0057.28 C

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Abstract

L'invention porte sur un procédé d'identification d'un modulateur d'un polypeptide de la famille EHD. Ledit procédé comprend (i) l'obtention d'un premier et d'un second échantillon d'un polypeptide EHD; (ii) la mise en contact dudit premier échantillon avec un modulateur candidat; (iii) la mise en contact desdits premier et second échantillons avec de l'ATP; et (iv) la surveillance de l'hydrolyse de l'ATP dans lesdits premier et second échantillons, une différence entre l'hydrolyse de l'ATP dans lesdits premier et second échantillons permettant d'identifier ledit modulateur candidat en tant que modulateur d'un polypeptide de la famille EHD. L'invention porte également sur des procédés de modélisation de molécules et sur divers polypeptides mutants EHD.
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