WO2017174762A1 - Structure cristalline de bêta-hydroxylase de dopamine humaine - Google Patents

Structure cristalline de bêta-hydroxylase de dopamine humaine Download PDF

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WO2017174762A1
WO2017174762A1 PCT/EP2017/058334 EP2017058334W WO2017174762A1 WO 2017174762 A1 WO2017174762 A1 WO 2017174762A1 EP 2017058334 W EP2017058334 W EP 2017058334W WO 2017174762 A1 WO2017174762 A1 WO 2017174762A1
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Pernille Hanne HARRIS
Hans Erik Mølager CHRISTENSEN
Trine Vammen VENDELBOE
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Danmarks Tekniske Universitet
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a crystalline form of dopamine ⁇ -hydroxylase.
  • Dopamine /3-hydroxylase (EC 1.14.17.1, dopamine ⁇ -monooxygenase) (DBH) catalyzes the hydroxylation of dopamine to norepinephrine, and is thus vital for regulation of these neurotransmitters.
  • DBH dopamine ⁇ -monooxygenase
  • the norepinephrine pathway the only source of norepinephrine and epinephrine, is believed to modulate many behavioral and physiological processes, such as mood, overall arousal, attention, sexual behavior as well as stress, and learning and memory.
  • the level of and balance between dopamine and norepinephrine are implicated in a large number of diseases of both physiological, neurological, and psychiatric character, such as hypertension (ranked as the world's largest disease burden), congestive heart failure, Alzheimer's disease, drug addiction as well as Parkinson ' s disease, Huntington ' s chorea, Tourette syndrome, depression, and attention deficit hyperactivity disorder (ADHD), for review see J. F. Cubells, C. P. Zabetian Psychopharmacology (Berl). 174, 463-476 (2004).
  • hypertension ranked as the world's largest disease burden
  • congestive heart failure ranked as the world's largest disease burden
  • Alzheimer's disease drug addiction as well as Parkinson ' s disease
  • Huntington ' s chorea Huntington ' s chorea
  • Tourette syndrome depression
  • depression attention deficit hyperactivity disorder
  • Dopamine /3-hydroxylase is a member of a small unique class of copper-containing hydroxylases that are found in eukaryotes and all play a critical role in biosynthesis of neurotransmitters and hormones.
  • This class of copper-containing hydroxylases are all multi-domain enzymes with a common catalytic core fused to different types of domains.
  • DBH has an N-terminal DOMON domain
  • DOMON domain (Dopamine /3-MOnooxygenase N-terminal domain), which belongs to the class of DOMON-like domains.
  • the DOMON domain class is divided into at least nine families that are distantly related by amino acid sequences.
  • the function of DOMON domains is largely unknown, but they are involved in ligand binding, so far either as heme and sugar-binding domains.
  • the catalytic core of DBH shows high sequence homology with the catalytic core of PHM (PHMcc). It consists of two domains, the Cu H and Cu M domains, each binding one copper, Finally, at the C-terminus there is an approximately 100 residues domain with no sequence resemblance to any known domains, which is referred to as a dimerization domain.
  • DBH is seen both as a homodimer and as a homotetramer.
  • DBH contains 15 cysteine residues of which many are conserved between DBH of different organisms. Based on studies of bovine DBH 14 cysteines are involved in disulfide bridge formation, six are intra- and two are inter-molecular bonds.
  • DBH is an ascorbate dependent glycoprotein that requires two type 2 bound copper per subunit in order to be active. The copper sites are labile and termed Cu H and Cu M , respectively. The Cu H is coordinated to three histidines and Cu M to two histidines and a methionine.
  • a crystalline form of dopamine ⁇ -hydroxylase is provided.
  • a method for identifying one or more modulators of dopamine ⁇ -hydroxylase is also provided.
  • a process for preparing the crystalline form of human dopamine ⁇ -hydroxylase is also provided.
  • Fig. 1 Structure of the human DBH dimer.
  • a and B Overall structure seen from two angles (90 degrees to each other). The DOMON domain is displayed in orange, the Cu H domain in dark green, the Cu M domain in light green and the dimerization domain in magenta. The inter domain regions are in gray.
  • C Secondary structure organization of DBH.
  • Fig. 2 The two conformations of the DBH catalytic core reveal a closed and an open active site.
  • A Same orientation as Fig. IB with chain A to the left.
  • B View from the back with chain A to the right.
  • C Closed conformation of the catalytic domain as seen in chain A.
  • D Open conformation of the catalytic domain as seen in chain B.
  • Cu M in chain A is modelled in the structure, whereas the three other coppers are inserted manually in a position indicated by the position of the conserved active site ligands. Same color coding as in Fig. 1.
  • Fig. 3. Alignment of the DBH DOMON domain with the cytochrome domain of cellobiose dehydrogenase.
  • DOMON chain A
  • DOMON chain A
  • the heme group in cellobiose dehydrogenase is shown in ball-and-stick.
  • the r.m.s.d. is 2.53 A for the backbone atoms, indicating an identical fold of the two domains.
  • Residues involved in hydrophilic interactions are shown as sticks and the disulfide bridges are shown in yellow.
  • Fig. 6 Binding pockets and channels in the vicinity of the closed active site seen in chain A.
  • CAVER identified binding pocket and channel (yellow) in the closed catalytic core (chain A).
  • the in the structure modelled Cu M and the manually inserted copper ion are in blue. Two different orientations are shown.
  • the pocket is of sufficient size to hold the substrate (dopamine).
  • Fig. 7. Proposed mode of action of DBH.
  • the closed conformation with the coupled binuclear copper site is the catalytically active site.
  • Fig. 8 Size exclusion analysis of purified DBH tetramer and dimer. To the left analysis of purified DBH tetramer. To the right analysis of purified DBH dimer. The absorbance at 280 nm is shown in blue. In both cases no conversion to the other form is seen, showing that in the purification buffer (10 mM hepes, 150 mM NaCI, pH 7.5) both the tetramer and the dimer are stable. Fig. 9.
  • Fig. 11 Mass spectrum of a non-separated sample containing a mixture of dimeric and tetrameric DBH.
  • the sample is 15 ⁇ DBH in 0.5 M ammonium acetate. Both tetramer DBH and dimer DBH are observed, confirming that both dimeric and tetrameric DBH are easily observed if present.
  • Fig. 12 SDS-Page analysis of dimeric and tetrameric DBH under non-reducing and reducing conditions. Under non-reducing conditions (to the left) dimeric and tetrameric DBH have the same mass corresponding to a dimer of ⁇ 150 kDa. To the right under reducing conditions (with ⁇ -mercaptoethanol) dimeric and tetrameric DBH both have the mass corresponding to a monomer of ⁇ 70 kDa. Markers (given in kDa) are Precision Plus Protein Standards and Low Range Standards from Bio-Rad. The SDS-Page analysis reveals that the dimer of dimers in the tetramer are not covalently bound, as no reducing agent is required to separate the tetramer into dimer under denaturation conditions.
  • Fig. 13 Structure of the human DBH dimer emphasizing the integrated structure created by the C-terminus interaction with both the Cu M domain and the DOMON domain.
  • the C-terminus is in violet.
  • Fig. 14 Structure of the human DBH dimer with the disulfide bridges and the glycosylation sites highlighted. The disulfide bridges are in yellow and the modelled glycosylation is shown as CPK's. DETAILED DISCLOSURE
  • DBH dopamine ⁇ -hydroxylase
  • native form cf. UniProt ID P09172
  • functional variants full-length forms as well as fragments thereof.
  • Particular variants include SeMet-labelled or glycosylated variants.
  • salts and other derivatives of DBH such as a K 2 PtCI 4 derivative.
  • Human DBH UniProt id P09172
  • Other species of interest bovine (Bos taurus) DBH (UniProt id P15101).
  • a crystalline form of dopamine ⁇ -hydroxylase is therefore provided .
  • the crystalline form belongs to the space group C222i having unit-cell dimensions a, b, c (A) of 102.8, 119.1, 224.8 or 102.4, 118.8, 225.2 or 102.5, 119.0, 225.5 or 103.0, 119.8, 225.4.
  • the crystalline form has an X-ray diffraction pattern substantially as in Annex 1.
  • the crystalline form of dopamine ⁇ -hydroxylase may be dimeric human DBH, in native or functional variant form .
  • the information can be used for identifying one or more modulators of dopamine ⁇ - hydroxylase, which can then be chemically synthesised and used in treatment.
  • a process for preparing the crystalline form of human dopamine ⁇ -hydroxylase is also provided .
  • the DBH expressed in eukaryotic cells such as mammalian cells, preferably human cells, such as H EK293S cells is present both as dimer and tetramer, which can be separated by size exclusion chromatography.
  • the dimer and tetramer do not interconvert in the pH interval 4 to 9 (see Fig . 8- 11) .
  • the tetramer converts to dimer, and upon addition of reducing agent the dimer converts to monomer (see Fig . 12) .
  • the overall three-dimensional structure of dimeric human DBH is shown in Fig . 1 and the overall architecture of the fold is shown in Fig . 1C.
  • the DOMON domain has an Ig-li ke /3-sandwich structure, the catalytic core : the Cu H - and Cu M -domains, has the same topology as the structure of peptidylglycine a-hydroxylating monooxygenase (PH M) (11 ) and the dimerization domains consisting of two antiparallel a- helices form a four helix bundle.
  • PH M a-hydroxylating monooxygenase
  • the dimeric structure is asymmetric.
  • the two catalytic Cu H and Cu M domains are in a closed conformation - and in the B-chain they adopt the same open conformation as seen in PH M .
  • the catalytic Cu H domain in chain A is moved away from the DOMON domain and closer to the catalytic Cu M domain .
  • the dimerization domain does, however, not overlay very well with itself (r.m.s.d. of 3.83 A for 53 atoms). Omitting the loop/helix from residues 525 to 538 improves the alignment and the r.m.s.d. becomes 0.87 A.
  • the black spheres represent positions of the copper ligands.
  • the a-helix marked * in the dimerization domain is only seen in chain A.
  • Detailed list of secondary structure assignment is provided in Table SI.
  • the Cu H domain contains two disulfide bridges.
  • the Cu M domain contains two disulfide bridges and forms an additional one with the dimerization domain.
  • the DOMON domain and the dimerization domain are linked via C154-C596.
  • Chain A is linked via two inter-molecular disulfide bonds with chain B in the dimerization domain. Glycosylation is observed at all four predicted sites; Asn64, Asnl84, Asn344 and Asn566. The position of the disulfide bridges and the
  • the DOMON super family structure is an Ig-like /3-sandwich with ten to eleven /3-strands and with a ligand-binding pocket.
  • the invention provides the experimental structure of the DBH DOMON domain.
  • the core -structure of the DOMON domain (residues 46-198) folds up in a crescent like structure consisting of two /3-sheets in a /3-sandwich containing five and six anti-parallel /3-strands, respectively as shown in Fig. 1A-C.
  • the C-terminal sheet includes a ⁇ - strand (residues 608-614) following the dimerization domain, see Fig. 1.
  • a Dali server search shows that the overall fold of the DBH DOMON domain is identical to the cytochrome domain of white-root fungus Phenerochaete chrysosporium cellobiose dehydrogenase (PDB ID 1D7B) and the Aromatoleum aromaticum ethylbenzene dehydrogenase o-subunit (PDB ID 2IVF) and to a lesser extent to the carbohydrate-binding module from Thermotoga maritima xylanase (10ACBM9-2) (PDB ID 1I8A).
  • the catalytic core consists of two domains an N-terminal domain where Cu H binds and a C- terminal domain where Cu M binds. Both domains consist primarily of /3-sheets and have the approximate dimensions of 37 A ⁇ 45 A ⁇ 33 A and 44 A ⁇ 45 A ⁇ 33 A for chain A and B, respectively. Both domains have the same topology as described for PHM.
  • the Cu H domain folds into a /3-sandwich formed of two antiparallel /3-sheets with 4 and 5 /3-strands in each (see Fig. 1C) .
  • Strands ⁇ 4 and ⁇ 6 are held closely together with disulfide bridge C269-C295, and strands ⁇ 2 and ⁇ 5 are held together by disulfide bridge C232-C283. Furthermore, glycosylation is observed in the B-chain at Asn344.
  • the Cu M domain folds into a /3-sandwich containing a four stranded antiparallel sheet and a five stranded mixed sheet. The two sheets are held together by a very hydrophobic interior and two disulfide bridges connecting strand ⁇ 8 and ⁇ 9 (C466-C488) and strand ⁇ 2 with ⁇ (C390-C503), respectively.
  • the C-terminal sheet is actually a six stranded sheet by addition of a /3-strand made of residues from the C- terminal of the protein (residues 561-566) following the dimerization domain, as described above.
  • the domain is further stabilized by a disulfide bridge to the additional strand C394- C565, see Fig. 1C-D.
  • glycosylation is seen at Asn366 in both chains.
  • a comparison of the A- and B-chain shows that the Cu H domain is positioned significantly differently in the two chains, as shown in Fig . 2.
  • the Cu H domain in chain A is moved away from the DOMON domain and closer to the Cu M domain.
  • the copper ligands appear to be pre- aligned for Cu binding, except His263 in chain B which is seen in a double conformation.
  • the copper binding sites are located at the interface between the Cu H and the Cu M domains. It is seen that the interatomic distance between the copper ions is different in the two chains.
  • the conformation in chain B is similar to what was observed in the structure of PHM, with a Cu-Cu distance of approximately 14 A.
  • the two copper ions are close together - approximately 4-5 A in what appears to be a closed active site with a coupled binuclear copper site.
  • the dimerization domain consists of two antiparallel a-helices from each chain with quite long loop regions. It has been proposed based on peptide mapping of bovine DBH that the domain should be held together by C528-C528 and C530-C530 disulfide bridges. However, the helices definitely are linked by C528A-C530B and C530A-C528B disulfide bonds. The four- helix bundle is furthermore stabilized by both hydrophobic and electrostatic interaction, as shown in Fig . 5.
  • the dimerization domain sequence (residues 508-560) has no sequence resemblance to any known domains and a Dali-server search did not reveal any closely related three-dimensional structures. However, the sequence is highly conserved among DBH from different organisms.
  • the extra helix (residues 535-542) in the dimerization domain of A molecule makes hydrophobic interactions with residues 376-379 and 478 in the Cu M domain. These are in close proximity to residues 481-483 stacking with the Cu H domain in the A molecule.
  • the dimerization domain is followed by a long C-terminal extension with two /3-strands.
  • the first /3-strand is part of the Cu M domain and the second /3-strand is part of the DOMON domain, see Fig . 1 and Fig . 13.
  • DBH attention deficit hyperactivity disorder
  • DBH is found in vesicles of central adrenergic and noradrenergic neurons, as well as peripheral noradrenergic neurons and is released to the blood in response to stimulation (35 and references therein) .
  • the DBH activity level is stable within individuals, but varies among individuals. In general, an association between lower plasma DBH activity and vulnerability to psychotic symptoms is observed (9) .
  • NE deficiency is a congenital disorder in which the patients suffer profound autonomic failure.
  • DBH non-synonymous coding region single-nucleotide polymorphisms
  • cSNPs non-synonymous coding region single-nucleotide polymorphisms of human DBH are known (NCBI dbSNP Build 142) and they are distributed over the entire sequence, however, none of the copper ligands or the glycosylation sites are affected.
  • Modulators of DBH include DBH inhibitors and DBH activators.
  • the herein described structure of DBH will facilitate further developments, as the modulator binding sites and modes of action can be elucidated in details.
  • Inhibitors of DBH are currently in clinical development for treatment of cocaine dependence, Post-traumatic stress disorder (PTSD), hypertension and heart failure and also here the described structure of DBH will facilitate further developments, as the inhibitor binding site and mode of action can be elucidated in details.
  • Activators of DBH DBH include acetate and fumarate, and also here the described structure of DBH will facilitate identification of activator molecules e.g. binding in the disclosed binding sites in the DOMON domain. Accordingly, a method for identifying one or more modulators of dopamine ⁇ -hydroxylase is provided, said method comprising : a.
  • the method may further comprise the steps of chemically synthesising the modulators, and testing their DBH-inhibiting activity.
  • said DBH modulators are selected from the group consisting of DBH inhibitors and DBH activators, preferably wherein said DBH modulators are DBH inhibitors.
  • Ligands identified by this method, and their use as a medicament are also provided. Such ligands can be used in the treatment, amelioration or prophylaxis of hypertension, depression, anxiety, stress, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD) congestive heart failure, Huntington ' s chorea, Tourette syndrome, and drug dependence.
  • ADHD attention deficit hyperactivity disorder
  • a pharmaceutical composition comprising such a ligand together with one or more pharmaceutically acceptable vehicles or excipients is also provided.
  • a method of preventing, treating or ameliorating hypertension, depression, anxiety, stress, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD) congestive heart failure, Huntington ' s chorea, Tourette syndrome, and drug dependence comprising administering to a patient in need thereof an effective amount of a ligand as defined herein or a pharmaceutical composition as defined herein.
  • Human DBH heterologously expressed in HEK293S cells exists as dimer and tetramer, which do not interconvert. Likewise, no conversion is seen between dimer and tetramer of isolated native forms of human DBH and human DBH expressed in D.
  • the two different conformations of the DBH catalytic core offer different options for possible catalytic mechanisms.
  • a possible mode of action is, that the closed form represents the catalytically active form and that the open form is catalytically inactive, but allows for loading of substrate, release of product, and recycling of the copper redox states.
  • the two subunits then alternate between an open form and a closed catalytically active form (see Fig. 7).
  • Other enzymes are known that have similar mode of action, with changes between two different conformations, known as a flip-flop mechanism.
  • the first structure of dopamine /3-hydroxylase the enzyme converting the neurotransmitter dopamine to norepinephrine.
  • the structure of the DOMON domain a domain found in more than 1600 other proteins, reveals a possible metal binding site and a possible ligand binding pocket.
  • the catalytic core structure shows both an open active site, similar to the structures of other enzymes in this family, and a closed active site, in which the two copper sites are only 4-5 A apart, in what is best described as a coupled binuclear copper site.
  • the dimerization domain adopts a conformation which bears no resemblance to any other known protein structure.
  • the structure of DBH provides new insights into numerous devastating disorders associated with the dopamine system.
  • a process for preparing the crystalline form of human dopamine ⁇ -hydroxylase comprising the steps of: a. inserting the DNA coding sequence for soluble human DBH into an eukaryotic cell; b. expressing human dopamine ⁇ -hydroxylase from said eukaryotic cells and purifying glycosylated dimeric human dopamine ⁇ -hydroxylase; c. dissolving the purified dimeric human dopamine ⁇ -hydroxylase in an aqueous solution of polyether compound and a salt of an inorganic or organic acid and allowing crystals of human dopamine ⁇ -hydroxylase to form.
  • the eukaryotic cell is a mammalian cell, preferably a human cell.
  • the human cell is a HEK 293 cell.
  • said polyether compound is selected from the group consisting of polyethylene glycol, propylene glycol, butylene glycol, preferably wherein said polyether compound is a polyethylene glycol.
  • said salt of an inorganic or organic acid is selected from the group consisting of salts of nitrate, perchlorate, formate, acetate, and propionate.
  • said process comprises the steps of: a. inserting the DNA coding sequence for soluble human DBH into a mammalian cell, preferably human cell such as HEK293; b. expressing human dopamine ⁇ -hydroxylase from said HEK cells and purifying glycosylated dimeric human dopamine ⁇ -hydroxylase; c. dissolving the purified dimeric human dopamine ⁇ -hydroxylase in an aqueous solution of polyethylene glycol and a nitrate salt and allowing crystals of human dopamine ⁇ -hydroxylase to form.
  • Table SI Secondary structure assignment in human DBH. Table S2. Domain - domain hydrogen bond contacts in chain A and chain B. In the A- chain there are 10 hydrogen bonds between the CU H domain and the CU M domain. In the B- chain the contacts are reduced to only 4 hydrogen bonds. Between the DOMON domain and the CU H domain there are no contacts in the A-chain while in the B-chain some none- hydrogen bond interactions are present.
  • the DNA coding sequence for soluble human DBH (GenBank accession No. P09172), residues 40 - 617 (e.g. the first 39 residues being a signal-anchor for type II membrane proteins was left out in order to get soluble DBH) was optimized for expression in HEK293 cells and synthesized by GenScript Corporation, USA.
  • the flanking sequences were modified by PCR.
  • the forward primer introduces an Agel restriction enzyme site, whereas, the reverse primer introduces a FLAG-tag, two stop codons and an Xhol site.
  • the obtained PCR modified gene was then cloned into the pHLsec vector by T4 ligase and transformed into E. coli DH5a.
  • Plasmid DNA for transfection was purified using GenElute HP Endotoxin-Free Plasmid Megaprep Kit from Sigma-Aldrich. Human DBH was then expressed by automated large scale transient protein expression in HEK293S GnTi " cells.
  • Purification Cells were removed by centrifugation at 5000 g and 4°C for 15 minutes and the supernatant filtered through a 0.22 ⁇ filter. Protein from 1.5 L of culture was purified at 4°C on a 5 mL anti-FLAG M2 affinity gel column equilibrated in 10 mM HEPES, 150 mM NaCI pH 7.5. DBH was eluted with 5 mg FLAG-tag peptide/mL in 10 mM HEPES, 150 mM NaCI pH 7.5 and concentrated to 2 mL by ultrafiltration.
  • DBH was present both as tetramer and dimer, which were separated on a Superdex 200 HR 16/60 in 10 mM HEPES, 150 mM NaCI pH 7.5 at 1 mL/min. Fractions containing the dimeric DBH were collected and pooled; the final concentration which was also used for crystallisation experiments was 6 mg/mL.
  • SeMet labelled protein was expressed and purified as described above with the exception that the media was exchanged every day and that the cells were harvested after three days. The yield was about 10 % of that in normal media.
  • Diffraction data were collected at 100 K at the Diamond Light Source, beamlines 102 and 124.
  • the structure was determined by a combination of MIR/MAD and molecular
  • Phenix.autosol were performed using native-1 and SeMet data. This revealed a domain movement in one of the two molecules (molecule A) and the resulting maps allowed a detailed model to be built.
  • the DOMON and the C-terminal domains were built manually in Coot. Met89 was used as a marker for the sequence together with the glycosylated Asn64 with the neighboring Trp63.
  • the amino acid sequence was changed according to a structural alignment. The initial refinement was performed in refmac5, later Phenix. refine was used. Non crystallographic symmetry between the individual domains and Torsion/libration/screw (TLS) motion were applied.
  • TLS domains in the two chains were differently defined using the TLS server.
  • Chain A was divided as follows: 46-78, 79-187, 188-236, 237-362, 363-523, 524-576, 577-611, and molecule B was divided as follows: 47-187, 188-523, 524-612.
  • COMPND 4 SYNONYM DOPAMINE BETA-MONOOXYGENASE ;
  • ATOM 24 CA TYR A 49 6.177 32. .804 42. .225 1. .00 60. .10
  • ATOM 46 CA ILE A 51 10.558 34. .203 36. .699 1. .00 54. .58

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Abstract

La présente invention décrit une forme cristalline de β-hydroxylase de dopamine. La cristallographie aux rayons X révèle le groupe spatial et les dimensions cellulaires, ainsi que les coordonnées atomiques. L'information peut être utilisée pour identifier un ou plusieurs modulateurs de la β-hydroxylase de dopamine, qui peut ensuite être chimiquement synthétisée et utilisée en traitement. Un procédé de préparation de la forme cristalline de β-hydroxylase de dopamine humaine est également décrit.
PCT/EP2017/058334 2016-04-07 2017-04-07 Structure cristalline de bêta-hydroxylase de dopamine humaine WO2017174762A1 (fr)

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Citations (1)

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WO2002072006A2 (fr) * 2001-03-07 2002-09-19 The Mclean Hospital Corporation Procedes et reactifs permettant d'identifier des composes et des mutations modulant l'activite de la dopamine $g(b)-hydroxylase

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