CN103865914B - Crystal and the growing method thereof of PDE2 catalyst structure domain/PDE2 specific inhibitor compound - Google Patents

Crystal and the growing method thereof of PDE2 catalyst structure domain/PDE2 specific inhibitor compound Download PDF

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CN103865914B
CN103865914B CN201210545326.3A CN201210545326A CN103865914B CN 103865914 B CN103865914 B CN 103865914B CN 201210545326 A CN201210545326 A CN 201210545326A CN 103865914 B CN103865914 B CN 103865914B
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pde2
crystal
catalyst structure
structure domain
albumen
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CN103865914A (en
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朱坚
王涛
彼得·雷瑟
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Shanghai Medicilon Inc
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04001—Phosphodiesterase I (3.1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

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Abstract

The crystal and the growing method thereof that the present invention relates to a kind of PDE2 catalyst structure domain/PDE2 specific inhibitor compound, this crystal has the space group P1 in monoclinic system, and this crystal has lattice paprmeter: α=109.59°±0.1%,β=91.93°±0.1%,γ=90.88°±0.1%。

Description

Crystal and the growing method thereof of PDE2 catalyst structure domain/PDE2 specific inhibitor compound
Technical field
The present invention relates to a kind of phosphodiesterase 2(PDE2) crystalline substance of catalyst structure domain/PDE2 specific inhibitor compoundBody, and the method for this crystal of growing.
Background technology
Cyclic adenosine monophosphate (cAMP) and cyclic guanylic acid (cGMP) are the second messenger molecules of Cellular Signaling Transduction Mediated, raw at cellIn life activity, have great importance. The regulation and control of its IC level are mainly by synthesis and the phosphorus of nucleotide cyclaseThe hydrolysis of acid diesters enzyme (PDE, Phosphodiesterase) realizes. Phosphodiesterase energy catalysis CAMP(cAMP) and the hydrolysis of the phosphodiester bond of cyclic guanosine monophosphate (cGMP), generate adenylate (AMP) and the guanylic acid of non-activity(GMP), the level of intracellular cAMP and cGMP is declined, thus the multiple metabolic function of regulating cell and even organism.
Phosphodiesterase is a huge protein families, has altogether so far 11 kinds of phosphodiesterases identified, nameFor PDE1-PDE11. The catalyst structure domain that di-phosphate ester enzyme family is held except C is guarded relatively, the regulatory domain phase not to the utmost of N endWith. The hydrolysis high special (PDE4, PDE7, PDE8) of some phosphodiesterases to cAMP, some are the hydrolysis height to cGMPSpecial (PDE5, PDE6, PDE9), phosphodiesterase 2(PDE2) there is mixing specificity, belong to Double bottom thing phosphodiesterase,, can catalyzing hydrolysis cGMP and can be hydrolyzed again cAMP. There are some researches show that substrate cGMP is bonded to the GAFB district activation of PDE2The activity of this enzyme. Phosphodiesterase 2(PDE2) total length is about 940 amino acid, and the catalysis of the GAFA/GAFB being held by N and C end is tiedStructure territory composition.
PDE2 is wide expression in tissue, but concentration is particularly high in brain and central nervous system. Large quantity researchDisclose the processes such as PDE2 participation in learning, memory and cognition, suppressed PDE2 simultaneously and can strengthen neuronic plasticity and antidepression. AlsoThere are some researches show that PDE2 can improve the permeability of endothelial cell. PDE2 is as potential drug targets, and its inhibitor also can be usedTreat central nervous system disease, improve permeability of memory and endothelial cell etc. In these researchs, all useThe specific inhibitor of PDE2 is illustrated the physiological function of PDE2.
In recent years, along with the development of structure biology, utilize X-ray crystallography to disclose increasing proteinSpace structure. By the space of analysing protein, contribute to understand the biological function of protein. Meanwhile, albumen is importantDrug targets. Utilize the three-dimensional structure information of disease-associated protein, can design for the specificity of this protein and suppressAgent, thus reach the object for the treatment of disease. It is multiple that WO2003/038080A discloses crystal and the PDE5/PDE5 part of a kind of PDE5The crystal of compound. The three-dimensional knot of compound of the existing holoenzyme about PDE2 and its catalyst structure domain and nonspecific inhibitor IBMXThe report of structure (Pandit, J., etal.ProcNatlAcadSciUSA, 2009.106 (43): p.18225-30), stillThe three-dimensional structure of PDE2 and specific inhibitor there is not yet report. Due to protein in the catalytic domain structure between each PDE familyFolding mode is close, and the binding pocket shape of holding substrate molecule is also similar, therefore understands the mutual of PDE and specific inhibitorFunction Characteristics seems particularly important for the PDE drug molecule of exploitation high selectivity. PDE2 and nonspecific inhibitor IBMX'sThe pattern of mutually combining can not fully provide the exploitation PDE2 useful information of specific inhibitor.
Therefore be necessary to illustrate the space structure of PDE2 and a certain specific inhibitor, utilize this information can instruct baseIn the drug design of structure.
BAY60-7550 is the powerful inhibitor of a kind of PDE2, is 4.7nM for the half inhibiting rate IC50 value of PDE2. SoAnd the binding pattern of BAY60-7550 and PDE2 is not yet illustrated. Before the present invention, people cannot know participate in PDE2 withThe amino acid residue of BAY60-7550 combination and corresponding combination power, as hydrogen bond, hydrophobic effect power, Van der Waals force etc. CauseLack above-mentioned information and cannot design, produce the better drug molecule for PDE2.
Summary of the invention
In the face of the problem that prior art exists, the present invention passes through PDE2 catalytic domain domain and its specificity mortifierThe structure elucidation of (for example BAY60-7550) compound crystal, has disclosed the space of PDE2 catalyst structure domain and BAY60-7550 and has tiedClose feature, know and participate in amino acid residue and the corresponding combination power that PDE2 is combined with BAY60-7550 based on this, as hydrogenKey, hydrophobic effect power, Van der Waals force etc.
At this, on the one hand, the invention provides a kind of crystalline substance of PDE2 catalyst structure domain/PDE2 specific inhibitor compoundBody, this crystal has the space group P1 in monoclinic system, and this crystal has lattice paprmeter: α=109.59°±0.1%,β=91.93°±0.1%,γ=90.88°±0.1%。
Three of the catalyst structure domain crystal structure that the present invention describes by atomic coordinates and its specific inhibitor compoundDimension structure, described atomic structure can obtain by the X-radiocrystallography of this compound. Can obtain about this based on this crystalThe interactional characteristic information of PDE2 catalyst structure domain and its specific inhibitor (for example BAY60-7550). Suppress for PDE2The qualification of agent and this class inhibitor are in screening, the evaluation of medicine as being used for the treatment of central nervous system disease. Be expected to useIn the drug design based on structure,
Preferably, in described crystal, have four molecules in an asymmetric cell, the molecular weight of each molecule is about 39± 0.5kD, solvent is about 61 ± 1%.
Preferably, the three-dimensional structure of described crystal meets the three-dimensional structure that table 1 limits:
Table 1:
In the structure demonstration PDE2 catalyst structure domain of having revised, the amino acid residue of participation and BAY60-7550 effect has:Gln859, Gln812, Ile826, Phe862, Met847, Ile822, Leu809, Leu770 and Ile870 etc.
Preferably, in the binding pocket of PDE2 catalyst structure domain, be combined with a PDE2 specific inhibitor molecule.
Preferably, PPDE2 catalyst structure domain is made up of 16 α spirals, has a Mg in the bottom of described binding pocket2+WithA Zn2+。
In the present invention, described PDE2 specific inhibitor can be BAY60-7550 compound,
Preferably, the amino acid sequence of described PDE2 catalyst structure domain derives from people.
Preferably, described PDE2 catalyst structure domain have the amino acid sequence listed in SEQIDNO:1 or its homologue,Fragment, variant.
On the other hand, it is compound that the present invention also provides the above-mentioned PDE2 catalyst structure domain/PDE2 specific inhibitor of a kind of growthThe method of the crystal of thing, comprises the following steps:
1) in buffer solution, prepare the albumen buffer solution containing the PDE2 catalyst structure domain albumen of 8-12mg/ml;
2) mix described albumen buffer solution and pond liquid as crystallization solution;
3) from described crystallization solution, grow containing the albumin crystal of PDE2 catalyst structure domain albumen; And
4) described albumin crystal is immersed in containing urging to obtain PDE2 in the pond liquid of 0.1-5mMPDE2 specific inhibitorChange the crystal of domain/PDE2 specific inhibitor compound.
Again, the preparation of described albumen buffer solution comprises:
A) PDE2 catalyst structure domain is cloned into pET32a prokaryotic expression carrier;
B) recombinant vector is transformed in prokaryotic BL21 (DE3) CodonPlus, IPTG induced protein is expressed;
C) purifying PDE2 catalyst structure domain albumen; And
D) the PDE2 catalyst structure domain albumen of purifying is immersed in buffer solution, be then concentrated into 8-12mg/ml.
Preferably, the pH value of described buffer solution is 7.5, and Hepes(hydroxyethyl piperazine 3 thiosulfonic acids that contain 25mM) (N-(2-ethoxy) piperazine-N '-(2-ethanesulfonic acid), the NaCl of 25mM and the TCEP(of 2mM tri-(2-carboxyethyl) phosphonium salt hydrochlorate).
Preferably, the pH value of described pond liquid is 8.5, and the Tris(trishydroxymethylaminomethane that contains 0.1M)-HCl,The MgCl of 0.2M2, and the PEG6000 of 16-20%.
Preferably, described albumin crystal can be grown by diffusion of vapor hanging drop method.
The present invention is by cultivating the crystal of PDE2 catalyst structure domain/PDE2 specific inhibitor compound and analyzing its three-dimensionalStructure shows PDE2 catalyst structure domain and its specific inhibitor, the combination feature of for example BAY60-7550. Urge about PDE2The information of changing the three-dimensional structure of domain and BAY60-7550 is design and produces little for the medicine for central nervous system of PDE2Molecular medicine (chemicals) has offered a kind of mode (based on the drug design of structure). Such as, soft by computer simulationParts etc. can suppress the small-molecule drug of PDE2 according to above-mentioned information virtual screening, design, thereby reach, treatment is depressed, improvement noteRecall, improve the objects such as cognitive function. Or utilize the mentioned PDE2 catalyst structure domain of the present invention and BAY60-7550 compoundThe crystal structure of crystal, by the method for computer modeling, designs for the small-molecule substance of PDE2 etc., then synthetic this kindMaterial, forms compound with PDE2, the methods analyst compound of recycling X ray crystal diffraction, thus obtain real combinationSite. The result of analyzing according to X ray crystal diffraction, can adjust small-molecule substance, until obtain optimized materialMolecule. And before the present invention, cannot specific aim design, produce owing to lacking the combining information of PDE2 and selective depressantGo out the better specific drug for PDE2.
Brief description of the drawings
Fig. 1 illustrates and in an asymmetric unit, contains four PDE2 catalyst structure domains that are equal to and BAY60-7550 compoundMolecule;
Fig. 2 illustrates PDE2 catalyst structure domain and the overview of BAY60-7550 composite structure;
Fig. 3 illustrates and shows that PDE2 catalyst structure domain is combined the graphics of feature with BAY60-7550.
Detailed description of the invention
Further illustrate the present invention below in conjunction with accompanying drawing and following embodiment, should be understood that following embodiment and/or attachedFigure is only for the present invention is described, and unrestricted the present invention.
Expression and the purifying of PDE2 catalytic domain domain:
The gene order of PDE2 catalytic domain domain is expressed: for example, PDE2 catalyst structure domain gene order is cloned into formerIn nuclear expression carrier pET32a. This PDE2 catalyst structure domain gene order has the order as indicated in SEQIDNo.1 after expressingRow or its homologue, fragment, variant.
The protein expression of PDE2 catalytic domain domain: for example, recombinant vector is transformed into E.coli cell BL21 (DE3)In CodonPlus bacterial strain, in LB culture medium, being cultured to OD600 value is 0.6～0.8 o'clock, adds IPTG inducible protein to express;
Protein purification, for example expressing protein utilizes the affine resin of Ni-NTA (Qiagene) and Superdex200(GEHealthcare) albumen that resin etc. is purified into enough purity is for crystallization, with the Trx fusion of PDE2 coexpression in this mistakeIn journey, removed with TEV enzyme.
Albumin crystal growth, complex crystallization:
The preparation of albumen buffer solution: the albumen dialysis of purifying is immersed in buffer solution, then concentrate and (for example use centrifugal establishingStandby (PALLCorp.) concentration tube is concentrated) can measure according to Bradford determination method to 8～12mg/ml(). The buffering usingLiquid can for example contain 25mMHepes ((N-(2-ethoxy) piperazine-N '-(2-ethanesulfonic acid), pH7.5,25mMNaCl,2mMTCEP(tri-(2-carboxyethyl) phosphonium salt hydrochlorate).
Albumin crystal growth: mix 1 μ L protein solution and 1 μ L pond liquid (reservoirbuffer) conduct knot at 18 DEG CBrilliant drop. Described pond liquid can contain 0.1MTris-HCl, pH8.5,0.2MMgCl2,16～20%PEG6000. After some days,Crystal grows to 100～200 μ m sizes.
Complex crystallization: the crystal of growing up is shifted and is dipped in the above-mentioned pond liquid that contains 0.5～2mMBAY60-7550.Soak and after 6 hours, collect crystal and transfer in the pond liquid that contains 20% glycerine as cryoprotection, subsequently by crystal quick-frozen in liquidIn nitrogen. Crystal can be diffracted into conventionallyLeft and right. Should be understood that and adopt BAY60-7550 to suppress as the specificity of PDE2 hereAgent part, but can adopt other suitable specific inhibitor.
Data Collection, processing and model optimization:
On the BL17U of Synchrotron Radiation SSRF bunch, carry out Data Collection, and use iMOSFLM to process. X penetratesLine wavelength isEvery a diffraction pattern of 1 ° of collection, collect altogether 180. Structure elucidation uses in CCP4 software kitMOLREP program is carried out molecular replacement, and the model that is numbered 1Z1L in employing PDB storehouse is as template. Obtain initial configuration laggardOne step is optimized in Refmac program. Produce at PRODRG server after the library file of compd B ay60-7550, by BAY60-7550 molecule constructions enter in corresponding cloud density figure. Composite structure is finally optimized in PHENIX program. DataProcess statistics and structure optimization numerical value as shown in table 1.
Table 1:
In an asymmetric unit of structure demonstration in Fig. 1 after explanation parsing, contain four PDE2 catalyst structure domains that are equal toWith BAY60-7550 complex molecule, in figure, arrow shows the BAY60-7550 molecule being incorporated in PDE2 catalyst structure domain.
Fig. 2 illustrates PDE2 catalyst structure domain and the overview of BAY60-7550 composite structure, shows PDE2 catalysis knot in this figureStructure territory is made up of 16 α spirals, has a Mg in binding pocket bottom2+With a Zn2+. Fig. 3 illustrates and shows PDE2 catalytic structureThe graphics of feature is combined in territory with BAY60-7550, show in PDE2 catalyst structure domain and participate in and BAY60-7550 effect in this figureAmino acid residue have: Gln859, Gln812, Ile826, Phe862, Met847, Ile822, Leu809, Leu770 andIle870 etc.
Can suppress according to above-mentioned information virtual screening, design the little molecule medicine of PDE2 by Computer Simulation Software etc.Thing, thus reach the objects such as treatment is depressed, improvement memory, raising cognitive function. Or utilize the mentioned PDE2 of the present invention to urgeChange the crystal structure of domain and BAY60-7550 compound crystal, by the method for computer modeling, design for PDE2Small-molecule substance etc., then synthetic this kind of material, forms compound with PDE2, the method for recycling X ray crystal diffraction is dividedAnalyse compound, thereby obtain real binding site. The result of analyzing according to X ray crystal diffraction, can enter small-molecule substanceRow is adjusted, until obtain optimized material molecule.
Sequence table
SEQIDNO:1
<110>Shanghai Medixi Biomedicine Co., Ltd.
<120>crystal and the growing method thereof of PDE2 catalyst structure domain/PDE2 specific inhibitor compound
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<170>PatentInversion3.5
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SerAspAspGluTyrThrLysLeuLeuHisAspGlyIleGlnPro
151015
ValAlaAlaIleAspSerAsnPheAlaSerPheThrTyrThrPro
202530
ArgSerLeuProGluAspAspThrSerMetAlaIleLeuSerMet
354045
LeuGlnAspMetAsnPheIleAsnAsnTyrLysIleAspCysPro
505560
ThrLeuAlaArgPheCysLeuMetValLysLysGlyTyrArgAsp
657075
ProProTyrHisAsnTrpMetHisAlaPheSerValSerHisPhe
808590
CysTyrLeuLeuTyrLysAsnLeuGluLeuThrAsnTyrLeuGlu
95100105
AspIleGluIlePheAlaLeuPheIleSerCysMetCysHisAsp
110115120
LeuAspHisArgGlyThrAsnAsnSerPheGlnValAlaSerLys
125130135
SerValLeuAlaAlaLeuTyrSerSerGluGlySerValMetGlu
140145150
ArgHisHisPheAlaGlnAlaIleAlaIleLeuAsnThrHisGly
155160165
CysAsnIlePheAspHisPheSerArgLysAspTyrGlnArgMet
170175180
LeuAspLeuMetArgAspIleIleLeuAlaThrAspLeuAlaHis
185190195
HisLeuArgIlePheLysAspLeuGlnLysMetAlaGluValGly
200205210
TyrAspArgAsnAsnLysGlnHisHisArgLeuLeuLeuCysLeu
215220225
LeuMetThrSerCysAspLeuSerAspGlnThrLysGlyTrpLys
230235240
ThrThrArgLysIleAlaGluLeuIleTyrLysGluPhePheSer
245250255
GlnGlyAspLeuGluLysAlaMetGlyAsnArgProMetGluMet
260265270
MetAspArgGluLysAlaTyrIleProGluLeuGlnIleSerPhe
275280285
MetGluHisIleAlaMetProIleTyrLysLeuLeuGlnAspLeu
290295300
PheProLysAlaAlaGluLeuTyrGluArgValAlaSerAsnArg
305310315
GluHisTrpThrLysValSerHisLysPheThrIleArgGlyLeu
320325330
ProSerAsnAsnSerLeuAspPheLeuAspGluGlu
335340

Claims (4)

1. a crystal for PDE2 catalyst structure domain and PDE2 specific inhibitor compound, is characterized in that, this crystal hasSpace group P1 in monoclinic system, this crystal has lattice paprmeter: α=109.59 ° ± 0.1%, β=91.93 ° ± 0.1%, γ=90.88 ° ± 0.1%, described PDE2 catalyst structure domainBe made up of the amino acid sequence of listing in SEQIDNO:1, described PDE2 specific inhibitor is BAY60-7550 compound,
In each asymmetric cell of the crystal of described PDE2 catalyst structure domain and PDE2 specific inhibitor compound, there are fourBe equal to molecule, the molecular weight of each molecule is 39 ± 0.5kD, and solvent is 61 ± 1%.
2. crystal according to claim 1, is characterized in that, in each binding pocket of PDE2 catalyst structure domain, is combined withA PDE2 specific inhibitor molecule.
3. crystal according to claim 2, is characterized in that, PDE2 catalyst structure domain is made up of 16 α spirals, describedA Mg is arranged at the bottom of binding pocket2+With a Zn2+。
4. growth, according to a method for the crystal described in any one in claim 1～3, is characterized in that, comprises following stepRapid:
1) in buffer solution, prepare the albumen buffer solution containing the PDE2 catalyst structure domain albumen of 8-12mg/ml;
2) mix described albumen buffer solution and pond liquid as crystallization solution, wherein mix 1 μ L albumen buffer solution and 1 μ L pond at 18 DEG CLiquid is as crystallization solution;
3) from described crystallization solution, grow containing the albumin crystal of PDE2 catalyst structure domain albumen; And
4) described albumin crystal is immersed in containing tying to obtain PDE2 catalysis in the pond liquid of 0.5-2mMPDE2 specific inhibitorThe crystal of structure territory and PDE2 specific inhibitor compound;
The preparation of described albumen buffer solution comprises:
A) PDE2 catalyst structure domain is cloned into pET32a prokaryotic expression carrier;
B) recombinant vector is transformed in prokaryotic BL21CodonPlus (DE3), IPTG induced protein is expressed;
C) purifying PDE2 catalyst structure domain albumen; And
D) the PDE2 catalyst structure domain albumen of purifying is immersed in buffer solution, be then concentrated into 8-12mg/ml;
The pH value of described buffer solution is 7.5, and the NaCl of the Hepes that contains 25mM, 25mM and the TCEP of 2mM;
The pH value of described pond liquid is 8.5, and the MgCl of the Tris-HCl that contains 0.1M, 0.2M2, and the PEG6000 of 16-20%;
Described albumin crystal is grown by diffusion of vapor hanging drop method.
CN201210545326.3A 2012-12-14 2012-12-14 Crystal and the growing method thereof of PDE2 catalyst structure domain/PDE2 specific inhibitor compound Active CN103865914B (en)

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CN108220269B (en) * 2017-12-29 2021-07-20 华南理工大学 Hydrogen peroxide-resistant lipase AflB crystal and preparation method thereof
CN110010199B (en) * 2019-03-27 2021-01-01 华中师范大学 Method for analyzing and identifying protein specific drug binding pocket

Citations (2)

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WO2005083069A1 (en) * 2004-01-30 2005-09-09 Pfizer Products Inc. Pde2 crystal structures for structure based drug design

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CN1585821A (en) * 2001-11-02 2005-02-23 美国辉瑞有限公司 Crystal structure of phosphodiesterase 5 and use thereof
WO2005083069A1 (en) * 2004-01-30 2005-09-09 Pfizer Products Inc. Pde2 crystal structures for structure based drug design

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Phosphodiesterase-2 inhibitor reverses corticosterone-induced neurotoxicity and related behavioural changes via cGMP/PKG dependent pathway;Ying Xu et al.;《International Journal of Neuropsychopharmacology》;20120731;第16卷;第835-847页 *
X-ray Crystal Structure of Phosphodiesterase 2 in Complex with a Highly Selective,Nanomolar Inhibitor Reveals a Binding-Induced Pocket Important for Selectivity;Jian Zhu et al.;《J.Am.Chem.Soc.》;20130730;第135卷;第11708-11711页 *
磷酸二酯酶2（PDE2）结构及其选择性抑制剂的研究进展;赵新筠等;《有机化学》;20091231;第29卷(第2期);第159-165页 *

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