CN103865914A - PDE2 catalytic structural domain/PDE2 specific inhibitor compound crystal, and growth method thereof - Google Patents
PDE2 catalytic structural domain/PDE2 specific inhibitor compound crystal, and growth method thereof Download PDFInfo
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- CN103865914A CN103865914A CN201210545326.3A CN201210545326A CN103865914A CN 103865914 A CN103865914 A CN 103865914A CN 201210545326 A CN201210545326 A CN 201210545326A CN 103865914 A CN103865914 A CN 103865914A
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Abstract
The invention relates to PDE2 catalytic structural domain/PDE2 specific inhibitor compound crystal, and a growth method thereof. Space groups P1 of the PDE2 catalytic structural domain/PDE2 specific inhibitor compound crystal belongs to monoclinic system; and lattice constants of the PDE2 catalytic structural domain/PDE2 specific inhibitor compound crystal are that: a=56.2 angstrom+-0.5%, b=73.8 angstrom+-0.5%, c=92.4 angstrom+-0.5%, alpha=109.59 angstrom+-0.1%, beta=91.93 angstrom+-0.1%, and gamma=90.88 angstrom+-0.1%.
Description
Technical field
The present invention relates to a kind of phosphodiesterase 2(PDE2) crystal of catalyst structure domain/PDE2 specific inhibitor mixture, and the method for this crystal of growing.
Background technology
Cyclic amp (cAMP) and cyclic guanosine monophosphate (cGMP) are the second messenger molecules of Cellular Signaling Transduction Mediated, in cell activities, have great importance.The regulation and control of its IC level are mainly realized by the Synthesis of nucleotide cyclase and the hydrolytic action of phosphodiesterase (PDE, Phosphodiesterase).The hydrolysis of the phosphodiester bond of phosphodiesterase energy catalysis cyclic monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), generate adenylic acid (AMP) (AMP) and the guanylic acid (GMP) of non-activity, 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, called after PDE1-PDE11.Phosphodiester enzyme family is except the catalyst structure domain of C end is guarded relatively, and the regulatory domain of N end is not quite similar.The hydrolysis high special (PDE4, PDE7, PDE8) of some phosphodiesterases to cAMP, some are the hydrolysis high specials (PDE5, PDE6, PDE9) to cGMP, phosphodiesterase 2(PDE2) there is mixing specificity,, can catalytic hydrolysis cGMP and can be hydrolyzed again cAMP belong to Double bottom thing phosphodiesterase.There are some researches show that GAFB district that substrate cGMP is bonded to PDE2 activates the activity of this enzyme.Phosphodiesterase 2(PDE2) total length is about 940 amino acid, and the catalyst structure domain of the GAFA/GAFB being held by N and C end forms.
PDE2 is wide expression in tissue, but concentration is particularly high in brain and central nervous system.Large quantity research has disclosed the processes such as PDE2 participation in learning, memory and cognition, suppresses PDE2 simultaneously and can strengthen neuronic plasticity and antidepressant.Also there are some researches show that PDE2 can improve the perviousness of endotheliocyte.PDE2 is as potential drug targets, and its inhibitor also can be used for treating central nervous system disease, improve perviousness of memory and endotheliocyte etc.In these researchs, all use the specific inhibitor of PDE2 to illustrate the physiological function of PDE2.
In recent years, along with the development of structure biology, utilize X-ray crystallography to disclose increasing protein steric structure.By the space of analysing protein, contribute to understand the biological function of protein.Meanwhile, albumen is important drug targets.Utilize the three-dimensional structure information of disease-associated protein, can design the specific inhibitor for this protein, thereby reach the object for the treatment of disease.WO2003/038080A discloses the crystal of a kind of PDE5 and the crystal of PDE5/PDE5 ligand complex.Report (the Pandit of the mixture three-dimensional structure of the existing holoenzyme about PDE2 and its catalyst structure domain and nonspecific inhibitor IBMX, J., et al.Proc Natl Acad Sci USA, 2009.106 (43): p.18225-30), but the three-dimensional structure of PDE2 and specific inhibitor there is not yet report.Because protein folding pattern in the catalytic domain structure between each PDE family is close, the binding pocket shape of holding substrate molecule is also similar, and the interaction characteristic of therefore understanding PDE and specific inhibitor seems particularly important for the PDE drug molecule of exploitation highly selective.The pattern of mutually combining of PDE2 and nonspecific inhibitor IBMX 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 the medicinal design based on structure.
BAY60-7550 is the powerful inhibitor of a kind of PDE2, is 4.7nM for the half inhibiting rate IC50 value of PDE2.But the binding pattern of BAY60-7550 and PDE2 is not yet illustrated.Before the present invention, people cannot be known amino-acid residue and the corresponding keying action power that participation PDE2 is combined with BAY60-7550, as hydrogen bond, hydrophobic interaction power, Van der Waals force etc.For want of above-mentioned information and cannot design, produce the better drug molecule for PDE2.
Summary of the invention
In the face of the problem of prior art existence, the present invention is by for example, structure elucidation to PDE2 catalytic domain structural domain and its specificity inhibition (BAY60-7550) compound crystal, disclose PDE2 catalyst structure domain and be combined feature with the space of BAY60-7550, know and participate in amino-acid residue and the corresponding keying action power that PDE2 is combined with BAY60-7550 based on this, as hydrogen bond, hydrophobic interaction power, Van der Waals force etc.
At this, on the one hand, the invention provides a kind of crystal of PDE2 catalyst structure domain/PDE2 specific inhibitor mixture, this crystal has the spacer P1 in oblique system, and this crystal has lattice parameter:
α=109.59 ° ± 0.1%, β=91.93 ° ± 0.1%, γ=90.88 ° ± 0.1%.
The catalyst structure domain crystalline structure that the present invention describes by atomic coordinate and the three-dimensional structure of its specific inhibitor mixture, described atomic structure can obtain by the X-radiocrystallography of this mixture.For example can obtain, about this PDE2 catalyst structure domain and the interactional characteristic information of its specific inhibitor (BAY60-7550) based on this crystal.For the qualification of PDE2 inhibitor and this class inhibitor in screening, the evaluation of medicine as being used for the treatment of central nervous system disease.Be expected to for the medicinal 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, and 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, Met 847, 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 pocket
2+with a Zn
2+.
In the present invention, described PDE2 specific inhibitor can be BAY60-7550 compound,
Preferably, the aminoacid sequence of described PDE2 catalyst structure domain derives from people.
Preferably, described PDE2 catalyst structure domain has the aminoacid sequence listed in SEQ ID NO:1 or its homologue, fragment, variant.
On the other hand, the present invention also provides the method for the crystal of the above-mentioned PDE2 catalyst structure domain/PDE2 specific inhibitor mixture of a kind of growth, comprises the following steps:
1) in damping fluid, prepare the albumen damping fluid containing the PDE2 catalyst structure domain albumen of 8-12mg/ml;
2) mix described albumen damping fluid 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 the crystal with acquisition PDE2 catalyst structure domain/PDE2 specific inhibitor mixture in the pond liquid of 0.1-5mM PDE2 specific inhibitor.
Again, the preparation of described albumen damping fluid comprises:
A) PDE2 catalyst structure domain is cloned into pET32a prokaryotic expression carrier;
B) recombinant vectors is transformed in prokaryotic cell prokaryocyte BL21 (DE3) Codon Plus, 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 damping fluid, be then concentrated into 8-12mg/ml.
Preferably, the pH value of described damping fluid is 7.5, and Hepes(hydroxyethyl piperazine 3 thiosulfonic acid that contain 25mM) (N-(2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid), the NaCl of 25mM and the TCEP(of 2mM tri-(2-propyloic) phosphonium salt hydrochlorate).
Preferably, the pH value of described pond liquid is 8.5, and the Tris(Tutofusin tris that contains 0.1M) MgCl of-HCl, 0.2M
2, and the PEG 6000 of 16-20%.
Preferably, described albumin crystal can be grown by vapor diffusion hanging drop method.
The present invention shows PDE2 catalyst structure domain and its specific inhibitor, the combination feature of for example BAY60-7550 by cultivating the crystal of PDE2 catalyst structure domain/PDE2 specific inhibitor mixture and analyzing its three-dimensional structure.Be design and produce and offered a kind of mode (based on the medicinal design of structure) for the medicine for central nervous system small-molecule drug (chemicals) of PDE2 about the information of the three-dimensional structure of PDE2 catalyst structure domain and BAY60-7550.Such as, can suppress the small-molecule drug of PDE2 according to above-mentioned information virtual screening, design by Computer Simulation Software etc., thereby reach treatment depressed, improve memory, improve the objects such as cognitive function.Or utilize the crystalline structure of the mentioned PDE2 catalyst structure domain of the present invention and BAY60-7550 compound crystal, by the method for computer modeling, design small-molecule substance for PDE2 etc., then synthetic this kind of material, form mixture with PDE2, the methods analyst mixture of recycling X ray crystalline diffraction, thus real binding site obtained.The result of analyzing according to X ray crystalline diffraction, can adjust small-molecule substance, until obtain optimized material molecule.And before the present invention, cannot specific aim design, produce the better specific drugs for PDE2 owing to lacking the combining information of PDE2 and selective depressant.
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 complex molecule;
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 three-dimensional plot of feature with BAY60-7550.
Embodiment
Further illustrate the present invention below in conjunction with accompanying drawing and following embodiment, should be understood that following embodiment and/or accompanying drawing are only for the present invention is described, and unrestricted the present invention.
Expression and the purifying of PDE2 catalytic domain structural domain:
The gene order of PDE2 catalytic domain structural domain is expressed: for example, PDE2 catalyst structure domain gene order is cloned in prokaryotic expression carrier pET32a.This PDE2 catalyst structure domain gene order has sequence as indicated in SEQ ID No.1 or its homologue, fragment, variant after expressing.
The protein expression of PDE2 catalytic domain structural domain: for example, recombinant vectors is transformed in E.coli cell BL21 (DE3) Codon Plus bacterial strain, and being cultured to OD600 value in LB substratum is 0.6～0.8 o'clock, adds IPTG inducible protein to express;
Protein purification, such as expressing protein utilizes the affine resin of Ni-NTA (Qiagene) and Superdex200(GE Healthcare) resin etc. be purified into enough purity albumen for crystallization, removed with TEV enzyme in this process with the Trx fusion rotein of PDE2 coexpression.
Albumin crystal growth, complex crystallization:
The preparation of albumen damping fluid: the albumen dialysis of purifying is immersed in damping fluid, and then concentrated (for example use centrifugation apparatus (PALL Corp.) evaporating pipe concentrated) can be measured according to Bradford assay method to 8～12mg/ml().The damping fluid using can for example contain 25mM Hepes ((N-(2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid), pH7.5,25mM NaCl, 2mMTCEP(tri-(2-propyloic) phosphonium salt hydrochlorate).
Albumin crystal growth: mix 1 μ L protein solns and 1 μ L pond liquid (reservoir buffer) as crystallization drop at 18 DEG C.Described pond liquid can contain 0.1M Tris-HCl, pH8.5,0.2M MgCl2,16～20%PEG 6000.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 0.5～2mM BAY60-7550.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 liquid nitrogen.Crystal can be diffracted into conventionally
left and right.Should be understood that and adopt the specific inhibitor part of BAY60-7550 as PDE2 here, but can adopt other suitable specific inhibitor.
Data gathering, processing and model optimization:
On the BL17U of Synchrotron Radiation SSRF bunch, carry out data gathering, and use iMOSFLM to process.X ray wavelength is
every a diffractogram of 1 ° of collection, collect altogether 180.Structure elucidation uses the MOLREP program in CCP4 software package to carry out molecular replacement, and the model that is numbered 1Z1L in employing PDB storehouse is as template.Obtain further in Refmac program, optimizing after original texture.Produce at PRODRG server after the library file of compd B ay60-7550, BAY60-7550 molecule construction is entered in corresponding cloud density figure.Composite structure is finally optimized in PHENIX program.Data processing statistics and composition optimizes numerical value are 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 to and 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 that PDE2 catalyst structure domain is made up of 16 α spirals in this figure, has a Mg in binding pocket bottom
2+with a Zn
2+.Fig. 3 illustrates and shows that PDE2 catalyst structure domain is combined the three-dimensional plot of feature with BAY60-7550, shows in PDE2 catalyst structure domain there be the amino-acid residue of participation and BAY60-7550 effect: Gln859, Gln812, Ile826, Phe862, Met847, Ile822, Leu809, Leu770 and Ile870 etc. in this figure.
Can suppress the small-molecule drug of PDE2 according to above-mentioned information virtual screening, design by Computer Simulation Software etc., thus reach treatment depressed, improve memory, improve the objects such as cognitive function.Or utilize the crystalline structure of the mentioned PDE2 catalyst structure domain of the present invention and BAY60-7550 compound crystal, by the method for computer modeling, design small-molecule substance for PDE2 etc., then synthetic this kind of material, form mixture with PDE2, the methods analyst mixture of recycling X ray crystalline diffraction, thus real binding site obtained.The result of analyzing according to X ray crystalline diffraction, can adjust small-molecule substance, until obtain optimized material molecule.
Sequence table
SEQ ID NO:1
<110> Shanghai Medixi Biomedicine Co., Ltd.
Crystal and the growth method thereof of <120> PDE2 catalyst structure domain/PDE2 specific inhibitor mixture
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 342
<212> PRT
The <213> mankind
<400> 1
Ser Asp Asp Glu Tyr Thr Lys Leu Leu His Asp Gly Ile Gln Pro
1 5 10 15
Val Ala Ala Ile Asp Ser Asn Phe Ala Ser Phe Thr Tyr Thr Pro
20 25 30
Arg Ser Leu Pro Glu Asp Asp Thr Ser Met Ala Ile Leu Ser Met
35 40 45
Leu Gln Asp Met Asn Phe Ile Asn Asn Tyr Lys Ile Asp Cys Pro
50 55 60
Thr Leu Ala Arg Phe Cys Leu Met Val Lys Lys Gly Tyr Arg Asp
65 70 75
Pro Pro Tyr His Asn Trp Met His Ala Phe Ser Val Ser His Phe
80 85 90
Cys Tyr Leu Leu Tyr Lys Asn Leu Glu Leu Thr Asn Tyr Leu Glu
95 100 105
Asp Ile Glu Ile Phe Ala Leu Phe Ile Ser Cys Met Cys His Asp
110 115 120
Leu Asp His Arg Gly Thr Asn Asn Ser Phe Gln Val Ala Ser Lys
125 130 135
Ser Val Leu Ala Ala Leu Tyr Ser Ser Glu Gly Ser Val Met Glu
140 145 150
Arg His His Phe Ala Gln Ala Ile Ala Ile Leu Asn Thr His Gly
155 160 165
Cys Asn Ile Phe Asp His Phe Ser Arg Lys Asp Tyr Gln Arg Met
170 175 180
Leu Asp Leu Met Arg Asp Ile Ile Leu Ala Thr Asp Leu Ala His
185 190 195
His Leu Arg Ile Phe Lys Asp Leu Gln Lys Met Ala Glu Val Gly
200 205 210
Tyr Asp Arg Asn Asn Lys Gln His His Arg Leu Leu Leu Cys Leu
215 220 225
Leu Met Thr Ser Cys Asp Leu Ser Asp Gln Thr Lys Gly Trp Lys
230 235 240
Thr Thr Arg Lys Ile Ala Glu Leu Ile Tyr Lys Glu Phe Phe Ser
245 250 255
Gln Gly Asp Leu Glu Lys Ala Met Gly Asn Arg Pro Met Glu Met
260 265 270
Met Asp Arg Glu Lys Ala Tyr Ile Pro Glu Leu Gln Ile Ser Phe
275 280 285
Met Glu His Ile Ala Met Pro Ile Tyr Lys Leu Leu Gln Asp Leu
290 295 300
Phe Pro Lys Ala Ala Glu Leu Tyr Glu Arg Val Ala Ser Asn Arg
305 310 315
Glu His Trp Thr Lys Val Ser His Lys Phe Thr Ile Arg Gly Leu
320 325 330
Pro Ser Asn Asn Ser Leu Asp Phe Leu Asp Glu Glu
335 340
Claims (13)
1. a crystal for PDE2 catalyst structure domain/PDE2 specific inhibitor mixture, is characterized in that, this crystal has the spacer P1 in oblique system, and this crystal has lattice parameter:
α=109.59 ° ± 0.1%, β=91.93 ° ± 0.1%, γ=90.88 ° ± 0.1%.
2. crystal according to claim 1, is characterized in that, has four molecules in each asymmetric cell, and the molecular weight of each molecule is 39 ± 0.5kD, and solvent is 61 ± 1%.
3. crystal according to claim 1 and 2, is characterized in that, the three-dimensional structure of described crystal meets the three-dimensional structure that table 1 limits.
4. according to the crystal described in any one in claim 1～3, it is characterized in that, in each binding pocket of PDE2 catalyst structure domain, be combined with a PDE2 specific inhibitor molecule.
5. according to the crystal described in any one in claim 4, it is characterized in that, PPDE2 catalyst structure domain is made up of 16 α spirals, has a Mg in the bottom of described binding pocket
2+with a Zn
2+.
6. according to the crystal described in any one in claim 1～5, it is characterized in that, described PDE2 specific inhibitor is BAY60-7550 compound,
7. according to the crystal described in any one in claim 1～6, it is characterized in that, the aminoacid sequence of described PDE2 catalyst structure domain derives from people.
8. crystal according to claim 7, is characterized in that, described PDE2 catalyst structure domain has the aminoacid sequence listed in SEQ ID NO:1 or its homologue, fragment, variant.
9. growth, according to a method for the crystal described in any one in claim 1～8, is characterized in that, comprises the following steps:
1) in damping fluid, prepare the albumen damping fluid containing the PDE2 catalyst structure domain albumen of 8-12mg/ml;
2) mix described albumen damping fluid 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 the crystal with acquisition PDE2 catalyst structure domain/PDE2 specific inhibitor mixture in the pond liquid of 0.5-2mM PDE2 specific inhibitor.
10. method according to claim 9, is characterized in that, the preparation of described albumen damping fluid comprises:
A) PDE2 catalyst structure domain is cloned into pET32a prokaryotic expression carrier;
B) recombinant vectors is transformed in prokaryotic cell prokaryocyte BL21 (DE3) Codon Plus, 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 damping fluid, be then concentrated into 8-12mg/ml.
11. according to the method described in claim 9 or 10, it is characterized in that, the pH value of described damping fluid is 7.5, and the NaCl of the Hepes that contains 25mM, 25mM and the TCEP of 2mM.
12. according to the method described in any one in claim 9～11, it is characterized in that, the pH value of described pond liquid is 8.5, and the MgCl of the Tris-HCl that contains 0.1M, 0.2M
2, and the PEG 6000 of 16-20%.
13. according to the method described in any one in claim 9～12, it is characterized in that, described albumin crystal is grown by vapor diffusion hanging drop method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220269A (en) * | 2017-12-29 | 2018-06-29 | 华南理工大学 | A kind of resisting hydrogen peroxide lipase A flB crystal and preparation method thereof |
| CN110010199A (en) * | 2019-03-27 | 2019-07-12 | 华中师范大学 | A method of analysis identification of protein specific drug binding pocket |
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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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2012
- 2012-12-14 CN CN201210545326.3A patent/CN103865914B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| JIAN ZHU ET AL.: "X-ray Crystal Structure of Phosphodiesterase 2 in Complex with a Highly Selective,Nanomolar Inhibitor Reveals a Binding-Induced Pocket Important for Selectivity", 《J.AM.CHEM.SOC.》 * |
| PANDIT J. ET AL.: "Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct", 《PNAS》 * |
| YING XU ET AL.: "Phosphodiesterase-2 inhibitor reverses corticosterone-induced neurotoxicity and related behavioural changes via cGMP/PKG dependent pathway", 《INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY》 * |
| 赵新筠等: "磷酸二酯酶2（PDE2）结构及其选择性抑制剂的研究进展", 《有机化学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220269A (en) * | 2017-12-29 | 2018-06-29 | 华南理工大学 | A kind of resisting hydrogen peroxide lipase A flB crystal and preparation method thereof |
| CN108220269B (en) * | 2017-12-29 | 2021-07-20 | 华南理工大学 | Hydrogen peroxide-resistant lipase AflB crystal and preparation method thereof |
| CN110010199A (en) * | 2019-03-27 | 2019-07-12 | 华中师范大学 | A method of analysis identification of protein specific drug binding pocket |
| CN110010199B (en) * | 2019-03-27 | 2021-01-01 | 华中师范大学 | Method for analyzing and identifying protein specific drug binding pocket |
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| CN103865914B (en) | 2016-05-25 |
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