CN105902537A - Lead compound for targeted human FKBP51 protein and screening method and application thereof - Google Patents
Lead compound for targeted human FKBP51 protein and screening method and application thereof Download PDFInfo
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Abstract
The invention provides a lead compound for a targeted human FKBP51 protein and a screening method and application thereof, belonging to the technical field of biochemical pharmacy. According to the lead compound and the screening method and application thereof, an FK1 structural domain of FKBP51 is used as a receptor; FK506 of the FKBP51 is selected to be combined with a sack; molecular docking is performed by using a Glide program; 150 small molecular compounds are obtained by virtual screening from more than 1.5 million of compounds and are used for fluorescence quenching experiments; according to the experiments, a combination action of the compounds and the FK1 is verified, and the binding property of a target protein and compounds with a strong docking effect is researched; two kinds of cells LNCaP and DU145 are selected to verify the cell viability of screened small molecular compounds resisting prostate cancer; meanwhile, the structural biology research on a compound formed by the FKBP51 and small molecular lead compounds is performed. According to the lead compound provided by the invention, a cell model and a mouse model of CRPC (Castration Resistant Prostate Cancer) are established, the effectiveness of the lead compound to the CRPC is evaluated, and an action mechanism and a rule of the lead compound and the target protein are explained at the molecular level; a foundation is provided for researching and developing new drugs for treating common prostate cancer and the CRPC.
Description
Technical field
The invention belongs to biochemical pharmacy technical field, more particularly to a kind of targeted human FKBP51 albumen, there is anti-prostatitis
The lead compound of adenocarcinoma activity and screening technique and the application in preparing anti-castration-resistant prostate cancer medicine.
Background technology
Androgen receptor (androgen receptor, AR) be considered carcinoma of prostate generation, develop and shift in rise
To pivotal role.Therefore, androgen withdrawal therapy (androgen deprivation therapy) becomes treatment prostatitis in late period
The most frequently used and the effective method of adenocarcinoma.But in 2 to three years for the treatment of, major part advanced prostate cancer will develop into
Castration-resistant prostate cancer (CRPC).The effective treatment means of shortage current for CRPC, becomes in prostate cancer therapy the most
Stubborn problem.Under androgen withdrawal environment, there is number of ways in the reactivation of AR, mainly includes that lesions position is androgenic
The Signal transduction abnormalities etc. of synthesis, the gene mutation of AR, multiplication and variable sheer and AR regulatory factor altogether.Chang et al. grinds
In CRPC, the synthesis of dihydrotestosterone can be with ind testosterone, and using adrenosterone as precursor to study carefully discovery.There are about
There is the up-regulated that the multiplication of AR gene causes in the CRPC of 25% so that it is the Androgen-sensitive to low concentration.And the sudden change of AR
Frequently can lead to its ligand binding region (ligand binding domain, LBD) decline with androgen antagonist binding ability, very
Transferred to agonism (such as the F876L mutant of AR) by antagonism to making androgen antagonist.Meanwhile, the variable sheer of AR generally may be used
Cause its expression product to lack LBD region, but still there is transcriptional activity and activate downstream target gene.Additionally, the common regulation of AR because of
Son such as SRC-1 and SRC-2 etc. all can make AR strengthen with androgenic binding ability or make its transcriptional activity not rely on androgen.
Therefore, how to suppress or block AR activation under androgen withdrawal environment become treatment CRPC key.
Rabphilin Rab FKBP51 and FKBP52 is as collaborative molecular chaperones (cochaperone), together with Hsp90 in immunity
Help the correct folding of AR, required for AR function, AR, AR can be reduced by suppression FKBP51/FKBP52 prominent
The effect of the transcriptional activity of variant and variable sheer product.FKBP51 Yu FKBP52 has the sequence similarity of 70%, and all has
Along proline isomerase activity (PPIase).The two structure is similar, all includes the FK506 binding structural domain of N end, this knot
Structure territory has PPIase activity, is commonly called FK1 domain.Adjacent FK1 domain is FK2 domain, it and FK1 structure
Territory there are about the sequence similarity of 19%, similar to the folding type of FK1, but owing to lacking critical active residue, does not have suitable
Proline isomerase activity, can not be in conjunction with FK506.C end is TRP (tetratricopeptide repeat) domain, it
Mediation FKBP51, FKBP52 interact with the C end of Hsp90.FKBP51 is suppression steroid receptor in most cells
Signal transduction and FKBP52 can promote the signal transduction of steroid receptor.TRP domain is because interacting with Hsp90, right
FKBP51 and FKBP52 function is indispensable.Although the PPIase activity of FK1 domain is for the regulation of steroid receptor
Not impact, but the FK506 binding pocket of FK1 is but most important to the function of FKBP51, has the pharmacology of highly significant to imitate
Should.
For FKBP51, it can be thin with cancer as the tumor markers during prostate cancer development, expression
The Gleason classification (Gleason grade) of born of the same parents becomes positive correlation.Existing two research groups report in prostate gland cancer cell,
FKBP51 promotes the activity of AR and improves the ability that AR is combined with androgen, and then makes the expression of downstream gene that AR regulated and controled
Raise, cause prostate gland cancer cell to be bred.Additionally, the expression of FKBP51 is also by the just regulation and control of AR, promote the formation of CRPC.Grind
Study carefully the growth of cancer cells also demonstrating that the inhibitor FK506 of FKBP51 can significantly suppress Androgen-dependent, this inhibitory action
Depend on AR.Research to FKBP51 knock out mice further demonstrates that, specific blocking-up or the function of suppression FKBP51
Potential side effect will not be produced.Additionally, to the broad spectrum inhibitors FK1706 (institute coming into clinical phase test FKBP family
The target spot of suppression includes FKBP51 and FKBP52) toxicologic study result show, even if for a long time suppression FKBP51 with
Human body will not be caused the most serious consequence by the activity of FKBP52.As can be seen here, FKBP51, FKBP52 are the non-of CRPC treatment
Normal promising drug target.
Summary of the invention
It is an object of the invention to provide lead compound and the screening technique thereof of a kind of targeted human FKBP51 albumen and making
Application in standby anti-castration-resistant prostate cancer medicine.
To achieve these goals, the technical solution used in the present invention is as follows:
The lead compound of a kind of targeted human FKBP51 albumen, has a chemical constitution of formula (I):
Present invention also offers the screening technique of the lead compound of described targeted human FKBP51 albumen, including following step
Rapid:
(1) high flux virtual screening
Utilize Schrodinger's program bag to carry out high flux virtual screening, download FKBP51's from Protein structure databases PDB
The crystal structure (PDB code:305R) of FK1 domain, is optimized structure, adds electric charge and hydrogen atom, choose its FK506
Binding pocket, utilizes molecular docking programs Glide, from Chemdiv company provided more than 150 ten thousand compounds carry out height
Flux virtual screening, utilizes QikProp to calculate the physico-chemical property of the little molecule lead compound screened, such as lipid
QPlogPo/w, filters out best front 150 the little molecules of docking score first according to Li Binsiji rule (Lipinski ' srule)
Lead compound, and buy little molecule lead compound from Chemdiv company；
(2) fluorescent quenching experiment
The FK1 domain of FKBP51 comprises only a tryptophan and is located exactly at FK506 binding pocket, owing to Trp is at egg
White matter has the strongest autofluorescence, and the intensity of its emission spectrum is affected by surrounding chemical microenvironment with wavelength, because of
It is strong and weak with the combination of little molecule lead compound that this can measure FK1 by the cancellation degree of monitoring Trp emission spectrum, will
The FK1 protein concentration that purification is good is set in 10 μMs, little molecule lead compound final concentration 100 μMs, reaction system 100 μ L, utilizes
The multi-functional microplate reader of Varioskan Flash of Thermo company measures little molecule lead compound to FK1 with 96 orifice plate forms
The cancellation degree of Trp fluorescence, excitation wavelength is set to 295nm, launches wavelength and is set to 335nm, every kind little molecule lead compound
Parallel do three times, using rapamycin as comparison, for fluorescent quenching degree higher than rapamycin or the little molecule suitable with it
Lead compound, utilizes PerkinElmer company LS55 spectrofluorophotometer to measure its dissociation constant Kd further, FK1's
Concentration is set to 10 μMs, and the concentration of little molecule lead compound arranges gradient, each little molecule lead compound between 0-100 μM
Parallel do three times, the data obtained utilizes Prism program carry out nonlinear fitting, tries to achieve dissociation constant Kd；
(3) expression and purification of albumen
Full genome synthesizes the FK1 domain (19-126) of FKBP51 and is cloned in pET28b carrier, uses escherichia coli bacterium
Strain BL21 (DE3) or Rosseta (DE3) expresses, and carries out thick purification with ni-sepharose purification, then hands over by anion or cation
Change column purification, finally with molecular sieve superdex75 column purification, build the FKBP51-FK1 without Histag simultaneously, the electricity such as utilization
The point character more than 8, uses cation exchange column SPFF to carry out thick purification, then with molecular sieve superdex75 column purification, purification
Good protein concentration, to subpackage after 40mg/mL, is placed on-80 DEG C of Refrigerator stores with liquid nitrogen flash freezer；
(4) surface plasma resonance (SPR) experiment
Utilize the power of SPR technique quantitative determination FK1 and the interphase interaction of micromolecular compound, will by amino coupled method
FK1 is coupled on CM5 chip be measured, and with NaOH or HCl or EDTA for chip regeneration condition, chooses the regeneration condition of optimum
It is circulated mensuration；
(5) cell and biochemical test
Utilize MTT/MTS experiment (Promega) measure micromolecular compound to prostate gland cancer cell LNCaP, PC3 and
The growth inhibited effect of DU145；For can relatively high inhibition LNCaP, but the little molecule that do not suppresses PC3 and DU145 is first
Lead compound, continue following several method and verify its suppression to AR signal path: 1. extract total mRNA of cell, fixed with fluorescence
Amount RT-PCR technology detection screening micromolecular compound to FKBP51, FKBP52, AR and AR Downstream regulatory gene such as PSA,
The impact of the transcriptional level of hK2, TMPRSS2；2. by the specific antibody of AR and PSA, by Western blot technology for detection
The micromolecular compound of screening on AR and downstream gene PSA in the impact of translation skill；3. steady turning is built with slow-virus transfection
The LNCaP cell line of ARELuc and ARE-EGFP, quantitative by Luciferase enzyme reporting system alive and fluorescence microscope respectively
The micromolecular compound of the research screening inhibitory action to AR transcriptional activity；4. it is total to slow-virus transfection structure and surely turns CRPC shearing
The DU145 cell line of AR-V7 and AR-NTD, i.e. DU145-V7 and DU145-NTD, by fluorescence quantitative RT-RCR technology and
The suppression to AR activity of the micromolecular compound of Western blot technology for detection screening；
(6) crystallization of albumen and structure elucidation
FK1 albumen and the little molecule lead compound cocrystallization sieved to FKBP51, screens various crystallization condition, it is thus achieved that
The crystal of high diffraction quality, utilizes synchrotron radiation light source to collect X-ray crystal diffraction data, uses molecular replacement technique to resolve FK1
Crystal structure with little molecule lead compound complex；
(7) structural analysis and molecular dynamics simulation
Software analysis FK1 and the crystal structure of little molecule lead compound complex, use the GPU version of Amber14 to enter
The molecular dynamics simulation of row FK1-little molecule lead compound complex, calculates little molecule lead compound by mmpbsa method
The free energy being combined with FK1, utilizes the program in Ambertools software kit to analyze conformation change and albumen and little molecule guide
The Interactions Mode of compound, the GPU version of Amber14 uses the GTX970 video card of Nvidia company to calculate, passes through
Above structural analysis, sums up the action rule of FK1 and little molecule lead compound, and the transformation further for lead compound carries
For instructing；
(8) CRPC zoopery
Concrete CRPC Animal Model: the male immunization deficient mice in 5-9 week is divided into two groups, one of which is real
Test group and carry out emasculation, by LNCaP cell (1 × 10 after recovering 5 days6Individual) subcutaneous injection is in the complete or immunodeficient mouse of emasculation
(BALB/C-nunu), Real Time Observation tumor size grows, and weighs Mouse Weight and tumor by digital calipers 2-3 time weekly
Volume is (wide2× long/2), 1cm is reached using mean tumour volume as reference, tumor growth 8 weeks and tumor size3It is successfully
The Transplanted tumor model set up；On the basis of CRPC animal model, be further divided into different administration groups, chemotherapeutics group: many west he
Match, carbadox is matched；Suppression androgen synthetic drug group: abiraterone group；Miscellaneous with androgen competitive inhibition AR active medicine grace
Shandong amine；And little molecule lead compound group, often 7 mices of group, within continuous 28 days, it is administered 1,10, or the medicine of 50mg/kg, and
If blank, by measuring the change of tumor size, weigh tumor weight, calculate tumour inhibiting rate, compare animal survival natural law, and petty action
Thing whole body optical imaging system, assesses the impact on the tumor of CRPC mice of the little molecule lead compound, observes after terminating, puts to death
Animal, takes transplanted tumor tissue, carry out with non-dosing and PCa mice after CRPC model mice dosing AR target gene PSA and under
Trip related gene such as TMPRSS2 expresses and contrasts；Metastatic CRPC mice and non-medication generation cancer is still there is after medicine effect
Transfer CRPC mice and PCa mice survival rate compare.
Present invention also offers Formulas I lead compound use in the medicine of preparation treatment castration-resistant prostate cancer
On the way.
The Advantageous Effects of the present invention: (1) known main thought for the drug discovery of FKBP51 be based on
The molecular modification of FK506 and rapamycin, but, the inhibitor of the FKBP51 designed based on FK506 and rapamycin is often
Higher with the binding ability of FKBP12, therefore obtained by compound there may be the strongest effect of missing the target, the present invention first with
FKBP51 albumen is target spot, the pilot compound of high flux virtual screening anti-prostate cancer, and in molecular level, cellular water
The flat effectiveness with animal individual these lead compound of level verification；(2) although CRPC shows supports androgen withdrawal
Resistance, but its progress usually still depends on AR, and FKBP51, FKBP52 are as collaborative molecular chaperones, and it is required to activate for AR, logical
Crossing cell model and the mouse model setting up CRPC, the present invention have studied the work screening the lead compound obtained to CRPC first
By effect and mechanism；(3) by resolving the crystal structure that target protein is combined with lead compound, target protein and guide's chemical combination are analyzed
The binding pattern of thing, combines rule, and by molecular dynamics simulation, studies the lead compound structure to FKBP51
The impact of elephant, transforms for medicines structure from now on and provides the foundation.
Accompanying drawing explanation
Fig. 1 is the structure of FKBP51.(A) the domain composition of FKBP51 albumen；(B) the FK1 domain of FKBP51 with
The interaction mode of FK506.
Fig. 2 is the interaction of FKBP51 and micromolecular compound.(A) with the FKBP51's of cation exchange purification acquisition
FK1 domain, the molecular weight of FK1 is about 14KDa；(B) fluorescent quenching of FK1 is tested by the 150 kinds of micromolecular compounds bought,
Redness is positive control rapamycin (a), and green is the buffer (b) without micromolecular compound.
Fig. 3 is that FKBP51 protein molecular sieves (FPLC) purification result.(A) coagulate with HiLoad 16/60Superdex75 pillar
The FKBP51 albumen that glue Purification by filtration obtains；(B) the FKBP51 electrophoretogram of 15%SDS-PAGE.
The growth inhibited effect to two kinds of prostate gland cancer cell LNCaP and DU145 of Fig. 4 position micromolecular compound.Upper figure is
The micromolecular compound growth inhibited curve to LNCaP cell, figure below is the little molecule lead compound growth inhibited to DU145
Curve.
Fig. 5 is the co-crystal thereof of little molecule lead compound and FK1.
Fig. 6 is the Technology Roadmap of little molecule lead compound screening.
Fig. 7 is the effect to CRPC of the little molecule lead compound and the Technology Roadmap of mechanism.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further elaborated with accompanying drawing explanation, but the present invention is not limited to following
Embodiment.
Embodiment 1
The chemical constitution of lead compound:
1, there is the high flux virtual screening method of the lead compound of formula (I) chemical constitution
Utilize Schrodinger's program bag to carry out, download the crystal of the FK1 domain of FKBP51 from Protein structure databases PDB
Structure (PDB code:305R), uses the programs such as Prime, QSite, Liasion and MacroModel to carry out excellent to structure
Change, add electric charge and hydrogen atom, choose its FK506 binding pocket, utilize molecular docking programs Glide, carried from Chemdiv company
Confession more than 150 ten thousand compounds in carry out high flux virtual screening, screening is carried out on the server of 4 core CPU, and every day is about
100,000 compounds can be screened, utilize QikProp to calculate the physico-chemical property such as fat water of the little molecule lead compound screened
Partition coefficient QPlogPo/w etc., filter out docking-score according to Li Binsiji rule (Lipinski ' s rule) best
Front 150 little molecule lead compound.Li Binsiji rule includes: 1. the molecular weight of compound is less than 500Da；2. compound
The quantity of the hydrogen-bond donor (including hydroxyl, amino etc.) in structure is less than 5；3. in compound, the quantity of hydrogen bond receptor does not surpasses
Cross 10；4. the logarithm value (logP) of the lipid of compound is between-2 to 5.Meet the chemical combination of Li Binsiji rule
Thing has more preferable pharmacokinetic properties, has more preferable bioavailability, it is easy to become oral medicine in metabolic process.Little molecule
Lead compound is bought in Chemdiv company.
2, fluorescent quenching experiment
The FK1 domain of FKBP51 comprises only a tryptophan and is located exactly at FK506 binding pocket, owing to Trp is at egg
White matter has the strongest autofluorescence, and the intensity of its emission spectrum is affected by surrounding chemical microenvironment with wavelength, because of
It is strong and weak, by pure with the combination of little molecule lead compound that this can measure FK1 by the cancellation degree of monitoring Trp emission spectrum
The FK1 protein concentration changed is set in 10 μMs, little molecule lead compound final concentration 100 μMs, reaction system 100 μ L, utilizes
The multi-functional microplate reader of Varioskan Flash of Thermo company measures little molecule lead compound to FK1 with 96 orifice plate forms
The cancellation degree of Trp fluorescence, excitation wavelength is set to 295nm, launches wavelength and is set to 335nm, every kind little molecule lead compound
Parallel do three times, using rapamycin as comparison, for fluorescent quenching degree higher than rapamycin or the little molecule suitable with it
Lead compound, utilizes PerkinElmer company LS55 spectrofluorophotometer to measure its dissociation constant Kd further, FK1's
Concentration is set to 10 μMs, and the concentration of little molecule lead compound arranges gradient, each little molecule lead compound between 0-100 μM
Parallel do three times,
The data obtained utilizes Prism program carry out nonlinear fitting, tries to achieve dissociation constant Kd.
| Dissociation constant Kd | 86.1 |
| Standard deviation | 64.8 |
3, the expression and purification of albumen
Full genome synthesizes the FK1 domain (19-126) of FKBP51 and is cloned into respectively in pET28b carrier, uses large intestine bar
Bacteria strain BL21 (DE3) or Rosseta (DE3) expresses, and carries out thick purification with ni-sepharose purification, then with anion or sun from
Sub-exchange column purification, finally with molecular sieve superdex75 column purification.Build the FKBP51-FK1 without Histag simultaneously, utilize
The character of isoelectric point, IP higher (more than 8), uses cation exchange column SPFF to carry out thick purification, then with molecular sieve superdex75 post
Purification, subpackage after the good protein concentration of purification to 40mg/mL, it is placed on-80 DEG C of Refrigerator stores with liquid nitrogen flash freezer.
4, surface plasma resonance (SPR) experiment
We utilize the power of the more accurate quantitatively FK1 of SPR technique and the interphase interaction of micromolecular compound.Use amino
FK1 is coupled to CM5 chip or utilizes the Histag on FK1 to be coupled on NTA chip by coupling method, finally chooses response relatively
Good chip is measured, and selects various different chip regeneration condition, such as NaOH, HCl, EDTA etc., finally chooses optimum
Regeneration condition.
5, cell and biochemical test
Utilize MTT/MTS experiment (Promega) measure micromolecular compound to prostate gland cancer cell LNCaP, PC3 and
The growth inhibited effect of DU145.For can relatively high inhibition LNCaP, but the little molecule that do not suppresses PC3 and DU145 is first
Lead compound, continue following several method and verify its suppression to AR signal path: 1. extract total mRNA of cell, fixed with fluorescence
Amount RT-PCR technology detection screening micromolecular compound to FKBP51, FKBP52, AR and AR Downstream regulatory gene such as PSA,
The impact of the transcriptional level of hK2, TMPRSS2 etc.；2. by the specific antibody of AR and PSA, examined by Western blot technology
Survey the micromolecular compound of screening to AR and downstream gene PSA in the impact of translation skill；3. steady turning is built with slow-virus transfection
The LNCaP cell line of ARELuc and ARE-EGFP, quantitative by Luciferase enzyme reporting system alive and fluorescence microscope respectively
The micromolecular compound of the research screening inhibitory action to AR transcriptional activity；4. it is total to slow-virus transfection structure and surely turns CRPC shearing
The DU145 cell line of AR-V7 and AR-NTD, i.e. DU145-V7 and DU145-NTD, by fluorescence quantitative RT-RCR technology and
The suppression to AR activity of the micromolecular compound of Western blot technology for detection screening.
6, the crystallization of albumen and structure elucidation
The FK1 albumen of FKBP51/FKBP52 is attempted and the little molecule lead compound cocrystallization sieved by we, and screening is each
Kind of crystallization condition (ScreenI&ScreenII, SaltRX, Index, PEGion and the MiTeGen company such as Hampton company
The conditions such as JBScreen Classic), attempt and improve various precipitant, buffer agent and additive, to obtain high diffraction quality
Crystal.Utilize synchrotron radiation light source to collect X-ray crystal diffraction data, utilize the softwares such as HKL2000, CCP4 and Phenix,
Molecular replacement technique is used to resolve the crystal structure of FK1 and little molecule lead compound complex.
7, structural analysis and molecular dynamics simulation
Use the crystal structure of the software analysis FK1 such as Pymol, Ligplot and little molecule lead compound complex, use
The GPU version of Amber14 carries out the molecular dynamics simulation of FK1-little molecule lead compound complex, with mmpbsa method meter
Calculate the free energy that little molecule lead compound is combined with FK1, utilize the program in Ambertools software kit to analyze conformation change,
Albumen and the Interactions Mode etc. of little molecule lead compound, the GPU version (i.e. CUDA version) of Amber14 uses
The GTX970 video card of Nvidia company calculates, and through our early stage test, finds that its computing capability is equivalent to 64 cores
Computer cluster.By above structural analysis, sum up the action rule of FK1 and little molecule lead compound, for guide's chemical combination
The transformation further of thing provides to be instructed.
8, CRPC zoopery
Concrete CRPC Animal Model: the male immunization deficient mice in 5-9 week is divided into two groups, one of which is experiment
Group carries out emasculation, by LNCaP cell (1 × 106) subcutaneous injection in the complete or immunodeficient mouse of emasculation after recovering 5 days
(BALB/C-nunu), Real Time Observation tumor size grows, and weighs Mouse Weight and tumor by digital calipers 2-3 time weekly
Volume (wide by 2 × long/2), using mean tumour volume as reference.Tumor growth about 8 weeks and tumor size reach 1cm3 i.e.
For the Transplanted tumor model being successfully established.On the basis of CRPC animal model, it is further divided into different administration groups.Chemotherapeutics group:
Docetaxel, carbadox is matched；Suppression androgen synthetic drug group: abiraterone group；With androgen competitive inhibition AR active drug
Thing grace miscellaneous Shandong amine；And little molecule lead compound group, often 7 mices of group, within continuous 28 days, it is administered 1,10, or 50mg/kg
Medicine, and set blank, by measuring the change of tumor size, weigh tumor weight, calculate tumour inhibiting rate, compare animal survival sky
Number, and small animal living body fluoroscopic imaging systems, assess the impact on the tumor of CRPC mice of the little molecule lead compound, observes knot
Shu Hou, puts to death animal, takes transplanted tumor tissue, carries out AR targeting base with non-dosing and PCa mice after CRPC model mice dosing
Contrast because PSA and downstream related gene such as TMPRSS2 expresses；Still occur metastatic CRPC mice with unused after medicine effect
Medicine generation cancerometastasis CRPC mice and PCa mice survival rate compare.
Claims (3)
1. the lead compound of the targeted human FKBP51 albumen of formula (I) structure use in preparing anti-prostate cancer tumour medicine
On the way,
2. in claim 1, the lead compound of targeted human FKBP51 albumen treats castration-resistant prostate cancer medicine in preparation
In application.
3. the screening technique of lead compound described in claim 1, it is characterised in that comprise the following steps:
(1) high flux virtual screening
Utilize Schrodinger's program bag to carry out high flux virtual screening, download the FK1 knot of FKBP51 from Protein structure databases PDB
The crystal structure (PDB code:305R) in structure territory, is optimized structure, adds electric charge and hydrogen atom, choose its FK506 combined mouth
Bag, utilizes molecular docking programs Glide, from Chemdiv company provided more than 150 ten thousand compounds to carry out high flux empty
Intend screening, utilize QikProp to calculate the physico-chemical property of the little molecule lead compound screened, such as lipid
QPlogPo/w, filters out best front 150 the little molecules of docking score first according to Li Binsiji rule (Lipinski ' srule)
Lead compound, and buy little molecule lead compound from Chemdiv company；
(2) fluorescent quenching experiment
The FK1 domain of FKBP51 comprises only a tryptophan and is located exactly at FK506 binding pocket, owing to Trp is at protein
In have the strongest poliosis fluorescence, and the intensity of its emission spectrum is affected by surrounding chemical microenvironment with wavelength, therefore may be used
FK1 is measured strong and weak with the combination of little molecule lead compound with the cancellation degree by monitoring Trp emission spectrum, purification is good
FK1 protein concentration be set in 10 μMs, little molecule lead compound final concentration 100 μMs, reaction system 100 μ L, utilize Thermo
It is glimmering to the Trp of FK1 that the multi-functional microplate reader of Varioskan Flash of company measures little molecule lead compound with 96 orifice plate forms
The cancellation degree of light, excitation wavelength is set to 295nm, launches wavelength and is set to 335nm, and every kind little molecule lead compound is parallel does three
Secondary, using rapamycin as comparison, for fluorescent quenching degree higher than rapamycin or the little molecule guide chemical combination suitable with it
Thing, utilizes PerkinElmer company LS55 spectrofluorophotometer to measure its dissociation constant Kd further, and the concentration of FK1 is set to
10 μMs, the concentration of little molecule lead compound arranges gradient between 0-100 μM, and each little molecule lead compound is parallel does three
Secondary, the data obtained utilizes Prism program carry out nonlinear fitting, tries to achieve dissociation constant Kd；
(3) expression and purification of albumen
Full genome synthesizes the FK1 domain (19-126) of FKBP51 and is cloned in pET28b carrier, uses coli strain
BL21 (DE3) or Rosseta (DE3) expresses, and carries out thick purification with ni-sepharose purification, then exchanges by anion or cation
Column purification, finally with molecular sieve superdex75 column purification, builds the FKBP51-FK1 without Histag simultaneously, utilizes isoelectric point, IP
Character more than 8, uses cation exchange column SPFF to carry out thick purification, then with molecular sieve superdex75 column purification, purification is good
Protein concentration to subpackage after 40mg/mL, be placed on-80 DEG C of Refrigerator stores with liquid nitrogen flash freezer；
(4) surface plasma resonance (SPR) experiment
Utilize the power of SPR technique quantitative determination FK1 and the interphase interaction of micromolecular compound, by amino coupled method by FK1
Being coupled on CM5 chip be measured, with NaOH or HCl or EDTA for chip regeneration condition, the regeneration condition choosing optimum enters
Row circulation measures；
(5) cell and biochemical test
MTT/MTS experiment (Promega) is utilized to measure micromolecular compound to prostate gland cancer cell LNCaP, PC3 and DU145
Growth inhibited effect；For can relatively high inhibition LNCaP, but little molecule guide's chemical combination that PC3 and DU145 is not suppressed
Thing, continues following several method and verifies its suppression to AR signal path: 1. extract total mRNA of cell, use fluorescent quantitation RT-
Round pcr detection screening micromolecular compound to FKBP51, FKBP52, AR and AR Downstream regulatory gene such as PSA, hK2,
The impact of the transcriptional level of TMPRSS2；2. by the specific antibody of AR and PSA, screened by Western blot technology for detection
Micromolecular compound on AR and downstream gene PSA in the impact of translation skill；3. build with slow-virus transfection and surely turn ARELuc
With the LNCaP cell line of ARE-EGFP, sieve by Luciferase enzyme reporting system alive and fluorescence microscope quantitative study respectively
The micromolecular compound of the choosing inhibitory action to AR transcriptional activity；4. it is total to slow-virus transfection structure and surely turns the CRPC AR-V7 of shearing
And the DU145 cell line of AR-NTD, i.e. DU145-V7 and DU145-NTD, with fluorescence quantitative RT-RCR technology and Western
The suppression to AR activity of the micromolecular compound of blot technology for detection screening；
(6) crystallization of albumen and structure elucidation
FK1 albumen and the little molecule lead compound cocrystallization sieved to FKBP51, screens various crystallization condition, it is thus achieved that Gao Yan
Penetrate the crystal of quality, utilize synchrotron radiation light source to collect X-ray crystal diffraction data, use molecular replacement technique to resolve FK1 with little
The crystal structure of molecule lead compound complex；
(7) structural analysis and molecular dynamics simulation
Software analysis FK1 and the crystal structure of little molecule lead compound complex, use the GPU version of Amber14 to carry out
The molecular dynamics simulation of FK1-little molecule lead compound complex, with mmpbsa method calculate little molecule lead compound with
The free energy that FK1 combines, utilizes the program in Ambertools software kit to analyze conformation change and albumen and little molecule guideization
The Interactions Mode of compound, the GPU version of Amber14 uses the GTX970 video card of Nvidia company to calculate, by with
On structural analysis, sum up the action rule of FK1 and little molecule lead compound, transform offer further for lead compound
Instruct；
(8) CRPC zoopery
Concrete CRPC Animal Model: the male immunization deficient mice in 5-9 week is divided into two groups, and one of which is that experimental group enters
Row emasculation, by LNCaP cell (1 × 10 after recovering 5 days6Individual) subcutaneous injection is in the complete or immunodeficient mouse (BALB/ of emasculation
C-nunu), Real Time Observation tumor size grows, and weighs Mouse Weight and gross tumor volume by digital calipers 2-3 time weekly
(wide2× long/2), 1cm is reached using mean tumour volume as reference, tumor growth 8 weeks and tumor size3It is and is successfully established
Transplanted tumor model；On the basis of CRPC animal model, it is further divided into different administration groups, chemotherapeutics group: docetaxel, card
Abbado is matched；Suppression androgen synthetic drug group: abiraterone group；With androgen competitive inhibition AR active medicine grace miscellaneous Shandong amine；
And little molecule lead compound group, often 7 mices of group, within continuous 28 days, it is administered 1,10, or the medicine of 50mg/kg, and sets sky
White comparison, by measuring the change of tumor size, weighs tumor weight, calculates tumour inhibiting rate, compares animal survival natural law, and toy is lived
Body fluoroscopic imaging systems, assesses the impact on the tumor of CRPC mice of the little molecule lead compound, observes after terminating, and puts to death dynamic
Thing, takes transplanted tumor tissue, carries out AR target gene PSA and downstream with non-dosing and PCa mice after CRPC model mice dosing
Related gene such as TMPRSS2 expresses and contrasts；Metastatic CRPC mice is still occurred to turn with non-medication generation cancer after medicine effect
Move CRPC mice and PCa mice survival rate compares.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108169195A (en) * | 2017-09-20 | 2018-06-15 | 大连民族大学 | Cationic-liposome/gene composite Dissociation detection method |
| CN110785428A (en) * | 2017-04-05 | 2020-02-11 | 锐新医药公司 | Methods and reagents for analyzing protein-protein interfaces |
| US11987590B2 (en) | 2015-01-09 | 2024-05-21 | Revolution Medicines, Inc. | Compounds that participate in cooperative binding and uses thereof |
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| WO2008017840A1 (en) * | 2006-08-08 | 2008-02-14 | Merlion Pharmaceuticals Sa | Cyclopentanecarboxylic acid derivatives and their use in the treatment of bacterial infectious diseases |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US11987590B2 (en) | 2015-01-09 | 2024-05-21 | Revolution Medicines, Inc. | Compounds that participate in cooperative binding and uses thereof |
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