CN1808106A - Method for screening acetylcholine esterase inhibitor by nuclear magnetic resonance - Google Patents

Abstract

The invention provides a method for screening cholinesterase inhibitor, which comprises: testing the relaxation rates of sample compounds in substrate before and after adding acetylcholinesterase to decide the sample compounds which relaxation rates being speed-up as the cholinesterase inhibitor; at the same time, employing double irradiation method to get the prolonged movement time of the substrate molecule; the nuclear magnetic resonance of the substrate proton before and after adding enzyme having no obvious change if the substrate molecule haven't combined with the enzyme.

Description

A kind of method with nuclear magnetic resonance screening acetylcholinesteraseinhibitors inhibitors

Technical field

The present invention relates to a kind of screening technique of acetylcholinesteraseinhibitors inhibitors, more particularly, relate to the method that a kind of using NMR screens acetylcholinesteraseinhibitors inhibitors.

Background technology

Alzheimer's disease (Alzheimer disease) be a kind of be the carrying out property mental illness of feature with the memory defects.The change of central cholinergic system transmission mechanism is the most significant pathology of alzheimer's disease patient central nervous system, and relevant with the seriousness of dementia.Present medicine is first-selected with the anticholinesterase.The function of acetylcholinesterase is the hydrolysis acetylcholine, makes its inactivation, and then the normal physiological function of the cynapse that affects the nerves.Anticholinesterase can delay the hydrolysis of acetylcholine, improves the content of acetylcholine in synaptic cleft, thereby keeps the normal physiological function of nervous centralis cynapse, produces result of treatment.But screening technique commonly used at present is, with from rat cerebral cortex, separating the acetylcholinesterase (AChE) obtain as screening model, add sample compound, develop the color with Ellman ' s reagent again, compare by colourimetry, try to achieve inhibiting activity of acetylcholinesterase, No. the 1417209th, Chinese invention patent application specification (for example, can referring to) thus.

Yet the shortcoming of colorimetric method is need with more enzyme amount, and colour developing to be interfered easily.

Summary of the invention

The objective of the invention is, be to seek a kind of fast and simple, method of not being subject to the screening acetylcholinesterase inhibitor that disturbs.

The screening technique of acetylcholinesteraseinhibitors inhibitors of the present invention adopts the means of nuclear magnetic resonance, screens effective acetylcholinesteraseinhibitors inhibitors by the bonding state of observing sample compound molecule and acetylcholinesterase.

Promptly, the present invention finishes on the basis of following experiment: add acetylcholinesterase in the solution as the sample compound of substrate, observe the variation of the relaxation properties of the sample compound proton that adds the acetylcholinesterase front and back, combine with enzyme as substrate molecule, then the selectivity relaxation rate of substrate proton accelerates, simultaneously, it is elongated to record the relevant run duration of substrate molecule by the double irradiation method; Combine with enzyme as substrate molecule, then the proton of substrate does not have significant change in the nuclear magnetic resonance character of enzyme-added front and back.

Therefore, the invention provides a kind of like this method of screening acetylcholinesteraseinhibitors inhibitors, it determines that by measuring the relaxation rate of sample compound before and after the adding acetylcholinesterase as substrate the sample compound that the selectivity relaxation rate accelerates is an acetylcholinesteraseinhibitors inhibitors.

In said method, described assay method can adopt the upset restoring method.Promptly use 180 degree pulses and make the proton magnetic vector upset that will observe, the recovery extent of observation magnetic vector after the different stand-by period is according to judging magnetic Henan speed the release time of magnetic vector.

The non-selective spin lattice speed of proton (hereinafter to be referred as the speed of relaxing) R ^NsThe relaxation rate of each proton when shining all protons in the molecule simultaneously.The selectivity spin lattice speed R of proton ^sBe the relaxation rate of this proton during specific proton in the irradiation molecule.R ^NsAnd R ^sSatisfy following equation:

$R_i^{\mathrm{ns}}=\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\ρ}}_{\mathrm{ij}}+\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\σ}}_{\mathrm{ij}}+{\mathrm{\ρ}}_i^{*}...\left(1\right)$

$R_i^s=\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\ρ}}_{\mathrm{ij}}+{\mathrm{\ρ}}_i^{*}...\left(2\right)$

In equation (1), (2), ρ ij is H _iWhen after proton is illuminated energy being given other proton of its mutual dipole to the contribution of relaxation rate.σ ij is that two protons of mutual dipole influence each other to the contribution of relaxation rate, ρ after simultaneously illuminated _i ^*For other relaxation mechanism to H _iThe influence of relaxation rate.Relatively (1), (2) can be found out, in (2) formula, and R ^sOwing to only shine H _iProton is not so have other illuminated proton to H _iInfluence item σ ij.

Be further to detect huperzine and the intermolecular interaction of torpedo acetylcholinesterase TnAChE, the method that we use double irradiation has been measured the relevant run duration of molecule.From equation (1), (2) as can be known, the fast σ ij=R that relaxes of the intersection between proton _i ^Ij-R _i ^s, R _i ^IjFor shining H simultaneously _iReach proton H with its coupling _jThe time H that records _iThe speed of speeding of proton.R ^sFor shining H in the irradiation molecule separately _iThe time this proton relaxation rate.When enzyme-to-substrate mutually combined, exists a mobile equilibrium system, set up following equation (3) and (4):

${\mathrm{\σ}}_{\mathrm{obs}}^{\mathrm{ij}}=p_{\mathrm{free}}{\mathrm{\σ}}_{\mathrm{free}}^{\mathrm{ij}}+p_{\mathrm{bound}}{\mathrm{\σ}}_{\mathrm{bound}}^{\mathrm{ij}}...\left(4\right)$

In the formula, p _FreeBe the substrate ratio of desmoenzyme not in the solution, p _BoundBe the ratio of the substrate of desmoenzyme, σ _Free ^IjObserved cross-relaxation rate when not enzyme-added, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates.

When considering σ with the most conservative method _Bound ^IjWith σ f _Ree ^Ij, promptly set enzyme-to-substrate combination completely, then p taken place _Bound=(protein)/(ligand), p _Free=1-p _BoundCalculate σ according to equation (4) again _Bound ^IjValue, use molecular motion τ correlation time _Bound ^IjWith σ _Bound ^IjBetween relational equation (5), can draw substrate and enzyme in conjunction with after the relevant run duration of molecule.

In the formula, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates, σ _Free ^IjObserved cross-relaxation rate when not enzyme-added, P _BoundBe the ratio of the substrate of desmoenzyme, γ is the magnetic rotation ratio of proton,

Be the modified value of Plank ' s constant, r _IjBe proton H _iAnd the distance between Hj, be H _iMove correlation time with Hj, ω is the Larmor frequency of proton.

In the method for the invention, can revolve lattice speed R in vain according to the proton selectivity of substrate behind the adding acetylcholinesterase ^sIncrement whether greater than its non-selective spin lattice speed R ^NsIncrement determine effective acetylcholinesteraseinhibitors inhibitors.

In addition, in the method for the invention, can be when adding acetylcholinesterase the proton selectivity spin lattice speed R of substrate ^sProton selectivity spin lattice speed R during greater than the adding butyrylcholine esterase ^sDetermine effective acetylcholinesteraseinhibitors inhibitors.

In addition, in the method for the invention, can also determine effective acetylcholinesteraseinhibitors inhibitors according to the relevant run duration ω τ of molecule that adds substrate behind the acetylcholinesterase is whether elongated.

The relevant run duration ω τ of described molecule is elongated for example to be from being converted into greater than 1 less than 1.

The advantage of this method is 1, and is easy to be quick: testing sample and enzyme are put together, and directly using NMR instrument test can be seen obvious result in 10 minutes.Need not harsh test condition and reagent etc.2, real-time: be dissolved in the observation of carrying out in the damping fluid of internal milieu with testing molecule that combines because of enzyme, thus the method can detect enzyme under coenocorrelation, solution state with the combining of substrate.3, can once screen the situation that a plurality of compositions combine with enzyme, according to the change situation of the NMR signal of each enzyme-added front and back of composition, find out the molecule that combines with enzyme.

Embodiment

Huperzine is at first to separate a kind of natural products that obtains by China scientist from the Chinese herbal medicine Huperzia serrata, after measured, it is the reversible inhibitor of acetylcholinesterase, characteristics such as have the selectivity height, toxicity is low and EDD is long are to treat the comparatively desirable drug candidate of senile dementia so far in the world.

In the present invention, the present inventor with huperzine, the same alkaloid Huperzine B that obtains and the 8-hydroxyl phlegmariurine B of from Huperzia serrata, separating as sample compound.

The huperzine Huperzine B

8-hydroxyl phlegmariurine B

Embodiment 1: the preparation of analytical sample and test

Huperzine, Huperzine B and 8-hydroxyl phlegmariurine B system extract from the medicinal plant serrate clubmoss herb and obtain, and purify with HPLC, and purity is all more than 99%.

Used acetylcholinesterase is Chinese Ding Shi Narcine brasiliensis acetylcholinesterase, is obtained through the separation and purification of affinity chromatography method by the electric ray electric organ, and it decomposes the acetylcholine activity and passes through colorimetric method for determining.

Butyrylcholine esterase: purchase company in Sigma.

It is pure that used analytical reagent is all analysis.

The sample preparation: huperzine is dissolved in the heavy water phosphate buffer of 20-100mM pH7.0, adding contains the 50mM pH7.0 heavy water phosphate buffer of acetylcholinesterase again, is mixed with to contain huperzine 1 μ mol/ml+ acetylcholinesterase 5 * 10 ^-3The enzyme-added sample of μ mol/ml.In addition, replace above-mentioned enzyme liquid as blank, make not enzyme-added sample contain huperzine 1 μ mol/ml with the 50mM pH7.0 heavy water phosphate buffer that does not contain acetylcholinesterase.

Use the same method and prepare the analytical sample of Huperzine B and 8-hydroxyl phlegmariurine B.

NMR analyzes: all experiments are carried out with 298K on the Varian Inova of U.S. Varian company 600NMR spectrometer.Chemical shift is calibrated with DSS (4,4-dimethyl-4-sila penta sodium sulfonate).It (is effective, the easiest method that the relaxation time of proton is used upset restoring method mensuration.Relaxation rate is tried to achieve by indicial equation.

Embodiment 2: substrate is measured determining of concentration

The difference of the polymerization state of substrate molecule in solution can cause the change of chemical shift, and simultaneously polymerization state also can influence the relaxation time of proton in the substrate, thereby has influence on the mensuration of the relaxation rate after enzyme-added.

For this reason, the chemical shift of substrate molecule under the variable concentrations when needing measurement not enzyme-added is to determine the substrate polymerization state.Change (Fig. 2) by the H3 chemical shift of proton of observing the huperzine in 0.2～30.0mM scope, discovery is increased with concentration, and the H3 proton chemical shifts is to high field displacement.The generation of aggregative state is described, but under≤1mM concentration, molecule exists with single poly-attitude.So determine under the concentration of≤1mM, to measure the relaxation rate of huperzine, so that the relaxation rate that records is not subjected to the influence of molecule aggregation state.Huperzine B and 8-hydroxyl phlegmariurine B are also measured under similarity condition.

Embodiment 3: the mensuration of substrate molecule movement rate

The non-selective spin lattice speed of proton (hereinafter to be referred as the speed of relaxing) R ^NsThe relaxation rate of each proton when shining all protons in the molecule simultaneously.The selectivity spin lattice speed R of proton ^sBe the relaxation rate of this proton during specific proton in the irradiation molecule.R ^NsAnd R ^sSatisfy following equation:

$R_i^{\mathrm{ns}}=\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\ρ}}_{\mathrm{ij}}+\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\σ}}_{\mathrm{ij}}+{\mathrm{\ρ}}_i^{*}...\left(1\right)$

$R_i^s=\underset{j\≠i}{\mathrm{\Σ}}{\mathrm{\ρ}}_{\mathrm{ij}}+{\mathrm{\ρ}}_i^{*}...\left(2\right)$

In equation (1), (2), ρ ij is H _iWhen after proton is illuminated energy being given other proton of its mutual dipole to the contribution of relaxation rate.σ ij is that two protons of mutual dipole influence each other to the contribution of relaxation rate, ρ after simultaneously illuminated _i ^*For other relaxation mechanism to H _iThe influence of relaxation rate.Relatively (1), (2) can be found out, in (2) formula, and R ^sOwing to only shine H _iProton is not so have other illuminated proton to H _iInfluence item σ ij.

In the present embodiment, we have observed huperzine, Huperzine B and acetylcholinesterase, the H2 when butyrylcholine esterase combines in huperzine, the Huperzine B, H3, H7/H8, the relaxation rate of M10 and M16 proton.Other proton is because chemical shift overlapped (as H8, H11) or be multiplet type (as H6, H14 is an AB system peak type with carbonaceous son), can't select suitable 180 to spend soft pulses and shine and ignore.The relaxation speed value of tested proton is shown in the table 1,2.Experimental result shows, enzyme-added front and back, and chemical shift of proton does not have significant change, that is and, chemical shift of proton is not subjected to enzyme-added influence (concrete data not shown goes out), but the relaxation rate of nearly all proton is enhanced after enzyme-added.And, R ^sStrengthen obviously greater than R ^NsThis be because when micromolecule with after big molecule combine, σ ij value often is a negative value, due to the feasible fast enhancing of relaxation that is brought by ρ ij is cancelled.R ^sObviously strengthen and illustrate that substrate huperzine, Huperzine B have all taken place to combine with enzyme.R ^sInfluence be H7=H3＞H2＞M16＞M10 in proper order, show that the unsaturated carbonyl conjugate ring part in huperzine, the Huperzine B is strong with the albumen effect.Relatively huperzine, Huperzine B respectively with acetylcholinesterase, R when butyrylcholine esterase combines ^s, can find the R that huperzine, Huperzine B combine with acetylcholinesterase ^sThe R that value generally combines with butyrylcholine esterase greater than huperzine, Huperzine B ^sValue illustrates that the bond strength of huperzine, Huperzine B and acetylcholinesterase is bigger.

Embodiment 4: the mensuration of cross relaxation speed and the relevant run duration of molecule

Be further to detect huperzine and the intermolecular interaction of torpedo acetylcholinesterase TnAChE, the method that we use double irradiation has been measured the relevant run duration of molecule.From equation (1), (2) as can be known, the fast σ ij=R that relaxes of the intersection between proton _i ^Ij-R _i ^s, R _i ^IjFor shining H simultaneously _iReach proton H with its coupling _iThe time H that records _jThe speed of speeding of proton.R ^sFor shining H in the irradiation molecule separately _iThe time this proton relaxation rate.When enzyme-to-substrate mutually combined, exists a mobile equilibrium system, set up following equation (3) and (4):

${\mathrm{\σ}}_{\mathrm{obs}}^{\mathrm{ij}}=p_{\mathrm{free}}{\mathrm{\σ}}_{\mathrm{free}}^{\mathrm{ij}}+p_{\mathrm{bound}}{\mathrm{\σ}}_{\mathrm{bound}}^{\mathrm{ij}}...\left(4\right)$

In the formula, p _FreeBe the substrate ratio of desmoenzyme not in the solution, p _BoundBe the ratio of the substrate of desmoenzyme, σ _Free ^IjObserved cross-relaxation rate when not enzyme-added, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates.

When considering σ with the most conservative method _Bound ^IjWith σ _Free ^Ij, promptly set enzyme-to-substrate combination completely, then p taken place _Bound=(protein)/(ligand), p _Free=1-p _BoundCalculate σ according to equation (4) again _Bound ^IjValue, use molecular motion τ correlation time _Bound ^IjWith σ _Bound ^IjBetween relational equation (5), can draw substrate and enzyme in conjunction with after the relevant run duration of molecule.

In the formula, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates, σ _Bound ^IjBe the cross-relaxation rate under the pure enzyme bonding state that calculates, σ _Free ^IjObserved cross-relaxation rate when not enzyme-added, p _BoundBe the ratio of the substrate of desmoenzyme, γ is the magnetic rotation ratio of proton,

Be the modified value of Plank ' s constant, r _IjBe proton H _iAnd the distance between Hj, be H _iMove correlation time with Hj, ω is the Larmor frequency of proton.In this test, and our three group protons adjacent to huperzine (H2-H3, H7-H8 H10-H11) has carried out the double irradiation test, the results are shown in Table 3, wherein, cross-relaxation rate variable Δ σ ^IjBe σ _Obs ^IjWith σ _Free ^IjBe the difference of the two.As can be seen, cross-relaxation rate σ ^IjNext less from unbound state on the occasion of a bigger negative value that changes under the bonding state, illustrate that huperzine combines its molecular motion related rate of back and slows down with TnAChE, make that ω τ is that motion correlation time under the unit interval is from being converted into greater than 1 less than 1, here we calculate molecular motion correlation time as a reference with the H2-H3 proton ${\mathrm{\σ}}_{\mathrm{bound}}^{23}=-11.2s^{-1},$ According to equation [5], calculate ${\mathrm{\τ}}_{\mathrm{bound}}^{23}=40.5$ Nanosecond (ns) is compared (τ is generally in psec (ps) scope) with combined state not, illustrates that huperzine and TnAChE have taken place to combine.

Comparative example 1: negative control test

When biological micromolecule adheres to protein surface, but not when being incorporated into the avtive spot of albumen, be subjected to the influence of albumen, influence micromolecular relaxation properties thereby can cause micromolecular motility that certain change takes place equally.In order to observe the avtive spot whether huperzine is incorporated into albumen veritably, get rid of because the illusion that the relaxation properties that adhesion causes changes, we observed from serrate clubmoss herb, get equally another with the huperzine molecular size is similar but the alkaloid that structure is different-8-hydroxyl phlegmariurine B in the change of the proton relaxation time that adds electric ray acetylcholinesterase front and back.Use and above-mentioned same experimental technique, the result shows, add before and after the TnAChE, and the 7H of 8-hydroxyl phlegmariurine B, the relaxation properties end of 16Me changes, when 8-hydroxyl phlegmariurine B does not combine with TnAChE, the R of 7H ^s=1.211 ± 0.052s ^-1, the R of 16Me ^s=1.584 ± 0.064s ^-1, 8-hydroxyl phlegmariurine B is with after TnAChE combines, the R of 7H ^s=1.119 ± 0.0346s ^-1, the R of 16Me ^s=1.546 ± 0.076s ^-1, relatively do not have significant difference in conjunction with the front and back result.Illustrate that 8-hydroxyl phlegmariurine B with TnAChE combination does not take place.

Table 1. have or do not have TnAChE (5 μ M) and butyrylcholine esterase (5 μ M) in the presence of the huperzine (1mM) that records ¹H NMR P parameter (600MHz, solvent: D ₂O, pH 7.0, T=298K)

		Contrast		Acetylcholinesterase		Butyrylcholine esterase
								Proton	δ(ppm)	R ns(s -1)	R s(s -1)	R ns(s -1)	R s(s -1)	R ns(s -1)	R s(s -1)
H2	6.57	0.51	0.36	0.54	0.57	0.52	0.44
								H3	7.89	0.66	0.48	0.68	0.74	0.57	0.58
H7	3.82	1.24	0.95	1.27	1.31	1.29	1.06
								M10	1.76	1.26	1.04	1.21	1.23	1.17	1.10
M16	1.60	1.27	1.15	1.22	1.37	1.24	1.20

Table 2. have or do not have TnAChE (5 μ M) and butyrylcholine esterase (5 μ M) in the presence of the Huperzine B (1mM) that records ¹H NMR P parameter (600MHz, solvent: D ₂O, pH 7.0, T=298K)

		Contrast		Acetylcholinesterase		Butyrylcholine esterase
								Proton	δ(ppm)	R ns(s -1)	R s(s -1)	R ns(s -1)	R s(s -1)	R ns(s -1)	R s(s -1)
H2	6.57	0.51	0.36	0.54	0.57	0.52	0.44
								H3	7.89	0.66	0.48	0.68	0.74	0.57	0.58
H7	3.82	1.24	0.95	1.27	1.31	1.29	1.06
								M10	1.76	1.26	1.04	1.21	1.23	1.17	1.10

Table 3. have or do not have acetylcholinesterase (5 μ M) in the presence of the huperzine (1mM) that records ¹The relevant run duration of H NMR cross relaxation speed (600MHz, solvent: D with molecule ₂O, pH 7.0, T=298K)

Proton is right	σ free ij(s -1)	σ obs ij(s -1)	Δσ ij(s -1)	σ bound ij(s -1)
					H 2-H 3	0.050	-0.006	-0.056	-11.2
H 7-H 8	0.055	0.032	-0.023	-4.6
					H 10-H 11	0.115	0.020	-0.095	-19.0

Claims (8)

1. method of utilizing nuclear magnetic resonance screening acetylcholinesteraseinhibitors inhibitors, it is characterized in that, it is by in the solution of mensuration as the sample compound of substrate, add acetylcholinesterase, the relaxation rate of enzyme acetylcholine front and back is changed, measure substrate molecule movement rate, cross-relaxation rate, the relevant run duration of molecule, determine that the sample compound that the selectivity relaxation rate accelerates is an acetylcholinesteraseinhibitors inhibitors.

2. method according to claim 1 is characterized in that, is heavy water phosphate buffering liquid concentration 20～100mM of 50 μ MPH=7.0 as the solution of the sample compound of substrate.

3. method according to claim 1 is characterized in that, adds acetylcholine esterase before the solution of the sample compound of substrate, measures the chemical shift of substrate molecule under variable concentrations when not enzyme-added, determines the substrate polymerization state.

4. method according to claim 1 is characterized in that, described assay method is the upset restoring method.

5. method according to claim 2 is characterized in that, behind the adding acetylcholinesterase, the increment of the proton selectivity spin lattice speed Rs of described acetylcholinesteraseinhibitors inhibitors is greater than its non-selective spin lattice speed R ^NsIncrement.

6. method according to claim 1 is characterized in that, when adding acetylcholinesterase, and the proton selectivity spin lattice speed R of the proton selectivity spin lattice speed Rs of described acetylcholinesteraseinhibitors inhibitors when adding butyrylcholine esterase ^s

7 methods according to claim 1 is characterized in that, behind the adding acetylcholinesterase, described acetylcholinesteraseinhibitors inhibitors is elongated with the relevant run duration ω τ of molecule that the double irradiation method records.

8. method according to claim 5 is characterized in that, described ω τ is from being converted into greater than 1 less than 1.