CN111595888B

CN111595888B - Method for detecting polypeptide drug structure based on high-field nuclear magnetic resonance technology

Info

Publication number: CN111595888B
Application number: CN202010326534.9A
Authority: CN
Inventors: 李红卫
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2021-03-23
Anticipated expiration: 2040-04-23
Also published as: CN111595888A

Abstract

The invention discloses a method for detecting a polypeptide drug structure based on a high-field nuclear magnetic resonance technology, which relates to the field of detection of polypeptide drugs and other functional polypeptide structures.

Description

Method for detecting polypeptide drug structure based on high-field nuclear magnetic resonance technology

Technical Field

The invention relates to the field of detection of polypeptide drugs and other functional polypeptide structures, in particular to a method for detecting a polypeptide drug structure based on a high-field nuclear magnetic resonance technology.

Background

Over 7000 natural polypeptides have been found to play a key role in physiological functions such as human hormonal activities, neuronal conduction, growth factors, ion channel ligands and infection resistance. Polypeptides generally interact as highly selective and potent signaling molecules with cell surface receptors or ion channels, etc., to trigger the biological function of molecules within cells (Padhi a., et al. tubericulosis, 2014,94, 363-. The polypeptide is used as a medicine, and has the characteristics of high safety, high tolerance and high efficiency compared with the traditional small molecule medicine; its product complexity is lower compared to protein drugs. Therefore, polypeptide drugs are an emerging field with strong attractive force. Currently, more than 140 polypeptide drugs are clinically tested (Fosgerau K., et al., Drug Discovery Today,2015,20, 122-128). The research and development and declaration of the medicine are the premise to clearly analyze the structure of the medicine. The polypeptide drug has three-dimensional spatial structure information besides primary sequence structure information, and the spatial structure information can be maintained in a certain system, so that the polypeptide drug has practical use significance only when the spatial structure of the polypeptide drug is obtained under the similar physiological environment.

Liquid nuclear magnetic resonance technology has been widely used in many fields such as biology, chemistry, medicine, agriculture, etc. as a conventional means for identifying compounds. Compared with other structural research means such as an X-ray crystal diffraction technology, a low-temperature cryoelectron microscope technology and the like, a liquid nuclear magnetic resonance technology research system is a liquid environment and is closer to a real system of a research object, which is particularly important for polypeptide drugs, namely, the liquid nuclear magnetic resonance technology can complete the research on the spatial structure of the polypeptide drugs in an approximate physiological environment. At present, methods for researching polypeptide drug structures by using nuclear magnetic resonance instruments mainly comprise two main types: one category is to consider it as a traditional drug small molecule, which is done using unlabeled samples and simple one-and two-dimensional nuclear magnetic experiments. The method has the advantages that the samples and experiments are simple, the cost is low, but compared with small molecules, the molecular weight of the polypeptide is large, the types of amino acids are not many, the signal overlapping is serious, a three-dimensional structure calculation method is lacked, a real three-dimensional space structure cannot be obtained finally, and the result is often expressed in the form of a nuclear magnetic resonance spectrogram of a comparison sample and a standard product. The other is to study the polypeptide structure by a method for studying the protein structure by nuclear magnetic resonance, and analyze the three-dimensional structure by using an isotope-labeled sample and a nuclear magnetic resonance three-dimensional experiment. Such methods have the advantage that a good three-dimensional structure is obtained once the sample is taken, but the polypeptide sample needs to be isotopically labelled. If the labeling is carried out by a biosynthesis method, not only the cost is high, but also the purification process is often more complicated than the protein purification, if the labeling is carried out by a chemical synthesis method, all reagents used need to be labeled by isotopes, and the cost is too high for most researchers to bear in consideration of the synthesis yield of each step. In addition, the method needs to acquire a three-dimensional nuclear magnetic resonance experiment, the used time is more, more than one week is needed for one sample, and the test cost is higher.

Disclosure of Invention

The invention aims to provide a method for detecting a polypeptide drug structure based on a high-field nuclear magnetic resonance technology, which can obtain a three-dimensional space structure of a polypeptide drug by using a low-cost, simple and unmarked sample and simple one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance experiments.

Aiming at the above purpose, the invention is realized by the following technical scheme:

a method for detecting a polypeptide drug structure based on a high-field nuclear magnetic resonance technology comprises the following steps:

and (3) spectrogram acquisition and analysis: nuclear magnetic resonance 2D for collecting polypeptide sample¹H-¹H TOCSY Spectrum, 2D¹H-¹H COSY spectrum, 2D¹H-¹³C HSQC spectra and 2D¹H-¹H NOESY spectrum; analyzing the spectrogram, extracting chemical shift attribution and spatial distance constraint of the polypeptide, and extracting dihedral angle constraint according to the chemical shift attribution

And (3) structural calculation: analyzing distance constraint information between hydrogen atoms extracted from the NOESY spectrogram, calculating to obtain a structure of the polypeptide sample in vacuum as an initial structure, and optimizing the initial structure to obtain a spatial structure of the polypeptide sample in the presence of a solvent; and counting the use condition of the distance constraint according to the space structure, calculating the root mean square deviation, the energy data and the Laplace graph data of the structure, and counting the angle of the structure and the default condition of the distance constraint to obtain the three-dimensional structure of the polypeptide medicament.

Further, the polypeptide sample collection method comprises the following steps: dissolving the polypeptide solid powder in different buffer systems to prepare polypeptide samples of different buffer systems, and finishing the nuclear magnetic resonance 1D of all the polypeptide samples¹H spectrum experiment, analyzing and comparing 1D under different buffer systems¹H spectrum, selecting the sample with the signal meeting the requirement to perform the subsequent experiment, and improving the resolution of the acquired 2D spectrogram.

Further, the dihedral angle constraints were extracted from the chemical shift assignments using TALOS (Cornilescu G.et al, J.biomol.biol.,1999,273,283-

Further, the distance constraint information between hydrogen atoms extracted in the NOESY spectra was analyzed using the method of SANE (Duggan B., et al., J.Biomol.biol.,2001,19, 321-329); optimization of CYANA (Guntert P, et al. J. mol. biol.,1997,273, 283) software and AMBER (Pearlman D.A., et al., Compult. Phys. Co., Ltd.)mmun.,1995,91,1-41) software input file format to enable adaptation to 2D NOESY¹H-¹And H distance constraint is used, the structure of the polypeptide sample in vacuum is calculated by using CYANA software to be used as an initial structure, and AMBER software is used for optimizing the initial structure to obtain the spatial structure of the polypeptide sample in the presence of a solvent.

Further, the pH of the buffer system should be neutral or weakly acidic, and it is preferable that the system does not contain H atom-containing molecules other than water molecules, and it is preferable to use water or PBS buffer system of different salt concentration as it is.

Further, all the distance information including NH and CH is used in extracting the distance constraint information between hydrogen atoms.

Further, the concentration of the polypeptide sample is greater than 1 mM.

Further, depending on the polypeptide sample, the instrumentation is typically required to be above 700 MHz.

Further, the signal compliance includes: the full width at half maximum of the H signal is narrow, namely the value of the H signal is matched with the molecular weight of the polypeptide; good dispersibility, i.e. less signal overlap, in particular backbone amino groups having chemical shifts in the range of 6-10ppm¹The number of H signals should exceed 80% of the number of amino acids, and overlap is particularly small.

Further, the mixing time of the TOCSY experiment is set to 80ms, the mixing time of the NOESY experiment is about 300ms (200- & 400ms), the mixing time is reduced when the molecular weight is larger, or vice versa, spectrograms of different mixing times can be collected for comparison during the experiment collection, the NOE signal of the experiment with strong NOE signal and no signal obviously more than the mixing time is selected as the best, and the NOESY experiment is replaced by the ROESY experiment if the NOESY signal is weak and the number of samples is small.

Furthermore, when chemical shift assignment is carried out, the types and chemical shifts of amino acid residues are identified by using TOCSY, COSY and HSQC, and main chain amino H and front and back amino acid alpha positions of all amino acid residues can be found in NOESY spectrogram¹The NOE signal of H, combined with NOESY experiments to determine sequence linkage information between amino acid residues.

Further, the using condition of the distance constraint comprises the using quantity of the determined constraint and the using quantity of the distance constraint of the fuzzy calculation, and the using quantity of the determined constraint comprises the using quantity of the medium-range and long-range distance constraints; the rms deviation of the structure includes the rms deviation of the main chain atoms and heavy atoms in the molecule; the energy data includes calculated amber energies, breach energies of distance constraints and angle constraints.

The method firstly uses a high-field nuclear magnetic resonance instrument (not less than 700MHz, and specifically according to the conditions of the molecular weight of the sample and the like) to collect 1D (1D) of the sample under different buffer systems¹H spectrum, the condition of the buffer system of the space structure of the polypeptide drug can be best maintained by analyzing the spectrogram. Secondly, 2D collection of polypeptide drugs¹H-¹H TOCSY Spectrum, 2D¹H-¹H COSY spectra and 2D¹H-¹³CHSQC spectrum, and analyzing the spectrum to obtain the chemical shift attribution of the polypeptide drug structure. Acquisition of 2D¹H-¹And extracting distance constraint information from the H NOESY spectrogram. And finally, optimizing input scripts of CYANA and AMBER, so that the input scripts can be suitable for data analysis and calculation of a two-dimensional spectrogram, and finally analyzing a three-dimensional polypeptide drug structure and summarizing result statistics results.

Compared with the prior art, the invention has the following beneficial effects:

compared with the two traditional methods mentioned above, the method uses a low-cost and easily-obtained polypeptide sample for detection, the polypeptide sample does not need to be specially marked, and the method can meet the requirement of analyzing the three-dimensional structure by only spending less time to collect a small amount of spectrogram, so that the time and cost for preparing the sample and analyzing the nuclear magnetic spectrogram are greatly saved. And by optimizing the specific calculation method, the analysis of the three-dimensional structure of the unmarked sample can be completed by using the software for calculating the protein structure, so that the three-dimensional space structure of the polypeptide drug which is closer to the physiological condition is obtained, and reliable three-dimensional structure information is provided for the research, development, declaration, examination and approval and the like of the drug.

Drawings

FIG. 1 is a flow chart for analyzing the three-dimensional structure of a polypeptide drug.

FIG. 2 is the 1D of the ziconotide tested in the examples¹And H, spectrum.

FIG. 3 is an example of a distance constraint file after CYANA and AMBER software optimization.

Figure 4 is a three-dimensional block diagram of ziconotide tested in the examples.

Detailed Description

The present invention will be further described with reference to the following examples.

With the development of nuclear magnetic resonance hardware, especially the increase of the field intensity of an instrument, the resolution of a nuclear magnetic resonance experiment is greatly improved, and the signal overlapping can be effectively reduced, so that the invention adopts a high-field instrument to collect high-resolution one-dimensional and two-dimensional nuclear magnetic resonance experiments, optimizes the method for calculating the protein structure and introduces the optimized method into the experiment, and can possibly use a low-cost simple unmarked sample to obtain the three-dimensional space structure of the polypeptide drug, as shown in figure 1. Nuclear magnetic resonance 1D¹The H spectrum can measure the chemical shift of hydrogen signal, 2D¹H-¹The H TOCSY spectrum can measure the related signals of all hydrogen and hydrogen in a spin system, 2D¹H-¹H COSY spectrum can detect the related signal of hydrogen and hydrogen within three chemical bonds, 2D¹H-¹³The C HSQC spectra can measure all hydrogen-carbon related signals directly connected by chemical bonds, and the experiments can be completed within two days by using low-cost, simple and unmarked samples. By analyzing the spectra, the main chain and the side chain of the polypeptide sample can be analyzed¹H and¹³chemical shifts of the C atom are assigned. Recombination of 2D that can measure all hydrogen atom spatial distance information¹H-¹The H NOESY experiment result can finally calculate the three-dimensional space structure of the polypeptide by using CYANA, AMBER and other calculation software. In this process, although the high-field nuclear magnetic resonance apparatus increases the time cost per unit time, the total number of hours used is greatly reduced, and thus the overall test cost is not high.

An example is listed below:

1. sample preparation:

the unlabelled ziconotide salt-containing sample 100mg was dissolved in 500. mu.L of pure water containing 10% by weight of water and the internal standard DSS, and since the sample provided already contained salt, the supplier required that the structure be determined according to the salt-containing conditions given, optimization of the buffer system was no longer performed.

2. Sample data collection and analysis:

1D acquisition on a 800MHz NMR instrument¹H spectrum, shown in FIG. 2, amino groups on the polypeptide backbone¹The signal intensity of the H (6-10 ppm) area is strong, the signal quantity is consistent with the number of amino acids, the signal dispersion is good, no overlapping exists, the half-height width accords with the molecular weight of ziconotide, no obvious broadening exists, and aggregation does not exist, so that the quality of a subsequent two-dimensional spectrogram is influenced, and therefore the sample is judged to have a good state and is suitable for further three-dimensional structure analysis. Further acquisition of 2D¹H-¹H TOCSY,¹H-¹HCOSY and¹H-¹³c HSQC and¹H-¹h NOESY spectrum, experiment temperature 298K, TOCSY experiment and NOESY experiment mixing time of 80ms and 300ms respectively. The chemical shift of the hydrogen atom and carbon atom of the main chain and side chain of the amino acid residue can be assigned by using the results of TOCSY, COSY and HSQC experiments, and the information of the amino acid sequence can be obtained by using the results of NOESY experiments, particularly NOESY signals between the main chain amino hydrogen and alpha side chain hydrogen, so that the chemical shift assignment of the polypeptide medicament is completed. And extracting all distance constraint information in the NOESY spectrogram for later use.

Except for the moiety on the side-chain aromatic ring¹H and¹³all main and side chains other than the C atom¹H atoms and all but C.alpha.of C8, C15, C25 (signals are suppressed due to their H.alpha.chemical shifts close to that of water)¹³The chemical shifts of the C atoms are assigned. Wherein the chemical shifts of C beta of C1, C8, C15, C16, C20 and C25 are 39.47ppm,40.06ppm,37.73ppm,37.58ppm,41.07ppm and 42.16ppm respectively, which shows that the cysteines exist in the form of disulfide bonds. Whether the 6 Cys residues of ziconotide form disulfide bonds directly determines the structural stability and further determines the activity of the drug, the chemistry of the C beta of cysteineThe shift can reflect whether or not it exists in the form of a disulfide bond.

3. Structural calculations and statistical data summarization:

constraints for structural computation include passing through 2D¹H-¹Spatial distance constraints from H NOESY and dihedral angle constraints predicted by chemical shifts using TALOS software

The spatial distance constraint only contains NOE information of HH in two dimensions, and the text format is modified for subsequent structure calculation. The structure was calculated using CYANA software and further optimized using AMBER software, and FIG. 3 is an example of a post-optimization distance constraint file. The 20 initial structures with the lowest energy are generated by calculation by using a CANDID module in CYANA software, and are applied to NOE attribution using SANE software. The CYANA calculation results in 200 structures, and 100 structures with the lowest energy are selected as initial structures for refining the structures in the AMBER. Finally, 20 structures were selected to represent the structure of the sample. The final structure was analyzed using MOLMOL and PROCHECK NMR software.

The resulting three-dimensional structure is shown in fig. 4. On the left, the screened 20-structure sets are represented, and on the right, one of the structures is represented by a drift band diagram, in which the side chain information of all cysteines is marked. The statistics of the structure are shown in table 1. From the three-dimensional structure chart, the ziconotide exists in a loop form, and the loop is firmly fixed by three pairs of disulfide bonds, so that the whole structure presents a better convergence state. The disulfide bonds are formed between cysteine C1 and C16, C8 and C20, C15 and C25, and the distances between the S gamma are respectively

And

the bond length range of disulfide bonds is met.

TABLE 1 statistics of three-dimensional Structure calculations for ziconotide

The above-described embodiments are not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for detecting a polypeptide drug structure based on a high-field nuclear magnetic resonance technology is characterized by comprising the following steps:

nuclear magnetic resonance 2D for collecting polypeptide sample¹H-¹H TOCSY Spectrum, 2D¹H-¹H COSY spectrum, 2D¹H-¹³C HSQC spectra and 2D¹H-¹H NOESY spectrum, extracting chemical shift attribution and space distance constraint of the polypeptide by analyzing the spectrogram, and extracting dihedral angle constraint according to the chemical shift attribution; identifying the types and chemical shifts of amino acid residues by using TOCSY, COSY and HSQC when assigning chemical shifts, and finding out main chain amino H and front and back amino acid alpha positions of all amino acid residues from NOESY spectrogram¹H, determining sequence connection information among amino acid residues by combining NOESY experiments;

analyzing distance constraint information between hydrogen atoms extracted from the NOESY spectrogram, calculating to obtain an initial structure of the polypeptide sample in vacuum, and obtaining a spatial structure of the polypeptide sample in the presence of a solvent according to the initial structure;

and counting the use condition of the distance constraint, the angle of the structure and the default condition of the distance constraint according to the space structure, and calculating the root mean square deviation, the energy data and the Laplace graph data of the structure to obtain the three-dimensional structure of the polypeptide medicament.

2. The method of claim 1, wherein dihedral angle constraints are extracted from chemical shift assignments using TALOS software; analyzing distance constraint information between hydrogen atoms extracted from the NOESY spectrogram by using a SANE method; optimizing input file formats of CYANA software and AMBER software, calculating by using the CYANA software to obtain an initial structure of the polypeptide sample in vacuum, and optimizing the initial structure by using the AMBER software to obtain a spatial structure of the polypeptide sample in the presence of a solvent.

3. The method of claim 1, wherein the polypeptide sample is prepared by dissolving the polypeptide solid powder in different buffer systems, and performing nuclear magnetic resonance 1D on all the polypeptide samples¹H spectrum experiment by comparing 1D under different buffer systems¹H spectrum, selecting samples with signals meeting the requirements; the buffer system has neutral or weakly acidic pH and contains no H atom-containing molecules other than water molecules, and comprises water or PBS buffer system.

4. The method of claim 1, wherein all distance information including NH and CH is used in extracting distance constraint information between hydrogen atoms.

5. The method of claim 1, wherein the concentration of the polypeptide sample is greater than 1 mM.

6. The method of claim 1, wherein the nmr cycle is above 700 MHz.

7. The method of claim 3, wherein signal compliance comprises: the half-height width of the H signal is narrow, the dispersity is good, the half-height width of the H signal means that the half-height width of the H signal is matched with the molecular weight of the polypeptide, and the dispersity is good and comprises main chain amino groups with chemical shift of 6-10ppm¹The number of H signals is more than 80% of the number of amino acids.

8. The method of claim 1, wherein the mixing time for the TOCSY experiment is set to 80ms and the mixing time for the NOESY experiment is set to 200-400 ms.

9. The method of claim 1, wherein the usage of the distance constraints comprises a number of uses of determined constraints and a number of uses of fuzzy-computed distance constraints, the number of uses of determined constraints comprising a number of uses of medium-and long-range distance constraints; the rms deviation of the structure includes the rms deviation of the main chain atoms and heavy atoms in the molecule; the energy data includes calculated amber energies, breach energies of distance constraints and angle constraints.