WO2018059088A1

WO2018059088A1 - Dna encoding molecular library and compound screening method

Info

Publication number: WO2018059088A1
Application number: PCT/CN2017/093564
Authority: WO
Inventors: 李笑宇
Original assignee: 深圳劲宇生物科技有限公司
Priority date: 2016-09-30
Filing date: 2017-07-19
Publication date: 2018-04-05
Also published as: CN107130299A

Abstract

Disclosed is a DNA encoding molecular library, comprising a short-strand DNA of 8-12 bases, wherein one end of the short-strand DNA is connected to an optical cross-linking group, and the short-strand DNA binds to the PCR primer region of the DNA encoding molecule in the DNA encoding molecule library. Also disclosed is a compound screening method of the DNA encoding molecule library: performing a light treatment after incubating the DNA encoding molecule library with a protein target point, then adding a DNA exonuclease for degradation, and obtaining a DNA tag binding to the protein target point; performing a PCR amplification and a DNA sequencing of the DNA tag, and reading the compound binding to the protein target point. The method removes the compound not binding to the protein target point from the system using the selective enzymic degradation method induced by a ligand, and thus can be applied to any protein target point.

Description

DNA coding molecular library and compound screening method

Technical field

The invention belongs to the technical field of biochemistry, and particularly relates to a DNA coding molecular library and a compound screening method.

Background technique

In contemporary drug development, high-throughput, large-scale screening is an indispensable tool for the development of new drugs by constructing a large library of drug candidates for drug targets. The major pharmaceutical companies in the world today have large molecular libraries and large-scale screening platforms for new drug development. However, traditional molecular libraries and screening platforms are costly, have high technical thresholds, and are complicated to manage, seriously restricting the development and application of high-throughput screening. In the past five years, DNA-encoding molecular library technology has gradually developed and become an emerging screening method in drug development. In the library of DNA-encoding molecules, each compound is linked to a specific DNA strand to form a specific barcode that enables specific encoding of the compound. The DNA-encoded molecular library enables high-throughput screening of millions or even billions of scales in a very small system. The screening results can be decoded and analyzed by PCR amplification and DNA sequencing to obtain lead compounds for further drug development. In recent years, DNA coding molecular libraries have been widely recognized and applied in the field of new drug research and development, and become an important supporting technology in the development of new drugs.

The DNA-encoding molecular library is used for drug screening. Most of the targets used are purified proteins. After the protein target is modified, it is immobilized on a solid phase such as magnetic beads, and then incubated with the molecular library. Small molecules that cannot bind to the target protein are eluted and separated from small molecules bound to the protein target. The bound small molecule is eluted, PCR amplified, and DNA sequenced under protein denaturation conditions to read the coding sequence to obtain the chemical structure of the small molecule bound to the target. However, the use of purified, immobilized protein targets limits the range of applications of DNA-encoding molecular libraries. Many other types of drug targets, such as membrane proteins, protein complexes, living cells, pathological tissues, etc., are difficult or impossible to purify. And immobilization, which cannot be used for the screening of DNA-encoding molecular libraries, has become a bottleneck problem in the field.

technical problem

The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a DNA coding molecular library and a compound screening method, aiming at solving the existing DNA coding molecular library for drug screening, only using purified and immobilized protein targets, thereby The technical problem of the application of the DNA-encoding molecular library is limited.

Technical solution

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

In one aspect, the invention provides a DNA encoding molecular library comprising a short chain of 8-12 bases DNA, one end of the short-stranded DNA is linked to a photocrosslinking group, and the short-stranded DNA is combined with a PCR primer region of a DNA-encoding molecule in the DNA-encoding molecule library.

Another aspect of the present invention provides a method for screening a compound encoding a DNA encoding molecule, comprising the steps of:

The above-mentioned DNA encoding molecular library is incubated with a protein target, followed by light treatment, and then added by exonuclease to decompose the DNA tag bound to the protein target;

The DNA tag is subjected to PCR amplification and DNA sequencing, and a compound that binds to the protein target is read.

Beneficial effect

A unique short-chain DNA (PC-DNA) is introduced into the DNA-encoding molecular library provided by the present invention, since all DNA coding has the same PCR primer region at both ends in the DNA-encoding molecular library, PC-DNA is capable of binding to the PCR primer region of all compounds. In this way, it is more advantageous to screen a compound that binds to a protein target from a library of DNA-encoding molecules.

The screening method of the compound of the DNA encoding molecular library provided by the invention completely eliminates the requirement for purification and immobilization of the protein target in the screening of the traditional DNA encoding molecular library. The method no longer relies on physical elution to separate compounds that bind to the protein target and not to the protein target, but instead uses ligand-induced selective enzymatic degradation to remove compounds that do not bind to the protein target from the system. Therefore, in principle, the method can be applied to any protein target. The method has been experimentally proven to be useful for the screening of DNA-encoding molecular libraries of various complex systems such as non-loaded proteins, protein complexes, living cell surface membrane proteins, and cell lysates.

DRAWINGS

1 is a conventional DNA encoding molecular library compound screening method in which a protein target is purified and modified;

2 is a schematic flow chart of a method for screening a DNA-encoding molecular library compound of the present invention;

3 is a schematic view showing the flow of DNA coding carried out by a small molecule capable of binding to a protein target in Example 1 of the present invention;

4a is a molecular library having 1027 different series synthesized in Example 2 of the present invention, which comprises a GLCBS, a CBS, and a CBM in addition to a large number of background sequences; wherein GLCBS is a protein target CA-II High binding force small molecule, CBS is medium combined with small molecules, while CBM is not combined with protein target CA-II, used as a negative control;

4b is a data result of screening a molecular library compound screening method according to Example 2 of the present invention;

Figure 5a is a molecular library having 1027 different series synthesized in Example 3 of the present invention, the library comprising 4800 DNA-encoded macrocyclic polypeptide compounds, and a GLCBS for positive control;

Fig. 5b is a data result of screening the molecular library compound screening method according to Example 3 of the present invention.

Embodiments of the invention

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In one aspect, the embodiments of the present invention provide a DNA encoding molecular library comprising a short-chain DNA (PC-DNA) having 8-12 bases, the structure of which is shown below, and one end of the short-chain DNA is linked A photocrosslinking group, and the short-stranded DNA is combined with a PCR primer region of a DNA-encoding molecule in a DNA-encoding molecule library.

In the above-mentioned DNA-encoding molecular library provided by the embodiment of the present invention, a unique short-chain DNA (PC-DNA) is introduced, since in the molecular library, all DNA coding has the same PCR primer region at both ends, The PC-DNA is capable of binding to the PCR primer region of the DNA-encoding molecule of all compounds (as shown in Figure 2); thus, the use of a compound that binds to a protein target from a library of DNA-encoding molecules is utilized.

Further, in the DNA-encoding molecular library of the embodiment of the present invention, the photocrosslinking group includes at least one of phenyl azide, benzophenone, and propyl acridine. The photocrosslinking group is capable of sending a cross-linking reaction with the protein target to protect the DNA tag of the small molecule capable of binding to the protein target; and if the small molecule does not bind to the protein target, the protein target is cross-linked with the PC-DNA Union cannot happen. The photocrosslinking group of the present invention is not limited thereto.

In another aspect, the embodiment of the present invention further provides a screening method for a compound of a DNA encoding molecular library, and the flow thereof is as shown in FIG. 2, and includes the following steps:

S01: incubated the above-mentioned DNA-encoding molecular library with a protein target, and then irradiating the light, and then adding a DNA exonuclease to degrade a DNA tag that binds to the protein target;

S02: performing PCR amplification and DNA sequencing on the above DNA tag, and reading with a protein target Combined compounds.

The screening method of the above-mentioned DNA encoding molecular library provided by the embodiments of the present invention completely eliminates the requirement for purification and immobilization of protein targets in the screening of traditional DNA encoding molecular libraries (as shown in FIG. 1). The method no longer relies on physical elution to separate compounds that bind to the protein target and not to the protein target, but instead uses ligand-induced selective enzymatic degradation to remove compounds that do not bind to the protein target from the system. Therefore, in principle, the method can be applied to any protein target. The method has been experimentally proven to be useful for the screening of DNA-encoding molecular libraries of various complex systems such as non-loaded proteins, protein complexes, living cell surface membrane proteins, and cell lysates.

Further, in the above step S01, the protein target may be a purified protein and/or a non-purified protein, the protein target may also be a modified protein or a non-modified protein, and the protein target may also be a immobilized protein and/or a non-solid protein. Loading protein. In principle, the method can be applied to any protein target. After incubating a DNA-encoding molecular library with PC-DNA with a non-loaded, unmodified protein target, a portion of the small molecule in the DNA-encoding library can bind to the protein target, causing light on the PC-DNA The cross-linking group is located near the protein target. Under light conditions, the photo-crosslinking group can send a cross-linking reaction with the protein target to protect the DNA tag of the small molecule that can bind to the protein target; if the small molecule does not When the protein target binds, cross-linking of the protein target with PC-DNA cannot occur. The exonuclease I (exoI, exonuclease I) was added to the system. Among them, there is no molecular library compound cross-linked with a protein target, and the DNA tag carried by it will be degraded by exoI; while the molecular library compound cross-linked with the protein target, the DNA tag is protected by the protein target itself, Degraded by exoI. Therefore, after exoI treatment, the remaining DNA in the system is a DNA tag of a small molecule capable of binding to a protein target.

Further, in the above step S01, the protein target is CA-II, and the compound bound to the protein target is GLCBS.

Further, in the above step S02, PCR expansion and DNA sequencing are performed, and the chemical structure of the selected small molecule can be read. PCR amplification was performed by fluorescent quantitative PCR amplification. Real-time quantitative PCR (qPCR) analysis can give C _T values. The smaller the C _T value, the more DNA is retained; the larger the C _T value, the more the DNA has been hydrolyzed by the enzyme and difficult to be amplified by PCR.

The present invention has been subjected to a number of tests in succession, and a part of the test results are now described in further detail as a reference, and will be described in detail below in conjunction with specific embodiments.

Example 1:

This example demonstrates that when a small molecule and a protein target are combined, the DNA coding carried by the small molecule is not cleaved by the enzyme. hydrolysis.

This example selects a series of small molecule compounds whose structures are as shown above. They are ligated to the PC-DNA of the present invention. After these small molecule-DNA conjugates were incubated with their known protein targets, illumination, digestion, and real-time quantitative PCR (qPCR) analysis were performed according to the procedure in FIG. qPCR can give C _T value, the smaller the C _T value, the more DNA remains; the larger the C _T value, the more the DNA has been hydrolyzed by the enzyme, and it is difficult to be amplified by PCR. Table 1 shows the data obtained, wherein the ΔC _T values: No C _T value of the target protein - protein C _T values are target; the larger the value, the stronger the protective effect of the protein target. In Table 1: K _d : dissociation constant, K _i : inhibition constant, both are constants that quantitatively indicate the ability of small molecules to bind or inhibit proteins; nM means nanomolar, nanomolar.

Table 1

The data in Table 1 above shows that for many types of small molecules, their binding to protein targets can effectively protect DNA coding. Even the weaker numbers 4 and 5 have a good effect. This data verifies the feasibility of the method proposed in this embodiment in principle.

Example 2:

A library of DNA-encoding molecules: The library has 1027 different sequences. Except for a large number of background sequences In addition to the column, it contains a GLCBS, a CBS, and a CBM.

As shown in Figure 4a: GLCBS is a high binding small molecule of protein target CA-II, CBS is moderately bound to small molecules, and CBM is not combined with protein target CA-II and used as a negative control. In this example, the PC-DNA specific to the present invention was introduced into the molecular library for synthesis, and the non-fixed and unmodified protein target CA-II was screened according to the method flow of FIG. 2, and the selected molecules were selected. Library members were subjected to PCR extension and DNA sequencing, and the data is shown in Figure 4b.

It can be seen from the data plot of Figure 4b that after the first round of screening, the strong binding force of GLCBS is enriched by 38.4 times, and the medium binding CBS is enriched by 7.2 times, without binding to the CA-II target. CBM is basically not enriched. One of the characteristics of this screening method is that the molecular library selected in the first round can directly enter the next round for secondary screening to achieve further enrichment. Figure 4b shows that after a second round of screening, GLCBS and CBS were approximately 200-fold enriched.

Example 3:

A library of DNA-encoding molecules: This library contains 4,800 DNA-encoded macrocyclic polypeptide compounds, and a GLCBS for positive control, as shown in Figure 5a.

In this embodiment, after the PC-DNA specific to the present invention is introduced into the molecular library for synthesis, according to the method flow of FIG. 2, after molecular library synthesis and screening of the non-fixed unmodified protein target CA-II, PCR amplification and DNA sequencing were used to decode, and the final data was shown in Figure 5b as a scatter plot. It can be seen from Fig. 5b that the positive control GLCBS is enriched nearly 100 times (Fig. 5b); while other compounds in the molecular library are not substantially enriched due to the lack of chemical structure combined with CA-II (Fig. 5b) under). From the data in Figure 5b, the feasibility of the molecular library screening method proposed by the present invention for non-fixed, unmodified protein target screening was verified.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

A DNA-encoding molecular library comprising a short-stranded DNA having 8-12 bases, one end of the short-stranded DNA linked to a photo-crosslinking group, and the short-stranded DNA and the The PCR primer region of the DNA-encoding molecule in the DNA-encoding molecule library is combined.
The DNA-encoding molecular library according to claim 1, wherein the photocrosslinking group comprises at least one of phenyl azide, benzophenone, and propyl acridine.
A method for screening a compound of a DNA encoding molecular library, comprising the steps of:

The DNA encoding molecular library of claim 1 is incubated with a protein target, followed by light treatment, and then added by exonuclease degradation to obtain a DNA tag that binds to the protein target;

The DNA tag is subjected to PCR amplification and DNA sequencing, and a compound that binds to the protein target is read.
The method for screening a compound of a DNA-encoding molecular library according to claim 3, wherein the photocrosslinking group comprises at least one of phenyl azide, benzophenone, and propyl acridine.
The method for screening a compound of a DNA-encoding molecular library according to claim 3, wherein the protein target is a purified protein and/or a non-purified protein.
The method for screening a compound of a DNA encoding molecular library according to claim 3, wherein the protein target is a modified protein or a non-modified protein.
The method for screening a compound of a DNA-encoding molecular library according to claim 3, wherein the protein target is a immobilized protein and/or a non-loaded protein.
The method for screening a compound of a DNA-encoding molecular library according to any one of claims 3 to 7, wherein the protein target is CA-II, and the compound that binds to the protein target is GLCBS.
The method for screening a compound of a DNA-encoding molecular library according to any one of claims 3 to 7, wherein the PCR amplification is fluorescence quantitative PCR amplification.
The method for screening a compound of a DNA-encoding molecular library according to any one of claims 3 to 7, wherein the exonuclease is exonuclease I.