A method for the synthesis of a library of dual pharmacophore DNA-encoded compounds

Abstract

The present application relates to the technical field of DNA coded compound library, and particularly relates to a synthesis method of double-pharmacophore DNA coded compound library, a reversible covalent Y-shaped DNA starting fragment is constructed, and the double-pharmacophore DNA coded compound library is synthesized; controllable transformation of the double-pharmacophore DNA coded compound library and the single-pharmacophore DNA coded compound library is carried out; the DNA coded compound library is compatible with various screening systems; the single-pharmacophore DNA coded compound library is used for constructing a dynamic combination compound library, and a covalent crosslinking group is used for balancing and locking the dynamic structure; the single-pharmacophore DNA coded compound library is combined with other single-pharmacophore compound libraries to construct a self-assembly multi-pharmacophore library; and the single-pharmacophore DNA coded compound library is paired and combined with a functional DNA conjugate through DNA hybridization and covalent crosslinking. The method simultaneously improves the efficiency of synthesis of the DNA coded compound library and the flexibility of screening through the reversible covalent Y-shaped DNA starting fragment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of DNA encoded compound library, and particularly relates to a synthesis method of a dual-pharmacophore DNA encoded compound library. BACKGROUND

[0002] In the field of new drug development, high-throughput screening for biological targets is one of the main means to quickly obtain lead compounds. However, the traditional high-throughput screening based on a single molecule requires a long time, huge equipment investment, limited number of compound libraries (hundreds of millions), and decades of accumulation to build a compound library, which limits the efficiency and possibility of discovering lead compounds. In 1992, Professors Sydney Brenner and Richard Lerner of Scripps Research Institute proposed the concept of DNA encoded compound library (DNA Encoded Library, DEL for short). This method connects an organic small molecule reagent with a unique sequence of DNA at the molecular level (i.e., DNA labeling of small molecule reagents), uses the “combination-splitting” strategy of combinatorial chemistry to quickly construct a huge compound library through two or more cycles, each compound in the compound library is composed of different organic small molecule reagent residues, and has a corresponding unique base sequence of DNA identification. DNA encoded compound library combines combinatorial chemistry and molecular biology technology, adds a DNA tag to each compound at the molecular level, can synthesize a compound library with a size of up to one hundred million in a very short time, and the compounds can be identified by gene sequencing, greatly increasing the size and synthesis efficiency of the compound library, and becoming the trend of the next generation of compound library screening technology. DNA encoded compound library technology is beginning to be widely used in the pharmaceutical industry and has produced many positive effects (Accounts of Chemical Research, 2014, 47, 1247-1255).

[0003] In the development of DNA-encoded chemical library drug screening technology, the synthesis and screening of DNA-encoded chemical library are two key points, which often determine the effectiveness of drug screening. Generally, the higher the complexity of the compound library, that is, the more different structural elements and chemical structures exist, the greater the possibility of discovering molecules with target activity through the compound library. Therefore, during the synthesis of the compound library, companies such as GlaxoSmithKline (formerly Praecis) use a "headpiece" with a stable covalent "hairpin" structure as a starting fragment, combined with the "split-pool-split" strategy to realize effective encoding and synthesis of the compound library; Neri team developed the technology of DNA-encoded self-assembling library (encoded self-assembling chemical, ESAC), based on the complementary self-assembly of DNA strands between two or three single-stranded DNA-encoded chemical libraries, to realize the rapid expansion of chemical structure and chemical diversity of the compound library, and to realize affinity maturation, expand the screening target range, etc. However, due to the synthesis of single-stranded DNA-encoded chemical library in the ESAC technology, the cost and complexity of auxiliary DNA reagent chains are increased, the stability of the non-covalent binding system is low, DNA mismatches may occur, and information transfer operation is required for decoding operation, etc. Therefore, it is of great technical and application significance to solve the efficient synthesis technology of double pharmacophore DNA-encoded chemical library. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a synthesis method of double pharmacophore DNA-encoded chemical library, which improves the efficiency of DNA-encoded chemical library synthesis and the flexibility of screening by constructing a reversible covalent "Y-type" DNA starting fragment.

[0005] The present application solves the above technical problems by the following technical means:

[0006] The present application discloses a synthesis method of double pharmacophore DNA-encoded chemical library, comprising the following steps:

[0007] Constructing a reversible covalent "Y-type" DNA starting fragment, forming orthogonal encoding of DNA-encoded fragments with different cohesive ends, synthesizing double pharmacophore DNA-encoded chemical library;

[0008] Controlling the transformation of double pharmacophore DNA-encoded chemical library and single pharmacophore DNA-encoded chemical library, realizing effective decoding of double pharmacophore DNA-encoded chemical library and single pharmacophore DNA-encoded chemical library;

[0009] The DNA-encoded compound library is compatible with various screening systems, and the target screening range of the DNA-encoded compound library is expanded, which includes double-pharmacophore DNA-encoded compound library screening and single-pharmacophore DNA-encoded compound library screening;

[0010] The single-pharmacophore DNA-encoded compound library is dynamically combined to construct a dynamic DNA-encoded compound library, and the dynamic structure is balanced and locked by using a covalent reversible cross-linking group;

[0011] The single-pharmacophore DNA-encoded compound library is combined with other single-pharmacophore compound libraries to construct a self-assembled multi-pharmacophore library.

[0012] The single-pharmacophore DNA-encoded compound library is combined with functional compound DNA conjugates through DNA hybridization and covalent cross-linking to realize multifunctionalization of the DNA-encoded compound library.

[0013] In some optional embodiments, the construction of the reversible covalent "Y-type" DNA starting fragment comprises the following steps:

[0014] Three single-stranded DNAs with X cross-linking groups are synthesized, respectively, and the three single-stranded DNAs are single-stranded ssDNA-T, single-stranded ssDNA-B, and single-stranded ssDNA-R, and the X cross-linking group is a special cross-linking group or a natural base;

[0015] Strategy one for constructing the reversible covalent "Y-type" DNA starting fragment:

[0016] Two single-stranded DNAs containing special cross-linking groups and complementary single-stranded DNAs not containing special cross-linking groups are combined through covalent cross-linking chemical reactions to form a reversible covalent "Y-type" DNA starting fragment;

[0017] The "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction;

[0018] Strategy two for constructing the reversible covalent "Y-type" DNA starting fragment:

[0019] Three single-stranded DNAs with X cross-linking groups are synthesized, respectively, and the X cross-linking group is a special cross-linking group or a natural base;

[0020] One single-stranded DNA containing a special cross-linking group and a complementary single-stranded DNA not containing a special cross-linking group are combined through a covalent cross-linking chemical reaction to form an intermediate DNA with a covalent structure; and another complementary single-stranded DNA containing a special cross-linking base is combined through a covalent cross-linking chemical reaction to form a reversible covalent "Y-type" DNA starting fragment;

[0021] The "Y-type" DNA initiator fragment formed by covalent cross-linking is dissociated into single-stranded initial state by covalent dissociation chemical reaction;

[0022] The construction strategy of the reversible covalent "Y-type" DNA initiator fragment

[0023] Three single-stranded DNAs with X cross-linking groups are synthesized respectively, and the X cross-linking groups are special cross-linking groups or natural bases;

[0024] The three single-stranded DNAs containing special cross-linking groups are formed into a novel reversible covalent "Y-type" DNA initiator fragment by covalent cross-linking chemical reaction;

[0025] The "Y-type" DNA initiator fragment formed by covalent cross-linking is dissociated into single-stranded initial state by covalent dissociation chemical reaction;

[0026] The construction cycle of the reversible covalent "Y-type" DNA initiator fragment is not less than 50 times;

[0027] The reversible covalent "Y-type" DNA initiator fragment contains at least two reaction sites R for compound library synthesis, and the reaction site R is connected with single-stranded DNA through a linker L.

[0028] In some optional embodiments, each of the single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R has a complementary region, and the length of each complementary region is 0-30 bases. At least one of the single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R contains at least one X cross-linking group.

[0029] The X cross-linking group is located on the reversible covalent "Y-type" DNA initiator fragment, and the X cross-linking group is any one of the following special cross-linking groups 1-12:

[0030]

[0031] The natural base is any one of adenine, guanine, cytosine and thymine.

[0032] In some optional embodiments, the controllable conversion of the dual-pharmacophore DNA-encoded compound library and the single-pharmacophore DNA-encoded compound library includes the following steps:

[0033] The "split-pool-split" strategy is used to realize the synthesis of the DNA-encoded compound library;

[0034] Reversible covalent "Y-type" DNA initiator fragments are reacted with DNA-compatible chemistries at the chemical structure motifs, which are any one of carboxylic acid, aldehyde, alkene, amine, boronic acid, halide;

[0035] DNA tags corresponding to each chemical structure motif are added, and the DNA sticky ends are ligated by DNA ligase or chemical methods; or after adding one chemical structure motif at each of the two chemical reaction sites of the "Y-type" DNA initiator fragment, DNA tags with different sticky ends corresponding to the two are added together, and the DNA sticky ends are ligated by DNA ligase or chemical methods;

[0036] The connection order of DNA tags and chemical structure motifs in each dimension can be exchanged, and the same dimension samples are mixed and then divided, and the next dimension synthesis is performed;

[0037] After the synthesis of the dual pharmacophore DNA encoded compound library is completed, the special "Y-type" structure is opened by destroying the covalent bond, and selective degradation conditions are used to degrade or separate the DNA strands that do not connect the encoded compounds in the library construction process, and the conversion of the dual pharmacophore DNA encoded compound library and the single pharmacophore DNA encoded compound library is completed.

[0038] In some optional embodiments, the dual pharmacophore DNA encoded compound library screening includes the following steps:

[0039] The dual pharmacophore DNA encoded compound library screening strategy I:

[0040] The known active compounds are integrated into any single pharmacophore compound library to synthesize a dual pharmacophore DNA encoded compound library;

[0041] The dual pharmacophore DNA encoded compound library is incubated with the related target of the active compound for screening;

[0042] The compound library members that do not bind to the target or have weak affinity are separated by selective degradation conditions, and the compound library members that have stronger affinity to the target are enriched;

[0043] The structure information of the enriched compound library members is decoded by polymerase chain reaction and DNA sequencing technology.

[0044] The dual pharmacophore DNA encoded compound library screening strategy II:

[0045] The protein target is used as the screening substrate of the dual pharmacophore DNA encoded compound library;

[0046] The dual pharmacophore DNA encoded compound library is incubated with the target for screening;

[0047] The members of the compound library that do not bind to the target or have weak affinity are separated by selective degradation conditions, and the members of the compound library that have strong affinity to the target are enriched;

[0048] The structural information of the enriched members of the compound library is decoded by polymerase chain reaction and DNA sequencing technology.

[0049] In some alternative embodiments, the single-pharmacophore DNA-encoded compound library screening further comprises the following steps:

[0050] The single-pharmacophore DNA-encoded compound library screening strategy one:

[0051] The single-pharmacophore DNA-encoded compound library is hybridized with DNA probes with chemical reactivity;

[0052] When the compounds with affinity in the DNA-encoded compound library bind to the target, the distance between the DNA probe and the target is shortened, promoting the covalent cross-linking of the DNA probe and the target;

[0053] The compound DNA conjugates that do not bind to the target are separated by selective degradation conditions, and the DNA compound conjugates that bind to the target are enriched;

[0054] The structural information of the compounds is decoded by polymerase chain reaction and DNA sequencing technology;

[0055] The single-pharmacophore DNA-encoded compound library screening strategy two:

[0056] The single-pharmacophore DNA-encoded compound library is incubated with the target connected with PCR primer sequence DNA, the hybrid structure of the compound library members with affinity to the target and the DNA primer sequence connected to the target is PCR amplified, and the structural information of the compounds with high affinity to the target is obtained;

[0057] The single-pharmacophore DNA-encoded compound library screening strategy three:

[0058] The single-pharmacophore DNA-encoded compound library is hybridized with DNA probes with chemical reactivity and covalently cross-linked;

[0059] When the compounds with affinity in the compound library bind to the target, the distance between the DNA probe and the target is shortened, promoting the covalent cross-linking of the DNA probe and the target, realizing the covalent cross-linking complex of the single-stranded DNA connected with the compound library information, the DNA probe with chemical reactivity, and the target;

[0060] The target is used for in vitro binding experiment, and the covalent complex is separated, and the structure information of the compound with high affinity to the target is obtained by DNA decoding.

[0061] In some alternative embodiments, the selective degradation condition is any one of exonuclease, chemical reaction condition, separation and capture, and the exonuclease is any one of ExoI, ExoIII and lambda Exo.

[0062] In some alternative embodiments, the construction of the dynamic combination compound library further comprises the following steps:

[0063] The double-pharmacophore DNA-encoded compound library is converted into single-pharmacophore DNA-encoded compound sub-library A and sub-library B, which can be combined with corresponding single-stranded DNA-encoded compound sub-library C or sub-library D, respectively;

[0064] Under the promotion of the target, the DNA conjugates of the compounds with affinity in the two compound libraries are close to each other, and the hybridization of the complementary regions of the DNA is promoted;

[0065] The hybridized double-stranded DNA is covalently cross-linked by using the cross-linking property of the special cross-linking group X, and the combination of the specific target compound is stabilized;

[0066] The encoding information of the double-stranded DNA is integrated into the same single-stranded DNA, and the structure information of the enriched compound is decoded by PCR amplification and DNA sequencing.

[0067] In some alternative embodiments, the construction of the self-assembled multi-pharmacophore library further comprises the following steps:

[0068] The single-pharmacophore DNA-encoded compound library is self-assembled, and the structure of the self-assembled DNA is stabilized by the special cross-linking group X;

[0069] The single-stranded DNA-encoded compound sub-libraries A, B and C are hybridized to form a double-pharmacophore or triple-pharmacophore compound library, and the size of the compound library is expanded.

[0070] In some alternative embodiments, the multifunctionalization of the DNA-encoded compound library further comprises the following steps:

[0071] The double-pharmacophore DNA-encoded compound library is converted into a single-pharmacophore DNA-encoded compound library and hybridized with single-stranded DNA connected with different functional compounds;

[0072] The special cross-linking group X is used to form a stable hybridization structure, and the multifunctionalization of the DNA-encoded compound library is realized.

[0073] The application adopting the above technical solution has the following beneficial effects:

[0074] (1) The synthesis method of the dual-pharmacophore DNA-encoded compound library of the present application can break the synthesis limitation of single-pharmacophore DNA-encoded compound library, encode the chemical information of multiple sub-libraries, and realize the breakthrough of the convenient synthesis technology of dual-pharmacophore DNA-encoded compound library, by constructing a special reversible covalent “Y-type” DNA (oligonucleotide) starting fragment and constructing a “Y-type” DNA-encoded compound library with a bifurcated coding region.

[0075] (2) The synthesis method of the “Y-type” dual-pharmacophore DNA-encoded compound library of the present application can quickly expand the scale and diversity of the compound library in the form of bifurcated encoding DNA under the condition of ensuring the high efficiency of encoding.

[0076] (3) The synthesis method of the “Y-type” dual-pharmacophore DNA-encoded compound library of the present application does not need to separately synthesize sub-libraries and does not need to recombine between compound libraries, and the connection of double-stranded DNA sticky ends mediated by DNA ligase is more convenient and efficient than the connection of single-stranded DNA splint method.

[0077] (4) The synthesis method of the “Y-type” dual-pharmacophore DNA-encoded compound library of the present application can directly use commercial encoding DNA, and is more suitable for industrial application.

[0078] (5) The synthesis method of the “Y-type” dual-pharmacophore DNA-encoded compound library of the present application has a shorter DNA chain length than the traditional self-assembled compound library, which is more conducive to reducing the influence of the coding region on ligand binding.

[0079] (6) The dual-pharmacophore compound library constructed by the synthesis method of the “Y-type” dual-pharmacophore DNA-encoded compound library of the present application can be used for affinity maturation screening and other applications.

[0080] (7) Based on the design of the reversible covalent “Y-type” DNA (oligonucleotide) starting fragment, the strategy can realize the reversible interconversion between the dual-pharmacophore DNA-encoded compound library and the single-pharmacophore DNA-encoded compound library.

[0081] (8) After the dual-pharmacophore DNA-encoded compound library is converted into a single-pharmacophore DNA-encoded compound library, by cross-linking with DNA connected with functional compounds, the compound library screening, compound library detection, and compound library delivery functions can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 is a schematic diagram of the reversible covalent “Y-type” DNA starting fragment of the present application.

[0083] Figure 2Representative structure of special cross-linking group X of the present application;

[0084] Figure 3 Schematic diagram of reversible covalent cross-linking and dissociation of the present application;

[0085] Figure 4 Schematic diagram of the construction of DNA-encoded compound library of the present application Figure 1 ;

[0086] Figure 5 Schematic diagram of the conversion of dual-pharmacophore DNA-encoded compound library to single-pharmacophore DNA-encoded compound library of the present application;

[0087] Figure 6 Screening strategy one compatible with dual-pharmacophore DNA-encoding system of the present application;

[0088] Figure 7 Screening strategy two compatible with dual-pharmacophore DNA-encoding system of the present application;

[0089] Figure 8 Screening strategy one compatible with single-pharmacophore DNA-encoding system of the present application;

[0090] Figure 9 Screening strategy two compatible with single-pharmacophore DNA-encoding system of the present application;

[0091] Figure 10 Screening strategy three compatible with single-pharmacophore DNA-encoding system of the present application;

[0092] Figure 11 Schematic diagram of compatibility of DNA-encoding system with dynamic library of the present application;

[0093] Figure 12 Schematic diagram of compatibility of DNA-encoding system with self-assembled multi-pharmacophore library of the present application;

[0094] Figure 13 Schematic diagram of functionalization of DNA-encoded compound library of the present application;

[0095] Figure 14 DNA cross-linking chromatogram of the present application;

[0096] Figure 15 Chromatogram of repeated cross-linking and dissociation of DNA of the present application;

[0097] Figure 16 Sequencing results of the present application;

[0098] Figure 17 Dynamic combination compound library verification of the present application;

[0099] Figure 18 Cell delivery and fluorescence imaging suitable for the present application. DETAILED DESCRIPTION

[0100] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0101] The synthesis method of the dual-pharmacophore DNA encoded compound library provided by the embodiments of the present application comprises the following steps:

[0102] The reversible covalent "Y-type" DNA starting fragment is constructed to form the orthogonal coding of the DNA encoded fragment with different cohesive ends, and the dual-pharmacophore DNA encoded compound library is synthesized.

[0103] The controllable conversion of the dual-pharmacophore DNA encoded compound library and the single-pharmacophore DNA encoded compound library is performed to realize the effective decoding of the dual-pharmacophore DNA encoded compound library and the single-pharmacophore DNA encoded compound library.

[0104] The DNA encoded compound library is compatible with multiple screening systems to expand the target screening range of the encoded compound library, and the screening of the encoded compound library includes the screening of the dual-pharmacophore DNA encoded compound library and the screening of the single-pharmacophore DNA encoded compound library.

[0105] The single-pharmacophore DNA encoded compound library is dynamically combined to construct a dynamic DNA encoded compound library, and the dynamic structure is balanced and locked by using a covalent reversible cross-linking group.

[0106] The single-pharmacophore DNA encoded compound library is combined with other single-pharmacophore compound libraries to construct a self-assembled multi-pharmacophore library.

[0107] The single-pharmacophore DNA encoded compound library is combined with a functional compound DNA conjugate by DNA hybridization and covalent cross-linking to realize the multifunctionalization of the DNA encoded compound library.

[0108] The reversible covalent "Y-type" DNA starting fragment comprises the following steps:

[0109] Three single-stranded DNAs with X cross-linking groups are synthesized respectively, and the three single-stranded DNAs are single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R respectively, and at least one of the single-stranded DNAs contains at least one X cross-linking group. The ssDNA-T and the ssDNA-B have a complementary region 1, the ssDNA-T and the ssDNA-R have a complementary region 2, and the ssDNA-B and the ssDNA-R have a complementary region 3. The length of each complementary region is between 0 and 30 bases, and the length of the complementary region does not require to be consistent. The X cross-linking group is any one of the special cross-linking groups 1-12 shown in the formula (I) or a natural base, and the X cross-linking group is located on the reversible covalent "Y-type" DNA starting fragment, but is not fixed at a specific site. Figure 2 The natural base is any one of adenine, guanine, cytosine and thymine

[0110] Strategy 1 for constructing the reversible covalent "Y-type" DNA starting fragment:

[0111] Two single-stranded DNAs containing special cross-linking groups and a complementary single-stranded DNA not containing special cross-linking groups are combined to form a reversible covalent "Y-type" DNA starting fragment through a covalent cross-linking chemical reaction; and the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction.

[0112] Strategy 2 for constructing the reversible covalent "Y-type" DNA starting fragment:

[0113] Three single-stranded DNAs with X cross-linking groups are synthesized respectively, and the X cross-linking groups are special cross-linking groups or natural bases; one single-stranded DNA containing special cross-linking groups and a complementary single-stranded DNA not containing special cross-linking groups are combined to form an intermediate DNA with a covalent structure through a covalent cross-linking chemical reaction; and another complementary single-stranded DNA containing special cross-linking groups is combined to form a reversible covalent "Y-type" DNA starting fragment through a covalent cross-linking chemical reaction; and the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction.

[0114] Strategy 3 for constructing the reversible covalent "Y-type" DNA starting fragment:

[0115] Three single-stranded DNAs with X cross-linking groups are synthesized respectively, and the X cross-linking groups are special cross-linking groups or natural bases; the three single-stranded DNAs containing special cross-linking groups are combined to form a novel reversible covalent "Y-type" DNA starting fragment through a covalent cross-linking chemical reaction; and the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction.

[0116] The number of cycles for constructing the reversible covalent "Y-type" DNA starting fragment is not less than 50 times.

[0117] The reversible covalent "Y-type" DNA starting fragment comprises at least two reaction sites R for synthesizing the compound library, which are respectively reaction site R1 and reaction site R2, and the reaction site R is connected with the single-stranded DNA through a linker L, and the linker L comprises linker L1 and linker L2.

[0118] The controllable conversion of the dual-pharmacophore DNA-encoded compound library and the single-pharmacophore DNA-encoded compound library comprises the following steps:

[0119] The synthesis of the DNA-encoded compound library is achieved by using a "split-pool-split" strategy; the reversible covalent "Y-type" DNA starting fragment is subjected to a DNA-compatible chemical reaction with a chemical structure element, which is any one of a carboxylic acid, an aldehyde, an alkene, an amine, a boric acid, and a halide; a corresponding coding DNA tag is added for each chemical structure element, and the DNA sticky ends are connected by using a DNA ligase or a chemical method; or after adding one chemical structure element to each of the two chemical reaction sites of the "Y-type" DNA starting fragment, the corresponding coding DNA tags with different sticky ends are added together, and the DNA sticky ends are connected by using a DNA ligase or a chemical method; the connection order of the DNA tags and the chemical structure elements in each dimension can be exchanged, and the same dimension samples are mixed and then divided for the next dimension synthesis by using a combinatorial chemistry method; after the synthesis of the dual-pharmacophore DNA-encoded compound library is completed, the special "Y-type" structure is opened by breaking the covalent bond, and selective degradation conditions are used to degrade or separate the DNA strands that do not connect the coding compounds in the library construction process, thereby completing the conversion of the dual-pharmacophore DNA-encoded compound library and the single-pharmacophore DNA-encoded compound library.

[0120] The screening of the dual-pharmacophore DNA-encoded compound library comprises the following steps:

[0121] The screening strategy of the dual-pharmacophore DNA-encoded compound library is as follows: a known active compound is added to any single-pharmacophore compound library to synthesize a dual-pharmacophore DNA-encoded compound library; the dual-pharmacophore DNA-encoded compound library is incubated with a related target of the active compound for screening; the compound library members that are not combined with the target or have weak affinity are separated by using selective degradation conditions, and the compound library members that have stronger affinity with the target are enriched; the structure information of the enriched compound library members is decoded by using a polymerase chain reaction and a DNA sequencing technology.

[0122] The second screening strategy of the dual-pharmacophore DNA-encoded compound library is: taking a protein target as a screening substrate of the dual-pharmacophore DNA-encoded compound library; incubating the dual-pharmacophore DNA-encoded compound library with the target; separating the compound library members that are not combined with the target or have weak affinity from the target through selective degradation conditions, and enriching the compound library members that have strong affinity with the target; decoding the structural information of the enriched compound library members through polymerase chain reaction and DNA sequencing technology.

[0123] The single-pharmacophore DNA-encoded compound library screening further includes the following steps:

[0124] The first screening strategy of the single-pharmacophore DNA-encoded compound library is:

[0125] The single-pharmacophore DNA-encoded compound library is hybridized with a DNA probe having chemical reactivity; when a compound with affinity in the DNA-encoded compound library is combined with the target, the distance between the DNA probe and the target is shortened, promoting the covalent cross-linking of the DNA probe and the target; the compound DNA conjugate that is not combined with the target is separated through selective degradation conditions, and the DNA compound conjugate combined with the target is enriched; the structural information of the compound is decoded through polymerase chain reaction and DNA sequencing technology;

[0126] The second screening strategy of the single-pharmacophore DNA-encoded compound library is:

[0127] The single-pharmacophore DNA-encoded compound library is incubated with a target connected with a PCR primer sequence DNA, and the hybrid structure of the compound library member having affinity for the target and the DNA primer sequence connected with the target is subjected to PCR amplification to obtain the structural information of the compound having high affinity for the target;

[0128] The third screening strategy of the single-pharmacophore DNA-encoded compound library is:

[0129] The single-pharmacophore DNA-encoded compound library is hybridized with a DNA probe having chemical reactivity and covalently cross-linked; when a compound with affinity in the compound library is combined with the target, the distance between the DNA probe and the target is shortened, promoting the covalent cross-linking of the DNA probe and the target, realizing the covalent cross-linking complex of the single-stranded DNA connected with the compound library information, the DNA probe having chemical reactivity, and the target; the covalent cross-linking complex of the three is separated through a target in vitro binding experiment, and then the structural information of the compound having high affinity for the target is obtained through DNA decoding.

[0130] The selective degradation condition is any one of an exonuclease, a chemical reaction condition, and a separation capture, and the exonuclease is any one of ExoI, ExoIII, and λExo.

[0131] Example 1

[0132] Construction of reversible covalent "Y-type" DNA (oligonucleotide) initiation fragments:

[0133] like Figure 1 , 2 As shown, a reversible covalent chemical reaction is employed to achieve efficient and reversible covalent cross-linking between double-stranded DNA strands, forming a reversible covalent hairpin structure. Specific cross-linking groups 1, 2, 4, and 7 can undergo cycloaddition cross-linking reactions under ultraviolet light irradiation in the 200–400 nm range, and their cross-linking products can simultaneously undergo cyclocleavage reactions under corresponding ultraviolet light wavelengths. Specific cross-linking group 5 can react under oxidative conditions for SS bond formation (such as potassium carbonate, sodium hydroxide, bismuth nitrate catalytic oxidation, etc.) to generate SS cross-linking products, and its cross-linking products can simultaneously undergo cleavage-reduction reactions under disulfide bond reduction conditions (such as β-mercaptoethanol (β-ME), dithiothreitol (DTT), etc.). Specific cross-linking group 6 can react under free radical oxidation conditions (such as photocatalysis, high-energy radiation, etc.) to generate cycloaddition cross-linking products, and its cross-linking products can simultaneously undergo reduction reactions under reduction conditions. Figure 14 As shown, the process was confirmed by high performance liquid chromatography (HPLC).

[0134] Example 2:

[0135] like Figure 1 and Figure 3 As shown, two single-stranded DNA strands, each containing a specific cross-linking group 3-cyanoethylenecarbazole, and their complementary single-stranded DNA strands are subjected to a cycloaddition reaction under 340-380 nm light. The specific base 3-cyanoethylenecarbazole undergoes a cycloaddition reaction with the obliquely opposite T or C base of the complementary DNA, forming a stable covalent bond, thereby achieving covalent cross-linking between the three single-stranded DNA strands. Under 300-320 nm light, they are reduced to their initial state before the cycloaddition reaction. Figure 15 As shown, the process was detected by HPLC, and the results showed that this strategy can achieve multiple interconversions between single-stranded DNA and double-stranded DNA, greatly improving the flexibility of DNA-encoded compound libraries.

[0136] Example 3

[0137] Using the above construction method, the DNA cross-linking and dissociation process can be achieved in more than 50 cycles without damaging the DNA.

[0138] Example 4

[0139] After the verification of the initiation of the synthesis of the reversible cross-linking "Y-type" DNA (oligonucleotide), the DNA-encoded polypeptide compound library was further synthesized. Two dimensions x two dimensions correspond to the sequence and the corresponding structure group category respectively (151 x 95) x (156 x 97). According to the synthesis method shown in Figures 4-5 The 1 mM starting DNA fragment solution was dispensed into 381 consecutive wells of a 96-well plate, 10 μL per well, and 11 μL of the upper and lower strands of the first cycle of labeled nucleotide double strands with a concentration of 1 mM was added to each well for DNA tag ligation, ethanol precipitation purification, and the purified DNA was dissolved in 10 μL of a sodium borate buffer solution (pH = 9.5, 250 mM). The corresponding compound structure unit was added, and after the reaction was complete, all the reaction solutions were mixed and subjected to ethanol precipitation to obtain DNA precipitate dissolved in 200 μL of double distilled water. A 10K ultrafiltration tube with a size of 500 μL was used for desalting and purification of the product to obtain the first cycle product. The last two cycles were synthesized by the same "split-pool-split" to obtain a DNA-encoded polypeptide compound library of 151 x 95 x 156 x 97 = 217,068,540 compounds. After the synthesis of the compound library, the single-stranded compound library was converted and characterized by liquid chromatograph-mass spectrometer (LC-MS).

[0140] Example 5: Screening of a dual-pharmacophore DNA-encoded compound library

[0141] Using carbonic anhydrase Ⅸ (CA Ⅸ) as the target protein, the method shown in Figure 6 The dual-pharmacophore DNA-encoded compound library constructed in Examples 1-5 (containing known affinity molecules of the target protein in a certain pharmacophore library) was purified by DNA selective degradation conditions (λExo degrades the DNA of the unlinked compounds), and after 8 hours of incubation with the target protein (CA Ⅸ) in a buffer system, the members that were not bound or weakly bound to the target were removed, and the high-affinity library members of the target protein were enriched. The structure of the screened and enriched compounds was decoded by PCR amplification and DNA sequencing, and the affinity maturation of the known affinity molecules of the target protein was achieved.

[0142] Using kirsten rat sarcoma viral oncogene (KRAS) as the target protein, the method shown in Figure 7The method shown is used for screening. The dual-pharmacophore DNA-encoded compound library constructed in Examples 1-5 is purified by DNA selective degradation conditions (λExo degrades the DNA of the unlinked compounds), incubated with the target protein (KRAS) in a buffer system for 8 hours, and the members that do not bind or weakly bind to the target are removed to enrich the high-affinity library members of the target protein. The screened compounds are then structurally decoded by PCR amplification and DNA sequencing, and it is found that the high-affinity binding molecules of the large surface area target protein.

[0143] As shown in Figure 16 the sequencing data clearly shows that the sequences are mixed and expected to be consistent before screening; but after the screening is completed, the specific sequence (DE-2-493) is clearly enriched. The above data prove that the encoding technology of the present application using reversible covalent "Y-type" hairpin structure DNA as the starting fragment can be used for library construction and screening, and truly realizes the efficient synthesis and diversified application of the dual-pharmacophore DNA-encoded compound library. It should be noted that in practical application, the encoding dimension of the present application is not limited to the four dimensions in the above examples, so a larger scale compound library can be constructed; the type of the library is not limited to the construction of a polypeptide library, so a compound library with higher chemical diversity can be constructed; the target for screening is not limited to protein, so screening can be performed for other targets.

[0144] Example 6: Screening of single-pharmacophore DNA-encoded compound library

[0145] α1-acid glycoprotein (AGP) is used as the protein target, and the method shown in Figure 8 is used for screening. The dual-pharmacophore DNA-encoded compound library constructed in Examples 1-5 is converted into a single-pharmacophore DNA-encoded compound library by DNA selective degradation conditions (λExo degrades the DNA of the unlinked compounds). Then hybridized with a probe having a chemical reactive group (bis-aziridine), the target protein (AGP) is added, and incubated in a buffer system for 8 hours, and the active probe is covalently cross-linked with the protein under ultraviolet wavelength 365 nm irradiation. Then the two DNAs are photo-crosslinked to form a stable double-stranded structure under ultraviolet wavelength 360-400 nm irradiation through the special cross-linking group 7: 3-cyanovinyl carbazole in the DNA. Finally, the DNA tag of the compound DNA conjugate that does not have binding force with the target protein is degraded by adding exonuclease ExoI. The screened compounds are then structurally decoded by PCR amplification and DNA sequencing.

[0146] According to Figure 9The method shown is used for screening, chemically linking the target protein with DNA with primer sequence, incubating with the encoding compound library converted into single pharmacophore DNA in buffer solution for 8h, and then analyzing by PCR technique, and for the compound with binding force to the target protein, the encoding DNA can be amplified by PCR to be sequenced and decoded.

[0147] As shown in Figure 16 the sequencing data clearly shows that before screening, the sequence is mixed and expected to be consistent; but after screening is completed, the specific sequence (DE-3-117) is clearly enriched. The above data prove that the encoding technology of the present application using reversible covalent "Y-type" hairpin structure DNA as starting fragment can be used for library construction and screening, and truly realizes the interconversion of double pharmacophore DNA encoded compound library and single pharmacophore DNA encoded compound library. It should be pointed out that in practical application, the encoding dimension of the present application is not limited to the four dimensions in the above example, so larger scale compound library can be constructed; the type of library is not limited to the construction of polypeptide library, so compound library with higher chemical diversity can be constructed; the target of screening is not limited to protein, so screening can be carried out for other targets.

[0148] Example 7: Construction of a diverse compound library

[0149] As shown in Figures 6-10 two single-stranded DNAs were prepared, containing a short complementary region, and both were connected with a desthiobiotin (DB) group as a ligand, and streptavidin was used as a target protein for verification, and gel electrophoresis was used for characterization, as shown in Figure 17 At 0°C, the two single-stranded DNAs form stable double-stranded DNAs, and under cross-linking conditions (I'), a large amount of cross-linked double-stranded product can be seen. In contrast, at 30°C, the double-stranded DNA is dynamic, and no cross-linking (I) is observed. By adding the target protein streptavidin, the cross-linking product becomes visible (II), indicating that the binding of the target ligand promotes DNA hybridization, and in turn promotes the cross-linking of double-stranded DNA. Control experiments using non-target protein bovine serum albumin or DNA without DB ligand show no cross-linking product (III and IV). It shows that the cross-linking group X can specifically cross-link the target-promoted double-stranded DNA. Therefore, this encoding strategy can be compatible with dynamic combinatorial compound libraries, and can effectively facilitate the rapid construction of compound libraries with higher chemical diversity.

[0150] Example 8

[0151] After the conversion of the dual-pharmacophore DNA-encoded compound library into a single-pharmacophore DNA-encoded compound library, it is incubated with single-stranded DNA connected with a cell penetration peptide and a fluorescent group (fluorescein), and through the special cross-linking base 7: 3-cyanovinyl carbazole in the DNA on the compound DNA conjugate, the single-stranded DNA connected with the cell penetration peptide is covalently cross-linked between the DNA strands under ultraviolet light at a wavelength of 360-400 nm. Human normal breast cells are selected for cell penetration experiments, and the membrane penetration effect of the DNA-encoded compound library is observed by fluorescence confocal microscopy, as shown in FIG. 3, which proves that the functionalization of the DNA-encoded compound library can be achieved by hybridization with compounds with functional groups. Figure 18

[0152] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and all of them should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are all known technologies.​

Claims

1. A method for synthesizing a dual-pharmacophore DNA-encoded compound library, characterized in that, Includes the following steps: Construct a reversibly covalent "Y-type" DNA initiation fragment, form orthogonal coding of DNA coding fragments with different sticky ends, and synthesize a library of dual pharmacophore DNA-encoded compounds; Controllable transformation of dual-pharmacophore DNA-encoded compound libraries into single-pharmacophore DNA-encoded compound libraries was performed to achieve effective decoding of these libraries. The DNA-encoded compound library is compatible with multiple screening systems, expanding the target screening range of the encoded compound library. The screening of the encoded compound library includes screening of dual-pharmacophore DNA-encoded compound libraries and screening of single-pharmacophore DNA-encoded compound libraries. A dynamic DNA-encoded compound library is constructed by dynamically combining single pharmacophore DNA-encoded compound libraries, and the dynamic structure is balanced and locked using covalent reversible cross-linking groups. By combining single-pharmacophore DNA-encoded compound libraries with other single-pharmacophore compound libraries, a self-assembled multipharmacophore library can be constructed. By combining DNA hybridization and covalent cross-linking, a single pharmacophore DNA-encoded compound library is combined with a functional compound DNA conjugate to achieve multifunctionality of the DNA-encoded compound library; The construction of the reversibly covalent "Y-type" DNA initiation fragment includes the following steps: Three single-stranded DNAs with X crosslinking groups were synthesized respectively. The three single-stranded DNAs were single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R, respectively. The X crosslinking group was a special crosslinking group or a natural base. Each of the single-stranded ssDNA-T, single-stranded ssDNA-B, and single-stranded ssDNA-R has a complementary region between each other, and the length of each complementary region is 0 to 30 bases. At least one of the single-stranded DNAs of the single-stranded ssDNA-T, single-stranded ssDNA-B, and single-stranded ssDNA-R contains at least one X crosslinking group. The X crosslinking group is located on a reversibly covalent "Y-type" DNA initiation fragment, and the X crosslinking group is any one of the following special crosslinking groups 1-12: ； The natural base is any one of adenine, guanine, cytosine, and thymine.

2. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 1, characterized in that, Construction strategy one for the reversibly covalent "Y-type" DNA initiation fragment: Two single-stranded DNAs containing special cross-linking groups are combined with complementary single-stranded DNAs that do not contain special cross-linking groups through a covalent cross-linking chemical reaction to form a reversible covalent "Y-type" DNA initiation fragment. The covalently cross-linked "Y-type" DNA initiator fragment is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction; Strategy 2 for constructing the reversibly covalent "Y-type" DNA initiation fragment: Three single-stranded DNA strands with X crosslinking groups were synthesized, wherein the X crosslinking group is a special crosslinking group or a natural base; A single-stranded DNA containing a special cross-linking group is covalently cross-linked with a complementary single-stranded DNA that does not contain a special cross-linking group to form an intermediate DNA with a covalent structure; then, it is covalently cross-linked with another complementary single-stranded DNA containing a special cross-linking base to form a reversible covalent "Y-type" DNA initiation fragment. The covalently cross-linked "Y-type" DNA initiator fragment is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction; Strategy 3 for constructing the reversibly covalent "Y-type" DNA initiation fragment: Three single-stranded DNA strands with X crosslinking groups were synthesized, wherein the X crosslinking group is a special crosslinking group or a natural base; Three single-stranded DNAs containing special cross-linking groups are covalently cross-linked to form a novel reversible covalent "Y-type" DNA initiation fragment; The covalently cross-linked "Y-type" DNA initiator fragment is dissociated into a single-stranded initial state through a covalent dissociation chemical reaction; The number of construction cycles for the reversible covalent "Y-type" DNA initiation fragment shall not be less than 50. The reversibly covalent "Y-type" DNA initiation fragment contains at least two reaction sites R for compound library synthesis, and the reaction sites R are connected to single-stranded DNA by a linker L.

3. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 2, characterized in that, The controlled conversion between the dual-pharmacophore DNA-encoded compound library and the single-pharmacophore DNA-encoded compound library includes the following steps: A "split-pool-split" strategy was used to synthesize a DNA-encoded compound library; A reversible covalent "Y-type" DNA initiator fragment is subjected to a DNA-compatible chemical reaction with a chemical structural motif, wherein the chemical structural motif is any one of carboxylic acid, aldehyde, alkene, amine, boric acid, or halide. Add the corresponding coding DNA tag for each chemical structural motif, and then ligate the sticky ends of the DNA using DNA ligase or chemical methods; or add a chemical structural motif to each of the two chemical reaction sites of the "Y-type" DNA start fragment, and then add the corresponding coding DNA tags with different sticky ends, and ligate the sticky ends of the DNA using DNA ligase or chemical methods. The connection order of DNA tags and chemical structural motifs in each dimension is interchangeable. By using combinatorial chemistry, samples from the same dimension are mixed and then divided equally for synthesis in the next dimension. After the synthesis of the dual pharmacophore DNA-encoded compound library is completed, the covalent bonds are broken to open the special "Y-type" structure. Selective degradation conditions are used to degrade or separate the DNA strands that were not linked to the coding compounds during the library construction process, thereby completing the conversion between the dual pharmacophore DNA-encoded compound library and the single pharmacophore DNA-encoded compound library.

4. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 3, characterized in that, The screening of the dual pharmacophore DNA-encoded compound library includes the following steps: The first screening strategy for the dual pharmacophore DNA-encoded compound library: A dual-pharmacophore DNA-encoded compound library can be synthesized by incorporating known active compounds into any single-pharmacophore compound library. A library of dual pharmacophore DNA-encoded compounds was incubated and screened with relevant targets of active compounds. Members of the compound library that did not bind to the target or had a weak affinity were separated by selective degradation conditions, while members of the compound library that had a stronger affinity for the target were enriched. The structural information of the enriched compound library members was decoded using polymerase chain reaction and DNA sequencing technology. The second screening strategy for the dual pharmacophore DNA-encoded compound library: Protein targets were used as screening substrates for a dual-pharmacophore DNA-encoded compound library. A library of dual-pharmacophore DNA-encoded compounds was incubated and screened with targets; Members of the compound library that do not bind to the target or have a weak affinity are separated by selective degradation conditions, while members of the compound library that have a strong affinity for the target are enriched. The structural information of the enriched compound library members was decoded using polymerase chain reaction and DNA sequencing technology.

5. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 4, characterized in that, The screening of the single pharmacophore DNA-encoded compound library also includes the following steps: The first screening strategy for single pharmacophore DNA-encoded compound libraries: Hybridize a library of single-pharmacophore DNA-encoded compounds with chemically reactive DNA probes; When compounds with affinity in a DNA-encoded compound library bind to a target, they bring the DNA probe closer to the target, promoting covalent cross-linking between the DNA probe and the target. DNA conjugates that do not bind to the target are separated by selective degradation conditions, while DNA conjugates that bind to the target are enriched. Amplification is performed using polymerase chain reaction (PCR), and the structural information of compounds is decoded using DNA sequencing technology. The second screening strategy for single pharmacophore DNA-encoded compound libraries: A library of single-pharmacophore DNA-encoded compounds was co-incubated with a target DNA linked to PCR primer sequences. The hybridization structures of the library members with affinity for the target and the DNA primer sequences linked to the target were amplified by PCR to obtain the structural information of compounds with high affinity for the target. The third screening strategy for single pharmacophore DNA-encoded compound libraries: A library of single-pharmacophore DNA-encoded compounds was hybridized and covalently cross-linked with chemically reactive DNA probes; When compounds with affinity in the compound library bind to the target, they bring the DNA probe closer to the target, promoting covalent cross-linking between the DNA probe and the target, thus forming a covalent cross-linked complex connecting the single-stranded DNA containing compound library information, the chemically reactive DNA probe, and the target. Using in vitro binding experiments with the target, the covalently cross-linked complex of the three components was isolated, and then the structural information of the compound with high affinity to the target was obtained through DNA decoding.

6. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 5, characterized in that, The selective degradation conditions are any one of exonuclease, chemical reaction conditions, and separation and capture, and the exonuclease is any one of ExoI, ExoIII, and λExo.

7. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 6, characterized in that, The construction of the dynamic DNA-encoded compound library also includes the following steps: The dual-pharmacophore DNA-encoded compound library can be converted into single-pharmacophore DNA-encoded compound sublibraries A and B, which can be dynamically combined with the corresponding single-stranded DNA-encoded compound sublibraries C or D, respectively. With the help of the target, DNA conjugates with affinity for each other from the two compound libraries approach each other, promoting hybridization of complementary DNA regions; By utilizing the cross-linking properties of a special cross-linking group X, hybrid double-stranded DNA can be covalently cross-linked to stabilize combinations of specific target compounds; The coding information of double-stranded DNA is integrated into the same single-stranded DNA, and the structural information of the enriched compound is decoded by PCR amplification and DNA sequencing.

8. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 7, characterized in that, The construction of the self-assembled multiphasor library also includes the following steps: A library of single pharmacophore DNA-encoded compounds is self-assembled, and the structure of the self-assembled DNA is stabilized by a special cross-linking group X. Single-stranded DNA-encoded compound sublibraries A, B, and C are hybridized to form dual- or triple-pharmacophore compound libraries, and the size of the compound libraries is expanded.

9. The method for synthesizing a dual pharmacophore DNA-encoded compound library according to claim 8, characterized in that, The multifunctionalization of the DNA-encoded compound library also includes the following steps: The dual-pharmacophore DNA-encoded compound library was converted into a single-pharmacophore DNA-encoded compound library and hybridized with single-stranded DNA containing different functional compounds; Then, by utilizing special cross-linking groups X to form stable hybridization structures, the multifunctionality of DNA-encoded compound libraries can be achieved.

Images