WO2021115319A1 - 同时结合多个生物靶标的dna编码化合物库筛选方法 - Google Patents

同时结合多个生物靶标的dna编码化合物库筛选方法 Download PDF

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WO2021115319A1
WO2021115319A1 PCT/CN2020/134863 CN2020134863W WO2021115319A1 WO 2021115319 A1 WO2021115319 A1 WO 2021115319A1 CN 2020134863 W CN2020134863 W CN 2020134863W WO 2021115319 A1 WO2021115319 A1 WO 2021115319A1
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complementary
dna
tag
compound
screening
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French (fr)
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李进
窦登峰
巩晓明
万金桥
刘观赛
高森
龚珍
蔡龙英
张雪琴
穆雪梅
张丽芳
刘川
康静文
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成都先导药物开发股份有限公司
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    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

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  • the invention belongs to the field of drug screening, and specifically relates to a method for screening compounds that simultaneously bind multiple biological targets through a DNA-encoded compound library.
  • high-throughput screening for biological targets is one of the main means to quickly obtain lead compounds.
  • traditional high-throughput screening based on a single molecule requires a long time, a huge investment in equipment, a limited number of library compounds (several millions), and the establishment of a compound library requires decades of accumulation, which limits the efficiency and effectiveness of lead compound discovery. possibility.
  • DNA coding compound library technology (WO2005058479, WO2018166532, CN103882532), which combines combinatorial chemistry and molecular biology technology, adds a DNA tag to each compound at the molecular level, and can synthesize up to A compound library of hundreds of millions, and compounds can be identified through gene sequencing methods, greatly increasing the size and synthesis efficiency of the compound library, becoming the trend of next-generation 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).
  • PROTAC PROteolysis TArgeting Chimera, protein degradation targeted chimera
  • PROTAC is a bifunctional small molecule, one end binds the ligand of the target protein, and the other end binds the ligand of the E3 ubiquitin ligase, connected by a chain.
  • the target protein and E3 enzyme can be brought closer, so that the target protein is labeled with ubiquitin, and then degraded through the ubiquitin-proteasome pathway.
  • molecular glue targeting E3 ubiquitin ligase that is, smaller molecules can simultaneously bind E3 ubiquitin ligase and target protein to achieve the degradation of the target protein.
  • the present invention provides a method for screening compounds that simultaneously bind multiple biological targets through a DNA-encoded compound library, which includes the following steps:
  • step d Replace the DNA-encoding compound library in step b with the DNA-encoding compound obtained by dissociation, replace the other side of the first complementary tag in step c with the other side of the second complementary tag, repeat steps b and c, Until step c, the other party of the nth complementary tag is added;
  • n 2, 3, 4 or 5.
  • the biological target is protein, RNA, DNA.
  • one of the complementary tags is a biotin tag, a His tag, a GST tag, an MBP tag, a FLAG tag, a C-Myc tag, a SUMO tag, a SNAP tag, one of complementary DNA, and one of complementary RNA.
  • the other side of the complementary label is avidin, streptomycin, nickel, glutathione, polysaccharide resin, anti-FLAG, anti-C-Myc, SUMO protease, benzylguanine (benzylguanine, BG), the other side of complementary DNA, the other side of complementary RNA.
  • the unbound molecules are removed by physical, chemical or biological methods.
  • the present invention also provides a method for screening compounds that simultaneously bind E3 ubiquitin ligase and target protein through a DNA-encoded compound library, which includes the following steps:
  • step c Simultaneously incubate the E3 ubiquitin ligase of the party with the first complementary tag and the target protein of the party with the second complementary tag with the DNA encoding compound obtained by dissociation in step c;
  • one side of the first type of complementary tag is a His tag, and the other side of the first type of complementary tag is nickel.
  • one side of the second complementary label is a GST label, and the other side of the second complementary label is glutathione.
  • the other side of the first type of complementary label and the other side of the second type of complementary label are fixed on a solid-phase carrier; preferably, the solid-phase carrier is a magnetic bead.
  • step c nickel-coated magnetic beads are added, and unbound molecules are removed by centrifugation.
  • step e After removing unbound molecules in step e, an elution buffer is added, and the elution is repeated 3 to 5 times;
  • the steps c and e dissociate from the biological target by heating to obtain a DNA-encoded compound.
  • the heating temperature is 85-95°C.
  • the heating dissociation time is 10-20 minutes.
  • step e glutathione-coated magnetic beads are added, and unbound molecules are removed by centrifugation.
  • the step e after the DNA encoding compound is obtained by dissociating from the biological target, the number of DNA molecules is quantified by qPCR.
  • the DNA-encoding compound obtained by dissociation is used as a new round of DNA-encoding compound library, and the screening is repeated again until the number of molecules drops to 10 7 to 10 8 .
  • DNA encoding compound obtained by dissociation is sequenced, the sequencing result is decoded, and the enrichment signal is obtained.
  • the E3 ubiquitin ligase is CRBN, VHL, MDM2, IAPs, DCAF15, DCAF16, RNF4, and RNF114.
  • the method of the present invention can also be used to screen molecules that can simultaneously affinity tissue-specific proteins and therapeutic target proteins, for targeted medication, reduce toxic and side effects, improve drug absorption in target tissues, and enhance drug bioavailability.
  • the method of the present invention can also be used to screen drug molecules that act on dual targets at the same time for the treatment of diseases with dual mechanisms (Dual Mechanisms).
  • Figure 1 is the structure of 6 positive compounds with different DNA tags added to the DNA encoding compound library in Example 1 of the present invention
  • Figure 2 shows the signal intensities of 6 positive compounds in different samples screened by the DNA-encoded compound library in Example 1 of the present invention
  • Figure 3 is the structure of the protein degrading agent corresponding to dBET1 and 6 DNA-encoding positive compounds in Example 1 of the present invention
  • Figure 4 is an experimental diagram of the degradation of BRD4 protein by dBET1 and 6 protein degrading agents in MV4;11 cells in Example 1 of the present invention
  • Example 5 is a schematic diagram of the structure of the DNA-encoding compound library for CRBN E3 ligase in Example 2 of the present invention.
  • Figure 6 is a compound signal diagram obtained by screening CRBN and BRD4 proteins in Example 2 of the present invention, and a Tanimoto similarity comparison of the structures of 4 representative compounds (R 2 and R 3 are represented by specific reagent codes);
  • Fig. 7 is the result of the degradation test of BRD4 protein in MV4;11 cells obtained by screening CRBN and BRD4 proteins in Example 2 of the present invention
  • Figure 8 is the test result of compound 75-NX-1 and its corresponding compound 57-NX-1 which does not contain thalidomide and Linker on MV4;11 cell proliferation in Example 2 of the present invention
  • Example 1 Screening of compounds that can simultaneously bind CRBN and BRD4
  • the DNA encoding compound library was constructed according to the method described in WO2006135786. Add the positive control compound with DNA tag shown in Figure 1 to the DNA-encoded compound library.
  • the protein CRBN-His tag/DDB1 used in Example 1 was expressed internally by HitGen, and the protein BRD4-GST tag was from Active Motif (Cat. No. 81855).
  • the screening solution was incubated in a closed centrifuge tube on a low-speed shaker at room temperature for 1 hour. Then add nickel-coated magnetic beads to group 1, and add glutathione-coated magnetic beads to group 2, and incubate at room temperature for 30 min. Centrifuge 250g x 3min to remove the supernatant containing unbound molecules. Add 200 ⁇ 500 ⁇ L of elution buffer and incubate for 1min, and then centrifuge for elution, repeat 3 ⁇ 5 times. Add 50-100 ⁇ L of dissociation buffer, heat and dissociate at 85-95°C for 10-20 minutes, and collect the supernatant by centrifugation.
  • qPCR quantifies the number of DNA molecules in the supernatant. If the number of molecules exceeds 10 8 , use the supernatant as a new round of DNA-encoding compound library. After adjusting the pH and desalting the purification column to remove additional buffer components, repeat the screening again until The number of molecules is reduced to 10 7 to 10 8 .
  • Figure 2 shows the signal intensity (Sequence Count) of the 6 positive compounds in the 4 screening groups.
  • the results in the first and second groups b These 6 compounds are respectively for the binding of CRBN and BRD4 proteins, but it cannot be explained that these compounds bind to the best combination of CRBN and BRD4 proteins at the same time.
  • the third grouping result shows that Conjugate-4 is obviously the best compound for binding two proteins at the same time, and its chain length is exactly the same as dBET1.
  • PROTAC molecules corresponding to these 6 compounds (without DNA tags, but containing the formamide structure linked to DNA, the structures of dBET1 and 6 PROTAC molecules are shown in Figure 3) to further verify the correspondence between the 6 compounds in Figure 1 and the screening results , Using the Western Blot method to evaluate the degradation of BRD4 protein against MV4;11 (a cell line sensitive to the degradation of BRD4 protein), and the results are shown in Figure 4.
  • PROTAC-4 has the best protein degradation effect, which is consistent with the compound signal of Conjugate-4 in screening group 3.
  • Example 2 Obtaining novel protein degradation agents through screening of compounds that simultaneously bind CRBN and BRD4
  • a DNA-encoded compound library as shown in Figure 5 was constructed, and then the compound library was screened.
  • the protein CRBN-His tag/DDB1 used in this example is expressed internally by HitGen, and the protein BRD4-GST tag is from Active Motif (Cat. No. 81855).
  • the screening solution was incubated in a closed centrifuge tube on a low-speed shaker at room temperature for 1 hour. Then add nickel-coated magnetic beads to group 1, and add glutathione-coated magnetic beads to group 2, and incubate at room temperature for 30 min. Centrifuge 250g x 3min to remove the supernatant containing unbound molecules. Add 200 ⁇ 500 ⁇ L of elution buffer and incubate for 1min, and then centrifuge for elution, repeat 3 ⁇ 5 times. Add 50-100 ⁇ L of dissociation buffer, heat and dissociate at 85-95°C for 10-20 minutes, and collect the supernatant by centrifugation.
  • qPCR quantifies the number of DNA molecules in the supernatant. If the number of molecules exceeds 10 8 , use the supernatant as a new round of DNA-encoding compound library. After adjusting the pH and desalting the purification column to remove additional buffer components, repeat the screening again until The number of molecules is reduced to 10 7 to 10 8 .
  • Figure 6 shows that the compound library in Example 2 Group 3 is aimed at the enrichment of CRBN and BRD4 proteins.
  • the signal and the library compound molecule have a positive correlation with the binding stability of CRBN and BRD4.
  • the selected compound corresponds to the Sequence Count as shown in Figure 6.
  • the Tanimoto similarity between four representative compounds and the BRD4 inhibitor JQ1 shows that these compounds are screened by DNA-encoded compound library New structure.
  • the method of the present invention can screen compounds that simultaneously bind E3 ubiquitin ligase and target protein through a DNA-encoded compound library, discover novel protein degrading agents (PROTAC), and rank the protein degradability of these compounds , Reflects very good commercial application value.
  • This method also further broadens the application range of DNA-encoded compound libraries, has high screening efficiency and accuracy, and can be applied to various simultaneous screening of biological targets.

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Abstract

一种通过DNA编码化合物库筛选同时结合多个生物靶标的化合物的方法,以及一种通过DNA编码化合物库筛选同时结合E3泛素连接酶和靶标蛋白的化合物的方法。上述方法进一步拓宽了DNA编码化合物库的应用范围，适用于各类生物靶标的筛选。

Description

同时结合多个生物靶标的DNA编码化合物库筛选方法 技术领域
本发明属于药物筛选领域，具体涉及一种通过DNA编码化合物库筛选同时结合多个生物靶标的化合物的方法。
背景技术
在新药研发领域，针对生物靶标的高通量筛选是快速获得先导化合物的主要手段之一。然而，基于单个分子的传统高通量筛选所需时间长、设备投入巨大、库化合物数量有限(数百万)，且化合物库的建成需要数十年的积累，限制了先导化合物的发现效率与可能性。近年来出现的DNA编码化合物库技术(WO2005058479、WO2018166532、CN103882532)，结合了组合化学和分子生物学技术，在分子水平上将每个化合物加上一个DNA标签，能在极短的时间内合成高达亿级的化合物库，而且化合物能够通过基因测序的方法进行识别，大幅度地增加了化合物库的大小和合成效率，成为下一代化合物库筛选技术的趋势。DNA编码化合物库技术正开始在制药行业广泛应用，并产生了诸多积极的效果(Accounts of Chemical Research,2014,47,1247-1255)。
PROTAC(PROteolysis TArgeting Chimera，蛋白降解靶向嵌合体)是一种双功能小分子，一端结合靶蛋白的配体，另一端结合E3泛素连接酶的配体，通过一段链条连接。在体内可以将靶蛋白和E3酶拉近，使靶蛋白被打上泛素标签，然后通过泛素—蛋白酶体途径降解。近期有针对E3泛素连接酶的分子胶(molecular glue)的报道，即更小的分子即可实现同时结合E3泛素连接酶和靶标蛋白，实现靶标蛋白的降解。
基于新的药物靶向技术发展，需要筛选能同时结合两个或多个靶标的化合物，如PROTAC蛋白降解靶向嵌合体或者分子胶。因此需要开发对应的新型DNA编码化合物库化合物筛选技术，以提升DNA编码化合物库技术的应用价值。
发明内容
本发明提供了一种通过DNA编码化合物库筛选同时结合多个生物靶标的化合物的方法，包括以下步骤：
a.将n种生物靶标分别用n种不同的互补标签的一方进行标记；
b.将n种带有标签的生物靶标和DNA编码化合物库同时进行孵育；
c.加入第1种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物；
d.将解离得到DNA编码化合物替换步骤b中的DNA编码化合物库，将步骤c中将第1种互补标签的另一方替换为第2种互补标签的另一方，重复步骤b和步骤c，直至步骤c加入第n种互补标签的另一方；
其中，n为2、3、4或5。
进一步地，所述生物靶标为蛋白、RNA、DNA。
进一步地，所述互补标签的一方为生物素标签、His标签、GST标签、MBP标签、FLAG标签、C-Myc标签、SUMO标签、SNAP标签、互补DNA的一方、互补RNA的一方。
进一步地，所述互补标签的另一方为亲和素、链霉素、镍、谷胱甘肽、多糖树脂、anti-FLAG、anti-C-Myc、SUMO蛋白酶、苯甲基鸟嘌呤(benzylguanine,BG)、互补DNA的另一方、互补RNA的另外一方。
进一步地，步骤c和步骤d中加入第1～n种互补标签的另一方后，通过物理、化学或生物的方法除去未结合的分子。
本发明还提供了一种通过DNA编码化合物库筛选同时结合E3泛素连接酶和靶标蛋白的化合物的方法，包括以下步骤：
a、将E3泛素连接酶用第1种互补标签的一方进行标记，将靶标蛋白用第2种互补标签的一方进行标记，第1种互补标签和第2种互补标签不同；
b、将带有第1种互补标签的一方的E3泛素连接酶和带有第2种互补标签的一方的靶标蛋白与DNA编码化合物库同时进行孵育；
c、加入第1种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物；
d、将带有第1种互补标签的一方的E3泛素连接酶和带有第2种互补标签的一方的靶标蛋白与步骤c解离得到DNA编码化合物同时进行孵育；
e、加入第2种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物。
进一步地，第1种互补标签的一方为His标签，第1种互补标签的另一方为镍。
进一步地，第2种互补标签的一方为GST标签，第2种互补标签的另一方为谷胱甘肽。
进一步地，第1种互补标签的另一方、第2种互补标签的另一方固定在固相载体上；优选地，所述固相载体为磁珠。
进一步地，所述步骤b、步骤d中孵育是在筛选缓冲液中进行，所述筛选缓冲液含有鲑鱼精DNA；更进一步地，筛选缓冲液为1)20mM磷酸缓冲液，包含150mM氯化钠,0.3mg/ml 鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0，或者2)50mM HEPES((4-(2-羟乙基)-1-哌嗪乙磺酸)缓冲液，包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0。
进一步地，所述步骤c中加入镍包被磁珠，通过离心除去未结合的分子。
更进一步地，步骤c后加入洗脱缓冲液，重复洗脱5～10次；具体地，洗脱缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0，或者2)50mM HEPES缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7。
更进一步地，步骤e除去未结合的分子后，加入洗脱缓冲液，重复洗脱3～5次；
进一步地，所述步骤c、步骤e通过加热从生物靶标上解离得到DNA编码化合物。优选地，所述加热温度为85～95℃。优选地，加热解离时间为10～20分钟。
更进一步地，解离在解离缓冲液中进行；具体地，解离缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,pH＝5.5，或者2)10-50mM MES(2-(N-吗啉代)乙磺酸)缓冲液，包含150mM氯化钠,pH＝6，或者3)20mM磷酸缓冲液,包含150mM氯化钠,pH＝7.0；或者4)50mM HEPES缓冲液,包含150mM氯化钠,pH＝7.0。
进一步地，所述步骤e中加入谷胱甘肽包被磁珠，通过离心除去未结合的分子。
进一步地，所述步骤e中从生物靶标上解离得到DNA编码化合物后对DNA分子数进行qPCR定量。
更进一步地，如果qPCR定量结果分子数超过10 8，则将解离得到DNA编码化合物作为新一轮DNA编码化合物库使用，重复再一次筛选直至分子数降至10 7～10 8。
进一步地，对解离得到DNA编码化合物进行测序，将测序结果解码，得到富集信号。
在本发明的一些具体实施方案中，所述E3泛素连接酶为CRBN、VHL、MDM2、IAPs、DCAF15、DCAF16、RNF4和RNF114。
本发明方法还可用于筛选能同时亲和组织特异性蛋白和治疗靶点蛋白的分子，用于靶向用药、减低毒副作用、提高目标组织药物吸收，增强药物生物利用度等。本发明方法还可以用于筛选同时作用双靶点的药物分子，用于具有双重机制(Dual Mechanisms)的疾病治疗。显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步地详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的 技术均属于本发明的范围。
附图说明
图1是本发明实施例1中在DNA编码化合物库中加入的带有不同DNA标签的6个阳性化合物的结构；
图2是本发明实施例1中6个阳性化合物在DNA编码化合物库筛选不同样本中的信号强度；
图3是本发明实施例1中dBET1和6个DNA编码阳性化合物对应的蛋白降解剂结构；
图4是本发明实施例1中dBET1和6个蛋白降解剂在MV4；11细胞中对BRD4蛋白降解实验图；
图5是本发明实施例2中针对CRBN为E3连接酶的DNA编码化合物库结构示意图；
图6是本发明实施例2中通过对CRBN和BRD4蛋白的筛选获得的化合物信号图，以及4个代表性化合物结构的Tanimoto相似性比较(R 2、R 3用具体试剂代码表示)；
图7是本发明实施例2中对CRBN和BRD4蛋白的筛选获得代表性的4个PROTAC分子在MV4；11细胞中对BRD4蛋白降解试验结果；
图8是本发明实施例2中化合物75-NX-1和其不含沙利度胺和Linker的对应化合物57-NX-1对MV4；11细胞增殖抑制试验结果；
具体实施方式
实施例1、筛选能同时结合CRBN和BRD4的化合物
按照WO2006135786中所述的方法构建DNA编码化合物库。在DNA编码化合物库中额外加入图1所示的带有DNA标签的阳性对照化合物。实施例1中使用的蛋白CRBN-His tag/DDB1由HitGen内部表达，蛋白BRD4-GST tag为来自Active Motif(货号81855)。
本实施例使用的筛选缓冲液为1)20mM磷酸缓冲液，包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0；或者2)50mM HEPES((4-(2-羟乙基)-1-哌嗪乙磺酸)缓冲液，包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0。
本实施例使用的洗脱缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0；或者2)50mM HEPES缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7。
本实施例使用的解离缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,pH＝5.5；或 者2)10-50mM MES(2-(N-吗啉代)乙磺酸)缓冲液，包含150mM氯化钠,pH＝6；或者3)20mM磷酸缓冲液,包含150mM氯化钠,pH＝7.0；或者4)50mM HEPES缓冲液,包含150mM氯化钠,pH＝7.0。
按照下述分组对靶标蛋白和DNA编码化合物库在筛选缓冲液中进行孵育。
组号 靶标蛋白
1 CRBN/DDB1复合物(250pmol)
2 BRD4(250pmol)
3 CRBN/DDB1复合物/BRD4(各250pmol)
4 镍包被磁珠和谷胱甘肽包被磁珠
对于分组1、组2和组4，将筛选溶液在封闭的离心管中室温低速摇床孵育1小时。然后在分组1中加入镍包被磁珠，在分组2中加入谷胱甘肽包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入200～500μL洗脱缓冲液孵育1min，进一步离心洗脱，反复3～5次。加入50～100μL解离缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。qPCR定量上清液中DNA分子数，如分子数超过10 8，将上清液作为新一轮DNA编码化合物库使用，调节pH以及纯化柱脱盐去除额外缓冲液成分后，分别重复再一次筛选直至分子数降至10 7～10 8。使用Illumina NovaSeq6000或HiSeq2500及其配套标准试剂进行测序。将测序结果进一步分析解码，得到各组富集信号分布图。
对于分组3，将筛选溶液在封闭的离心管中室温低速摇床孵育1小时。然后加入镍包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入200～500μL洗脱缓冲液孵育1min，进一步离心洗脱，反复5～10次。加入50～100μL解离缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。将上清液调节pH以及纯化柱脱盐去除额外缓冲液成分后，作为新一轮DNA编码化合物库使用，再一次重复与CRBN/DDB1复合物/BRD4进行孵育，室温下低速摇床孵育1小时。加入谷胱甘肽包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入50～100μL洗脱缓冲液孵育1min，进一步离心洗脱，反复3～5次。加入50～100μL洗脱缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。qPCR定量上清液中DNA分子数，如分子数超过10 8，将上清液作为新一轮DNA编码化合物库使用，分别重复再一次筛选直至分子数降至10 7～10 8。使用Illumina NovaSeq6000或HiSeq2500及其配套标准试剂进行测序。将测序结果进一步分析解码，得到各组富集信号分布图。
图2展示了6个阳性化合物在筛选4个分组中分别的信号强度(Sequence Count)。第1、 2分组中的结果b这6个化合物分别针对于CRBN和BRD4蛋白的结合情况，但无法说明这些化合物同时结合CRBN和BRD4两个蛋白的最佳组合。第3分组结果显示Conjugate-4明显为同时结合两个蛋白的最佳化合物，其链长与dBET1完全相同。通过这6个化合物对应的PROTAC分子(不含DNA标签，但包含链接DNA的甲酰胺结构，dBET1和6个PROTAC分子的结构如图3)进一步验证图1中6个化合物与筛选结果的对应关系，使用Western Blot的方法针对MV4；11(对BRD4蛋白降解敏感的细胞系)进行BRD4蛋白降解的评价，结果如图4。图4中PROTAC-4蛋白降解效果最优，与筛选分组3中Conjugate-4的化合物信号一致。
实施例2、通过同时结合CRBN和BRD4的化合物筛选获得新颖的蛋白降解剂
按照WO2006135786中所述的方法构建如图5的DNA编码化合物库，然后对于该化合物库进行筛选。本实施例使用的蛋白CRBN-His tag/DDB1由HitGen内部表达，蛋白BRD4-GST tag为来自Active Motif(货号81855)。
本实施例使用的筛选缓冲液为1)20mM磷酸缓冲液，包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0；或者2)50mM HEPES((4-(2-羟乙基)-1-哌嗪乙磺酸)缓冲液，包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0。
本实施例使用的洗脱缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7.0；或者2)50mM HEPES缓冲液,包含150mM氯化钠,0.3mg/ml鲑鱼精DNA,0.1％吐温-20,10mM咪唑(仅对镍包被磁珠),pH＝7。
本实施例使用的解离缓冲液为1)20mM磷酸缓冲液,包含150mM氯化钠,pH＝5.5；或者2)10-50mM MES(2-(N-吗啉代)乙磺酸)缓冲液，包含150mM氯化钠,pH＝6；或者3)20mM磷酸缓冲液,包含150mM氯化钠,pH＝7.0；或者4)50mM HEPES缓冲液,包含150mM氯化钠,pH＝7.0。
按照下述分组对靶标蛋白和DNA编码化合物库在筛选缓冲液中进行孵育。
组号 靶标蛋白
1 CRBN/DDB1复合物(250pmol)
2 BRD4(250pmol)
3 CRBN/DDB1复合物/BRD4(各250pmol)
4 镍包被磁珠和谷胱甘肽包被磁珠
对于分组1、组2和组4，将筛选溶液在封闭的离心管中室温低速摇床孵育1小时。然后在分组1中加入镍包被磁珠，在分组2中加入谷胱甘肽包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入200～500μL洗脱缓冲液孵育1min，进一步离心洗脱，反复3～5次。加入50～100μL解离缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。qPCR定量上清液中DNA分子数，如分子数超过10 8，将上清液作为新一轮DNA编码化合物库使用，调节pH以及纯化柱脱盐去除额外缓冲液成分后，分别重复再一次筛选直至分子数降至10 7～10 8。使用Illumina NovaSeq6000或HiSeq2500及其配套标准试剂进行测序。将测序结果进一步分析解码，得到各组富集信号分布图。
对于分组3，将筛选溶液在封闭的离心管中室温低速摇床孵育1小时。然后加入镍包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入200～500μL洗脱缓冲液孵育1min，进一步离心洗脱，反复5～10次。加入50～100μL解离缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。将上清液调节pH以及纯化柱脱盐去除额外缓冲液成分后，作为新一轮DNA编码化合物库使用，再一次重复与CRBN/DDB1复合物/BRD4进行孵育，室温下低速摇床孵育1小时。加入谷胱甘肽包被磁珠，室温孵育30min。离心250g x 3min,去掉包含未结合分子的上清液。加入50～100μL洗脱缓冲液孵育1min，进一步离心洗脱，反复3～5次。加入50～100μL洗脱缓冲液，与85～95℃加热解离10～20分钟，离心收集上清液。qPCR定量上清液中DNA分子数，如分子数超过10 8，将上清液作为新一轮DNA编码化合物库使用，分别重复再一次筛选直至分子数降至10 7～10 8。使用Illumina NovaSeq6000或HiSeq2500及其配套标准试剂进行测序。将测序结果进一步分析解码，得到各组富集信号分布图。
图6展示了实施例2分组3中化合物库针对于CRBN和BRD4两个蛋白富集的情况，如实施例1中信号的关系，信号与库化合物分子与CRBN和BRD4结合稳定性有正相关关系。通过挑选分组3中不同信号强度的化合物进行验证，选定的化合物对应Sequence Count如图6所示。四个代表性的化合物与BRD4抑制剂JQ1(dBET1与BRD4结合的部分)的Tanimoto相似性(越接近于0越不相似，越接近于1越相似)显示这些化合物为通过DNA编码化合物库筛选出的新结构。通过合成4个PROTAC分子(75-NX-1，78-NX-1，123-NX-1，127-NX-1，如图6)进行针对于MV4；11细胞的BRD4蛋白降解实验。结果表明筛选信号的强弱与蛋白降解存在正向相关关系(图7)。进一步对化合物75-NX-1和其不含沙利度胺和Linker部分的化合物57-NX-1进行MV4；11细胞增殖抑制实验。结果表明，PROTAC分子与抑制剂相比，具有明显增强的细胞活性。
综上所述，本发明的方法可以通过DNA编码化合物库筛选同时结合E3泛素连接酶和靶标蛋白的化合物，进行新颖蛋白降解剂(PROTAC)的发现，并能将这些化合物的蛋白降解能力排序，体现很好的商业应用价值。这一方法也进一步拓宽了DNA编码化合物库的应用范围，具有较高的筛选效率和准确度，可适用于各类同时结合生物靶标的筛选。

Claims (15)

  1. 一种通过DNA编码化合物库筛选同时结合多个生物靶标的化合物的方法，包括以下步骤：
    a.将n种生物靶标分别用n种不同的互补标签的一方进行标记；
    b.将n种带有标签的生物靶标和DNA编码化合物库同时进行孵育；
    c.加入第1种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物；
    d.将解离得到DNA编码化合物替换步骤b中的DNA编码化合物库，将步骤c中将第1种互补标签的另一方替换为第2种互补标签的另一方，重复步骤b和步骤c，直至步骤c加入第n种互补标签的另一方；
    其中，n为2、3、4或5。
  2. 根据权利要求1所述的方法，其特征在于：所述生物靶标为蛋白、RNA、DNA。
  3. 根据权利要求1所述的方法，其特征在于：所述互补标签的一方为生物素标签、His标签、GST标签、MBP标签、FLAG标签、C-Myc标签、SUMO标签、SNAP标签、互补DNA的一方、互补RNA的一方。
  4. 根据权利要求1所述的方法，其特征在于：所述互补标签的另一方为亲和素、链霉素、镍、谷胱甘肽、多糖树脂、anti-FLAG、anti-C-Myc、SUMO蛋白酶、苯甲基鸟嘌呤(benzylguanine,BG)、互补DNA的另一方、互补RNA的另外一方。
  5. 根据权利要求1所述的方法，其特征在于：步骤c和步骤d中加入第1～n种互补标签的另一方后，通过物理、化学或生物的方法除去未结合的分子。
  6. 一种通过DNA编码化合物库筛选同时结合E3泛素连接酶和靶标蛋白的化合物的方法，包括以下步骤：
    a、将E3泛素连接酶用第1种互补标签的一方进行标记，将靶标蛋白用第2种互补标签的一方进行标记，第1种互补标签和第2种互补标签不同；
    b、将带有第1种互补标签的一方的E3泛素连接酶和带有第2种互补标签的一方的靶标蛋白与DNA编码化合物库同时进行孵育；
    c、加入第1种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物；
    d、将带有第1种互补标签的一方的E3泛素连接酶和带有第2种互补标签的一方的靶标蛋白与步骤c解离得到DNA编码化合物同时进行孵育；
    e、加入第2种互补标签的另一方，除去未结合的分子，从生物靶标上解离得到DNA编码化合物。
  7. 根据权利要求6所述的方法，其特征在于：第1种互补标签的一方为His标签，第1种互补标签的另一方为镍。
  8. 根据权利要求6所述的方法，其特征在于：第2种互补标签的一方为GST标签，第2种互补标签的另一方为谷胱甘肽。
  9. 根据权利要求6所述的方法，其特征在于：第1种互补标签的另一方、第2种互补标签的另一方固定在固相载体上；优选地，所述固相载体为磁珠。
  10. 根据权利要求6所述的方法，其特征在于：所述步骤b、步骤d中孵育是在筛选缓冲液中进行，所述筛选缓冲液含有鲑鱼精DNA。
  11. 根据权利要求6所述的方法，其特征在于：所述步骤c中加入镍包被磁珠，通过离心除去未结合的分子。
  12. 根据权利要求6所述的方法，其特征在于：所述步骤c、步骤e通过加热从生物靶标上解离得到DNA编码化合物；优选地，所述加热温度为85～95℃。
  13. 根据权利要求6所述的方法，其特征在于：所述步骤e中加入谷胱甘肽包被磁珠，通过离心除去未结合的分子。
  14. 根据权利要求6所述的方法，其特征在于：所述步骤e中从生物靶标上解离得到DNA编码化合物后对DNA分子数进行qPCR定量。
  15. 根据权利要求6所述的方法，其特征在于：对步骤e中解离得到DNA编码化合物进行测序，将测序结果解码，得到富集信号。
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