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DNA coding compound library and compound screening method

A compound library and screening method technology, which is applied in the field of DNA-encoded compound library and compound screening, can solve the problems of limited application range, degradation, loss of function of DNA tags, etc.

Active Publication Date: 2019-08-06
HITGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, due to the different properties of different protein targets, when DNA exonuclease is added, the exonuclease may degrade the DNA tag of the covalently cross-linked DNA-encoded compound, thus limiting its application range
However, if strong separation and strong elution conditions such as electrophoresis are directly used for the above-mentioned covalently cross-linked DNA-encoded compounds, it may not be sufficient because only a small amount of hydrogen bonds between the protein target part and the DNA tag part are connected. Tolerance, resulting in partial dissociation of the protein target from the DNA tag, rendering the DNA tag ineffective

Method used

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  • DNA coding compound library and compound screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1, the synthesis of the starting DNA raw material of DNA coding compound library

[0075] Synthetic method one:

[0076] Step 1, the synthesis of compound 1

[0077]

[0078] The compound HUB1 (82nmol) was dissolved in sodium borate buffer (82 μL, pH=9.4, concentration 250mM / L) to form an initial solution with a concentration of 1mM / L; then the DMA solution of 4-azido-benzoic acid ( 8.2μmol, concentration 200mM / L), the DMA solution of HATU (8.2μmol, concentration 400mM / L) and the DMA solution of DIPEA (8.2μmol, concentration 400mM / L) were respectively placed in -20 ℃ refrigerator and mixed after cooling, the The mixture was vortexed to mix thoroughly, and then stored in a refrigerator at 4°C for 5 minutes. The above mixed solution was added to the starting solution of compound HUB1, vortexed and thoroughly mixed, and reacted at room temperature for 12 hours. Add 100 μL dd-H to 1 μL reaction solution 2 O dilution, LCMS to monitor the reaction. After th...

Embodiment 2

[0089] Embodiment 2, DNA coding compound library and screening

[0090] Using the starting DNA material prepared in Example 1, a DNA-encoded compound library with the following structure was constructed:

[0091]

[0092] The above-mentioned DNA-encoded compound library is screened according to the following steps:

[0093] 1) Incubate the above DNA-encoded compound library with the target protein ROCK2 in 100 μL buffer system for 60 minutes, and then react under 365nm light conditions for 60 seconds;

[0094] 2) Heat the reaction solution to 90°C to denature the protein, separate it by preparative SDS-PAGE (12-15%), recover and extract the corresponding target protein-DNA-encoded compound covalently cross-linked complex by gel cutting the strip;

[0095] 3) Perform PCR amplification and DNA sequencing on the recovered samples, read the DNA sequences corresponding to the enriched small molecular compounds, and then obtain the structural information of the compounds.

Embodiment 3

[0096] Embodiment 3, DNA coding compound synthesis

[0097] Through the screening of the DNA-encoded compound library in Example 2, after analyzing the structure of the compound, a compound without a DNA tag was re-synthesized, and its inhibitory activity IC on ROCK2 50 About 50nM. Use it as a tool compound to synthesize DNA-encoded compounds, and further verify the covalent cross-linking.

[0098] Step 1: Synthesis of compound 5

[0099]

[0100]Compound 3 (5 μmol) was dissolved in sodium borate buffer (5mL, pH=9.4, concentration 250mM / L) to make a starting solution with a concentration of 1mM / L; then the DMA solution of SM01 (10μmol, concentration 20mM / L ), the DMA solution of HATU (10 μmol, concentration 20mM / L) and the DMA solution of DIPEA (10 μmol, concentration 20mM / L) were respectively placed in a -20°C refrigerator and mixed, and the mixed solution was vortexed and thoroughly mixed. Then store in a refrigerator at 4°C for 5 minutes. The above mixed solution was...

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Abstract

The invention provides a DNA coding compound, a compound library and a compound screening method. Purifying or fixing on target protein is not needed by using the DNA coding compound library to perform screening, so that the DNA coding compound library can be suitable for the screening of complex systems such as non-immobilized protein, cell transmembrane protein and cell lysate. In addition, after the DNA coding compound covalently crosslinks with protein, the protein and DNA label sections are all covalently linked, various separating conditions, such as electrophoretic separation and strongelution conditions, can be tolerated, and the protein and the DNA label sections cannot be separated due to the harsh separating conditions.

Description

technical field [0001] The invention relates to a DNA coding compound library and a compound screening method. Background technique [0002] In drug development, especially 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 investment in equipment, limited number of library compounds (millions), and the construction of a compound library requires decades of accumulation, which limits the efficiency and efficiency of lead compound discovery. possibility. The DNA-encoded compound library synthesis technology that has emerged in recent years combines combinatorial chemistry and molecular biology techniques to add a DNA tag to each compound at the molecular level, and can synthesize hundreds of millions of compound libraries in a very short period of time. Moreover, compounds can be identified by...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869G01N33/68
CPCC12Q1/6869G01N33/68C40B40/08C40B30/04
Inventor 李进刘观赛罗华东陈璞睿常咏陈秋霞陈浩东万金桥
Owner HITGEN INC
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