A DNA-encoded compound library drug molecule fishing method

A drug molecule and compound library technology, applied in the field of DNA-encoded compound library drug molecule fishing, can solve problems such as the inability to meet the protein interaction mechanism and the inability to function at the protein interaction level, and achieve sample cost savings, low cost, and high sensitivity. Effect

Active Publication Date: 2022-07-19
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional DEL only works on a single protein target and cannot work at the protein-protein interaction level
With the research on targeted inhibition of cancer, drug screening only targeting a single target obviously cannot meet the needs of protein interaction mechanism
At present, there is no report on the combination of DEL and SERS for molecular fishing and its application in small molecule drug screening

Method used

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  • A DNA-encoded compound library drug molecule fishing method
  • A DNA-encoded compound library drug molecule fishing method
  • A DNA-encoded compound library drug molecule fishing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of DNA-encoded compound library

[0035] 1. Oligonucleotide-compound ligation reaction

[0036] Dissolve a compound (1.25 μmol) in DMSO and dilute it to 230 μL, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (12 μL 100 mM in DMSO) and N-hydroxysuccinimide (NHS) (10 μL of 330 mM in DMSO / H2 O 2 : 1) After activation at 30°C for 20 minutes, add triethylamine (TEA) / HCl (50μL 500mM pH10.0) and corresponding oligonucleotides (30μL 500μM) and stir overnight at 30°C. Tris-HCl (20 μL, 500 mM, pH 8.0) was added to terminate the reaction after stirring at 30°C for 1 h, and then triethylamine acetic acid (TEAA) (500 μL, 100 mM, pH 7.0) was added. After HPLC purification, it was dried under reduced pressure, redissolved in 100 μL of H20, and then checked for concentration and quality with UV spectrophotometer and LC-ESI-MS. A total of 40 different oligonucleotide-compounds were ligated using the method described above. The structure o...

Embodiment 2

[0047] Example 2 Molecular Fishing

[0048] Synthesis of silver nano-ion probes (AgNPs): Take a 250mL three-necked flask and soak it in an acid tank overnight, take it out the next day, rinse it, and then wash it with water for injection three times to ensure that the wall is pure and free of impurities. Put in the oven to dry and cool to room temperature. During this period, 0.018 g of silver nitrate powder was accurately weighed and poured into a three-necked flask carefully. Add 100 mL of ultrapure water, shake well, connect the condenser tube and set the temperature of the electric heating mantle to 100 °C, stir and heat the solution to a slight boil, and immediately add 2 mL of 1% sodium citrate solution. Observe the color change of the solution: light yellow, dark yellow, and then gray-green, no change in color is observed, slightly lower the temperature to 90°C, continue magnetic stirring, and maintain for 40min to obtain a silver nanoparticle dispersion system. Measu...

Embodiment 3

[0050] Example 3 Compound structure determination

[0051] The information of the DNA fragments is determined by PCR amplification and DNA sequencing according to the oligonucleotide fragments connected to the compounds, thereby obtaining the structure information of the compounds.

[0052] Take 1 μL of the supernatant as a template, add 10 μM primers (the primer structures are 5'-GCC TCC CTC GCGCCA TCA GGG AGC TTG TGA ATT CTG G-3' (SEQ ID NO. 11), 5'-GCC TTG CCA GCC CGC TCAGGT AGT CGG ATC CGA CCA C-3' (SEQ ID NO. 12)) 4*2mM dNTP, 1.25U Taq enzyme and PCRbuffer. The PCR steps were as follows: DNA denaturation at 94°C for 2 min, 4 cycles of 94°C for 40s, 30 cycles of 54°C for 40s, 72°C for 40s, 94°C for 40s, 64°C for 30s, 72°C for 30s, and 72°C for 7 minutes. After the reaction, the corresponding samples were mixed and purified with an ion exchange column, and extracted with 50 μL of QE buffer.

[0053] The information of the DNA fragments is determined according to the resul...

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Abstract

In the present invention, the metal nanoparticle probe is coupled with the target protein or the target protein-binding receptor protein, and is added to a pre-prepared DNA-encoded compound library for incubation to carry out molecular fishing, and targeted drug information is obtained through PCR amplification and DNA sequencing. Then, metal nanoparticle probes and magnetic bead probes were prepared and coupled with Raman signal molecules, respectively, and the metal nanoparticle probes or magnetic bead probes were respectively coupled with target proteins or target protein-binding receptor proteins, and added The Raman signal is detected in the targeted drug obtained by screening to determine whether the compound to be screened is a targeting inhibitor, and the structure of the screened compound is obtained by PCR amplification and DNA sequencing. The present invention combines molecular fishing with a DNA-encoded compound library for the first time, performs drug screening at the protein interaction level, and combines surface-enhanced Raman scattering spectroscopy to form a simple and sensitive new drug screening method.

Description

technical field [0001] The invention belongs to the technical field of drug screening, and in particular relates to a drug molecule fishing method for a DNA-encoded compound library. Background technique [0002] Molecule Fishing is a high-throughput bioanalytical method based on the interaction of drug targets and active molecules to affinity select ligands from complex biological samples to realize the discovery of lead compounds, with strong specificity. , high efficiency, less requirements for sample pretreatment, etc., is an important technological progress on the basis of traditional high-throughput screening methods (High Throughput Screening, HTS). It generally consists of four parts: target immobilization (enzyme, cell, etc.), affinity fishing, ligand elution and LC-MS analysis. Molecular fishing technology includes molecular fishing technology in offline mode and molecular fishing technology in online mode. In molecular fishing in offline mode, affinity fishing a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B30/04C40B40/08
CPCC40B30/04C40B40/08
Inventor 梁重阳徐抒平戴璐
Owner JILIN UNIV
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