Application of Yeast Two-hybrid Technology in Screening Drugs and Finding Drug Targets

The yeast two-hybrid system is an important technical means for studying protein-protein interactions, and is now also being used in high-throughput screening of interacting proteins and other related molecules. Yeast transformation is simple and efficient. After the yeast two-hybrid library is constructed, it is relatively easy to directly screen out positive clones that interact between proteins from the eDNA library or genomic library. Yeast two-hybrid library construction generally includes nuclear system and membrane System two methods.

The yeast two-hybrid technique can be used to determine the interaction between peptide drugs used in treatment and proteins derived from tumors, bacteria, and viruses, which can be used to screen peptide drugs and find drug targets. Medicilon provides yeast two-hybrid services. It has independent high-quality laboratories and professional experimenters, can issue authoritative test data reports, and provide detailed yeast two-hybrid experiment procedures, original data and pictures, and result analysis.

Using yeast two-hybrid technology combined with important contemporary biophysical technologies such as SPR and In Cell technology, cell transcription activation experimental technology, etc., it can carry out the use of estrogen receptor (ER), farnesoid derivative X receptor (FXR) and liver X receptor (LXR) is a drug lead compound discovery study that is a target for drug screening. Through the high-throughput screening of the existing laboratory compound library, three small-molecule compounds with brand-new structures (including one natural product molecule and one marine natural product molecule) were discovered, which are the selectivity of ER. Agonist, antagonist of FXR and agonist of LXR. By studying their corresponding biological activities and physiological functions, it has been confirmed that they are brand-new nuclear receptor ligands. The results obtained in the research provide the discovery of lead compounds for anti-metabolic diseases based on these three types of nuclear receptors as targets. Important structural information.

The outstanding advantage of the yeast two-hybrid technology is that the “X” protein can be used as a “bait” to screen all the “Y” proteins and their genes that can bind to the “X” protein from the gene library. The two-hybrid system is used to screen the peptides that interact with the target protein. If the target protein is the target of drug design, some small-molecule peptide drugs with drug therapeutic effects can be obtained. For example, some researchers in my country constructed yeast expression vectors by fusing random DNA fragments with AD of GAL4, and successfully constructed a yeast two-hybrid random peptide library that can be screened and amplified, which can be used to screen and design target protein-interacting peptides (mainly Used for drug screening design). In addition, this technology can also be used to find compounds that regulate protein interactions. It can be used to determine the two interacting proteins. After adding a molecular compound to the two-hybrid system that restores transcriptional activity, the expression intensity of the reporter gene can be tested. Look for small molecule compounds that inhibit protein-protein interactions.

Now small molecules that can inhibit the Src SH2 domain and interact with ligands have been developed and used to treat osteoporosis, especially to inhibit osteoclast resorption. However, in the process of designing these small molecule inhibitors and modulators, a historical challenge was encountered: these modular domains are highly homologous in their respective families, and it is difficult to develop a highly specific inhibitor for a specific interaction. . Foreign researchers use the three-hybrid method to screen small molecule kinase inhibitors in the proteome, because various known CDK inhibitors, including purines and indenopyrazole analogs, are based on methotrexate-based hybrid ligands The form of cDNA library or yeast cell array-based screening can be used to identify kinase targets in the cell cycle, proving that this three-hybrid system can be used to find drug targets.

Although the yeast two-hybrid system has a wide range of applications, it also has some shortcomings, mainly in the following aspects:

(1) Non-universal applicability

Not all proteins are used in the yeast two-hybrid system. The proteins used in the system must be located in the nucleus to activate the expression of the reporter gene. Therefore, proteins that cannot be transported to the nucleus, such as cytoplasmic proteins and cell membrane proteins, etc. Apply this method.

(2) False positive

That is to say, the interaction of proteins observed by yeast two-hybrid may not necessarily occur in real situations, which is one of the important drawbacks that cannot be ignored in yeast two-hybrid.

(3) False negative

Certain expressed fusion proteins are usually toxic in yeast strains, thereby inhibiting the expression of the reporter gene and the growth of yeast, or the interaction between the proteins is weak, the reporter gene is not expressed or the expression level is so low that it cannot be detected, making Yeast two-hybrid showed false negative results.