Non-labeled Natural Product Target Identification and Validation

The structure of natural products is complex and rich, with a wide range of biological activities, and is an important source of new drug innovation. According to statistics, nearly half of the drugs approved by the U.S. Food and Drug Administration (FDA) for cancer treatment in the past 30 years Medicines are directly or indirectly derived from natural products and their derivatives. In 2015, abamectin and artemisinin derived from natural products were awarded the Nobel Prize in Medicine or Physiology, heralding the arrival of a new era of natural product medicines.

The target research of natural products is very important to clarify the mechanism of drug action and the development of new drugs. Many small molecule compounds exert corresponding pharmacological effects by acting on multiple targets. At present, the methods and technologies of natural product target recognition have made great progress. Progress, Yue et al. gave a detailed summary of traditional methods and technologies for natural product target identification from the aspects of genomics, proteomics, and computer-aided simulation, including affinity chromatography and activity protein expression profiles. based protein profiling, ABPP), etc. However, these technologies often require certain chemical structure modification tags for natural products, which may cause the natural products to lose their functional activity. In addition, the chemical structure modification sites of natural products are very limited and have become This type of method recognizes the interference factors of natural product targets, hindering the research and application of natural products. In view of the lack of label modification methods, in recent years, scientists have tried a variety of non-labeling methods on natural products (that is, there is no need to Methods for modifying product molecules) and have made certain progress. These new methods mainly include direct methods and indirect methods, including genomics, proteomics, and bioinformatics, which provide direct identification and confirmation of natural product targets. Or indirect evidence. This article reviews these non-labeled methods to provide methods and ideas for natural product target research.

1.Direct Method

With the development of technologies such as chemical biology, genomics, proteomics, chemical proteomics, and bioinformatics, more and more novel methods can be used to directly detect the interaction between natural products and target proteins. These technologies Mainly by using the principle of changing the stability of the drug after the drug is bound to the target protein, to identify the target that the natural product may act on. These novel drug target detection technologies include drug affinity responsive target stability (drug affinity responsive). target stability, DARTS), oxidation rate protein stability (stability of proteins from rates of oxidation, SPROX), protein thermal stability analysis (cellular thermal shift assay, CETSA), thermal proteome profiling (TPP), etc., And they have been widely used and recognized.

1.1DARTS

The concept of DARTS technology was first proposed by Lomenick and other related research in 2009. The research group proposed that the drug binds to the target protein to make the protein structure more stable and thus resistant to protease hydrolysis. In order to confirm this speculation, the researchers selected The FKBP12 protein combined with rapamycin and FK506 inhibitor activity was used as the research object. Subtilisin was used for hydrolysis, and the anti-hydrolysis effect of FKBP12 combined with natural products was observed by SDS-PAGE. The study confirmed that the drug and The protein is more stable after binding, and a systematic target recognition DARTS technology method has been established. The DARTS experiment requires a specific method to detect the enzymatic effect. The commonly used methods include SDS-PAGE and gel staining technology (such as Coomassie brilliant blue staining) In addition, protein two-dimensional electrophoresis (2D-PAGE), gel or non-gel mass spectrometry (LC-MS/MS) and other methods can also be used for detection. Through comparison, the obvious difference between the drug group and the control group can be identified. , And then find the candidate target of the drug for follow-up experimental confirmation. Because the DARTS technology is simple to operate and time-consuming, it has been widely used.

Kost et al. used DARTS technology to confirm that piperazine-like small molecule RX-5902 combines with phosphorylated p68RNA helicase to down-regulate the expression of c-Myc and other genes to inhibit the growth of cancer cells, revealing its anti-cancer mechanism. Jung Tanabe et al. used DARTS technology to identify the combination of KRIBB53 and OCT4 and degrade it, thereby causing OCT4-positive testicular germ cell cancer cells to undergo apoptosis. Tanabe et al. used DARTS technology to find that matrine can directly activate extracellular heat shock protein 90 (heat shock protein 90). shock protein 90, HSP90), which in turn enhances the axon growth and functional recovery of mice with spinal cord injury, confirming the feasibility of DARTS. DARTS technology does not require any chemical modification of natural products, and provides for the determination of the target that natural products directly bind to. It is convenient, but the ability of DARTS to identify targets with low protein abundance is very limited. The protease required for proteolysis in the DARTS experiment and the choice of cell lysate will affect the identification of natural product targets and interfere with the accuracy of DARTS technology.

1.2SPROX

Similar to DARTS, SPROX is another detection method based on ligand-induced target stability. Different from the DARTS technology to detect changes in proteolysis, SPROX mainly detects the oxidation level of methionine of the target protein, and the principle is to use drugs. After binding to the target, the antioxidant capacity of the target protein can be increased. When the protein complex is incubated with the natural product, the protein is oxidized with an oxidant such as hydrogen peroxide in the presence of a chemical denaturant. Then different types of mass spectrometry techniques (such as LC- MS/MS) to quantify selectively oxidized methionine. This technology uses the oxidation rate of methionine residues to analyze the thermodynamic properties of protein folding or unfolding reactions, and can detect and analyze multiple targets that interact with drugs Spot protein can also quantify the binding affinity of natural products to target protein.

DeArmond et al. used SPROX technology to find the target of resveratrol, the active ingredient of traditional Chinese medicine, and found that in addition to the previously identified cytoplasmic dehydrogenase, resveratrol also has 6 potential target proteins. Wallace et al. combined SPROX technology with iTRAQ Combined with mass spectrometry technology, 21 target proteins of the natural product ManassantinA were identified, laying the foundation for elucidating its mechanism of action. Xu et al. used SPROX technology to treat the natural product geldanamycin with good anti-tumor activity and its known targets The affinity of point HSP90 was studied, and it was confirmed that the dissociation constant (Kd value) depends on the time for geldanamycin and lysate to equilibrate before SPROX analysis. Although SPROX technology has good application potential, it also has some shortcomings: SPROX requires the oxidation of methionine residues to measure thermodynamic changes, but different methionine residues may show the same oxidation rate, which is not sufficient to provide sufficient information for the confirmation of the interaction between the natural product ligand and the target protein. ｡

1.3CETSA

The CETSA technology was first proposed and used by Molina in 2013. This method can be used to confirm the binding of drugs and targets in living cells, cell lysates and animal tissue samples. Similar to the above methods, CETSA is mainly based on natural products and The principle of the change in thermodynamic stability of the target protein after binding. After the natural product and the sample are incubated, they are heated separately under different temperature gradients. The target protein bound by the natural product has a higher thermal stability, and the unbound protein has a weaker stability It is more prone to degradation, and then use Western-Blot or mass spectrometry-based methods to analyze the thermal stability of the soluble protein according to its melting curve, so as to confirm the binding of the natural product to the intracellular protein.

Wang et al. used CETSA binding molecular docking and mass spectrometry and other technologies to identify the natural product curcumol extracted from the traditional Chinese medicine turmeric with multiple biological activities, which can bind to and degrade the NCL protein in nasopharyngeal carcinoma (NPC) cells. , Thus exerting an anti-cancer effect, confirming that NCL is one of the targets of curcumol in the treatment of nasopharyngeal cancer. Jin et al. combined CETSA technology with DARTS technology to confirm the relationship between the natural product geranynaringenin (CG902) and STAT3. It was found that CG902 inhibits the activity of STAT3 by activating SHP-2, thereby playing a role in cancer treatment. CETSA can also be used in animal tissues. Ishii et al. successfully evaluated mouse RIPK1 and drugs in the spleen and brain of mice using CETSA. The target engagement of CETSA indicates that CETSA can be effectively applied to preclinical and clinical drug development and provide effective tools for it. However, CETSA requires high drug concentration and is often used for target determination, which is useful for discovering new drugs. The target still has the disadvantage of insufficient flux.

1.4TPP

In order to overcome the shortcomings and challenges of CETSA’s low sensitivity and insufficient throughput, and increase its application in target and off-target recognition and biomarker recognition, Savitski developed TPP technology on the basis of CETSA in 2014. Natural products and After the solvent control was incubated with live cells or cell lysates, they were heated at 10 temperatures, and the undenatured soluble protein samples were incubated with TMT10 (isobaric tandem mass tag 10-plex) for LC-MS/MS detection and analysis Draw a melting curve after data standardization to find proteins with different stability. Franken et al. found that TPP can unbiasedly analyze the direct or indirect binding between natural products and target proteins in the temperature range of 37~67℃, and extract them with cells. The incubation of the liquid and the drug can detect the direct binding to the natural product. The TPP performed on the whole cell can be compared with the TPP result at the level of the cell extract to obtain the downstream target of the natural product. The key to the TPP method is to carry out the sample. TMT10 incubation and bioinformatics analysis.

Savitski et al. initially applied TPP to the target confirmation of the natural product staurophyllin extracted from bacteria, and identified more than 50 target proteins. Miettinen et al. used TPP technology to find that pabociclib can increase The thermal stability of 20S proteasome and the reduction of ECM29, which makes cells senescence, explains the mechanism of this CDK4/6 inhibitor in the treatment of breast cancer leading to cellular senescence, and provides ideas for the study of natural product targets. TPP technology has It has the advantages of good stability, large number of identification proteins, and no need to use antibodies for incubation detection. It is a broad-spectrum protein identification technology. However, TPP has problems such as time-consuming, high cost, and limited detection of membrane proteins.

2. Indirect Method

In recent years, methods for indirectly identifying and confirming the binding of non-labeled natural products to targets have also been gradually developed. Such as degradation tags (dTAG) and other protein degradation technologies, RNA interference (RNA interface, RNAi), and clustering rules Clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 gene editing technology and other genomics methods, and ConnectivityMap (CMAP) and other methods based on bioinformatics analysis (Figure 2). These technologies are mainly based on physiological Changes in response or biochemical characteristics infer drug targets. The following mainly describes these methods.

2.1 Protein degradation technology

Previously, protein degradation technologies such as phthalimide-based protein degradation technology and proteolysis targeting chimeras (PROTACs) have been reported, but these two technologies need to determine the target protein. Specific degradation ligands are not conducive to the identification and confirmation of natural product targets. You et al. summarized a variety of previously developed targeted and induced protein degradation technologies and found that more and more protein degradation technologies have been applied to natural product targets In the process of identification and confirmation. Nabet et al. recently invented a protein degradation technology called dTAG, which can be used as an important tool for drug target identification and confirmation. This technology uses CRISPR to combine an exogenous variant non-natural protein FKBP12F36V with a protein that needs to be degraded. The target protein is fused, and the membrane is better when used. The dTAG compound that can selectively bind with FKBP12F36V and E3 linkase (cereblon, CRBN) degrades the fusion protein and conducts biological research. dTAG technology is a broad-spectrum protein degradation Technology, a dTAG molecule can be used to accurately degrade a broad-spectrum protein. Nabet et al. proved that dTAG-13 is a highly selective, fast and efficient dTAG molecule. For candidate targets of natural products, dTAG molecules can be used for degradation. Perform analysis and confirmation. dTAG can be applied to cells, tissues and organisms, providing a very powerful tool for the identification and confirmation of natural product targets.

2.2 Genomics methods

In recent years, RNA-based technology has made great progress in the confirmation of drug targets. RNAi includes shRNA (short-hairpin RNA) and siRNA (small-interfering RNA), which are used as gene knockout tools to confirm natural products and targets Point interaction and screening of drug target genes. The expression level of target protein is manipulated by gene knockout methods to confirm the interaction between natural products and the target protein. These technologies have recently been combined with high-throughput methods to expand these The scope of application of genomics technology makes it possible to silence hundreds of genes in a single experiment, and it has become a powerful tool for indirect identification of natural product targets. Although RNAi has a very broad application prospect in natural product target identification, its The existing shortcomings such as off-target effects and false positive results still need to be overcome.

CRISPR/Cas9 is a gene editing technology developed in recent years, including CRISPRa and CRISPRi. Compared with RNAi technology, CRISPR/Cas9 technology can effectively overcome off-target effects. In 2014, the two research groups of Shalem and Wang reported that they can A CRISPR library for natural product target recognition, which has higher efficiency in natural product target screening. At present, scientists have established at least 13 CRISPR knockout files that can be used in humans libraries, 3 CRISPRa libraries and 2 CRISPRi libraries. These libraries can be used not only for drug target screening, but also for clinical research.

After random mutagenesis with the CRISPR library, the natural products or drugs such as anticancer drugs are incubated with the cells, the drug-resistant cells are collected, and their guide RNA (guide RNA, gRNA) sequence is analyzed and amplified and enriched, and the resulting gRNA is enriched. It is predicted to be the target gene of the drug. In order to make the candidate target gene obtained by screening meaningful, it is important to maintain the diversity of gRNA and the activity of Cas9. Kasap et al. used CRISPR/Cas9 with high-throughput sequencing and computational mutation discovery (computational mutation discovery) ) Developed a new technology named DrugTargetSeqR, and conducted experiments on ispinesib, an anticancer drug that has entered clinical trials. The experiment identified kinesin-5 as one of the candidate target genes of ispinesib, which is consistent with previous literature reports. Consistent. Using CRISPR/Cas9 technology, Wu et al. found that isobavachalcone, an active molecule derived from the traditional Chinese medicine psoralen, can regulate and inhibit the apoptosis and differentiation of acute myeloid leukemia cells by inhibiting dihydroorotate dehydrogenase (DHODH). The feasibility of this method.

2.3 Connectivity Map (CMAP) and other bioinformatics-based analysis methods

China Unicom Atlas CMAP is a biological application database based on gene expression profile data established by Broad Institute in 2006 (https://www.broadinstitute.org). The CMAP database is based on the principle of pattern matching, through a large-scale characteristic gene expression The Atlas database establishes the correlation between drugs, genes and diseases. The database was updated in 2017. There are more than 1.5 million genome-wide transcription data from about 5000 biologically active small molecules processed by human cells. Use of CMAP database Gene chip technology can obtain gene expression profiles of different small molecule drugs, and by comparing and analyzing the expression profile data of different samples, it can be widely used in the fields of new use of old drugs, new drug discovery, and drug action mechanism speculation.

In recent years, the CMAP database has also been gradually applied to the field of traditional Chinese medicine and natural products, mainly used in the identification of small molecule targets of natural products, revealing the mechanism of action of traditional Chinese medicine compounds, etc. In 2017, Lv and others of this research group established on the basis of the CMAP database. The first natural product small molecule gene expression profile database platform in China, which can be used in combination with CMAP to predict the pharmacological activity of natural product small molecules, natural product molecular targets and pathway identification, and natural product new drug creation and other fields.

The database platform initially selected 102 small molecules of common Chinese medicines to process standardized cells, obtained gene expression profiles and CMAP database for comparative analysis, and successfully applied to the natural product chlorinate chlorinate, and found that it has the ability to block α-adrenergic receptors. The new function of lowering blood pressure. In addition, Lv and others also used the database platform and the CMAP database to analyze and found that Tanshinone IIA can selectively act on PKCζ and PKCε targets, providing new ideas for understanding the anti-tumor mechanism of Tanshinone IIA.

3. Summary and Outlook

Traditional Chinese medicine and natural products are important treasure troves in the research of new drugs. However, the target research of natural products is not clear, which seriously hinders the development and internationalization of Chinese medicine. Clarify the role of natural product-target interactions in understanding natural products The mechanism and efficacy are very important, and it is urgent to find new target identification and validation methods. Compared with the method of labeling natural products, the method of non-labeling natural products can retain the structure and activity of natural products to the greatest extent, and expand its scope of application.

In the field of modern drug research and development, target recognition technology without chemical modification has been widely used, but there are still some shortcomings. In the case of unclear target information, first a large number of early experiments are required for the incubation concentration and incubation time of the drug It is more cumbersome to determine the conditions such as temperature and temperature. Secondly, the sensitivity of the target to non-labeled natural products is also an important limiting factor. For example, in the DARTS experiment, some proteins may also be affected by the combination of natural products. Protease hydrolysis makes the experimental results have a higher false negative rate. In CETSA and TPP technologies, there are also cases where natural products bind to the target to make the thermal stability of the target protein worse, so that many targets are not detected, causing experimental problems Error. In addition, it is not easy to find the target information of the corresponding natural product from the large amount of data generated by omics.

A better method for target identification and confirmation should meet the following conditions: one is to maintain the structure and activity of natural products to the greatest extent, and will not lead to a decrease or loss of activity; the other is to have broad-spectrum application It is not only suitable for high-abundance proteins, but also for low-content proteins, and is suitable for different tissues, cells, organisms, etc. With the development and update of proteomics, genomics, bioinformatics, and mass spectrometry technology , More new technologies will integrate the advantages of different technologies and reduce the cost and time of detection. In addition to being applied to target identification of natural products, these new technologies will also clarify the mechanism of action and toxicity of drugs more clearly, in order to solve New drug research and development, drug mechanism research, human disease marker identification and other difficult problems provide an important method basis.

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