The Necessity of Label-Free Target ID

Traditional ophthalmic drug discovery has focused on the functional modulation of druggable proteins associated with the fault of protein signaling networks in ocular diseases. Target protein identification (ID) is considered to be a significant bottleneck for further drug development with this method. Whether it is target-based drug discovery or phenotypic drug discovery, due to the limited binding affinity between the target protein and the hit compound, the synthesis of target ID probes is difficult, so there is an urgent need for label-free target identification methods that do not require structural modification. Label-free target ID methods monitor target engagement by following changes in the proteolytic, chemical, or thermal stability of the target protein. In these methods, researchers can examine the binding event of a small molecule to its target protein without structural modification of the original molecule.

Fig. 1. General schemes for label-free target identification. (Ha J, et al., 2021)

Ace Therapeutics' Label-Free Target Identification Services

Thanks to a deep understanding of ocular proteomics and researchers with decades of experience in ophthalmic drug discovery, Ace Therapeutics has developed a unique label-free protein target screening service to identify target proteins associated with ocular diseases and discover small molecules that bind to specific protein targets, enabling hit identification of targets not suitable for routine analysis.

At Ace Therapeutics, our protein scientists have developed a complete workflow for label-free target identification. Our label-free target identification protocol is considered an optimal solution for small molecules with low binding affinity to the target protein. Following the binding of a small molecule to its target protein, our protein scientists use a variety of strategies to analyze the thermal, proteolytic, or chemical stability of the protein to identify the target protein of interest.

Analysis Strategies for Label-Free Target Identification Services

Fig. 2. Analysis strategies for label-free target identification.

• Target Protein Stability Analysis

• Drug affinity reaction target stability (DARTS): Protein samples are separated by SDS-PAGE, and the intact target protein after protease degradation after drug treatment is detected by limited proteolysis (LiP) mass spectrometry.
• Stability of proteins from rates of oxidation (SPROX): The extent of protein denaturation is assessed by pulse proteolysis (PP), which is described by intact protein levels or oxidized methionine levels after proteolysis or hydrogen peroxide treatment, respectively.

• Target Protein Screening

Scientists at Ace Therapeutics use thermostability-shifted fluorescent difference two-dimensional gel electrophoresis (TS-FITGE) to screen target proteins. During the entire analysis process, the target protein group and the target protein-drug treatment group are separated by different dyes labeled, followed by two-dimensional gel electrophoresis. Based on the quantitative analysis of the fluorescent gel images, the fluorescence intensity ratio (target protein-drug treatment group/target protein group) of each protein was calculated, and finally the target protein was selected according to whether the ratio shifted.

• Target Protein ID

Thermal stability and dissolution stability of the target protein are the main criteria for identification. Throughout the analysis, our expert team tags peptides with a tandem mass tag (TMT), and then uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to illustrate the melting profile of each protein. Calculate the melting point shift (ΔT_m) or area under the curve (ΔS_m) from the melting curve, and identify the target protein based on the melting curve shift or area under the curve.

Ace Therapeutics' protein scientists have extensive experience in target ID. We provide global customers with high-quality label-free protein target identification services for the development of small molecule compounds in ophthalmology. If you are interested in our services or want to know more information, please feel free to contact us. We look forward to cooperating with you.

Reference

1. Ha J, Park H, Park J, et al. Recent advances in identifying protein targets in drug discovery. Cell Chem Biol. 2021:18;28(3):394-423.

• RNAi-Based Target Screening
• Computer-Aided Target Identification and Verification
• Gene Expression Profiling-Based Screening
• Genetic Engineering-Based Target Screening
• CRISPR/Cas9-Based Target Screening
• Mechanism of Action (MOA) Study
• Target Functional Annotation and ID
• Target Structure Determination

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