Methods for the Discovery and Identification of Small Molecules Targeting Oxidative Stress-Related Protein–Protein Interactions: An Update
Abstract
:1. Introduction
2. Proximity-Based Methods
2.1. Alpha Technology
2.2. Fluorescence Resonance Energy Transfer (FRET)
3. Affinity-Based Methods
3.1. Thermal Shift Assay
3.2. Isothermal Titration Calorimetry (ITC)
3.3. Surface Plasmon Resonance (SPR)
3.4. Biolayer Interferometry (BLI)
3.5. Microscale Thermophoresis (MST)
3.6. DNA-Encoded Library
4. Competition-Based Methods
4.1. Fluorescence Polarization (FP)
4.2. Pull-Down
4.3. Co-Immunoprecipitation (Co-IP)
4.4. Enzyme Linked Immunosorbent Assay (ELISA)
5. Structure-Guided Methods
5.1. Tethering
5.2. Fragment-Based Drug Design (FBDD)
5.3. Crystal Soaking
5.4. Computational Docking
6. Functional-Based Methods
Protein Fragment Complementation Assays (PCAs)
7. Case Study
7.1. Identification of Type I PPI Inhibitors
7.2. Identification of Type II PPI Inhibitors
7.3. Identification of Type III PPI Inhibitors
7.4. Identification of Type IV PPI Inhibitors
7.5. Identification of Type V PPI Inhibitors
8. Conclusions
9. Perspectives
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Assay | Alpha Technology | FRET Technology | |||
---|---|---|---|---|---|
Alpha- Screen | Alpha- LISA | FRET | TR-RET | HTRF | |
Excitation wavelength (nm) | 680 | 436 | 320 | ||
Emission wavelength (nm) | 520–620 | 615 | 528 | 665 | |
Distance (nm) | <200 | <10 | |||
Advantage |
|
| |||
Disadvantage |
|
| |||
Applicable PPI type | I, II | I, II | |||
HTS suitability | Yes | Yes |
Assay | Advantages | Disadvantages | Applicable PPI Type | HTS Suitability |
---|---|---|---|---|
Thermal Shift |
| Not suitable for some proteins that have no ideal thermal denaturation curve and stability information cannot be extracted | All types | No |
ITC |
|
| All types | No |
SPR |
|
| All types | No |
BLI |
|
| All types | No |
MST |
| No accurate information on stoichiometry can be obtained. | All types | No |
Assay | Advantage | Disadvantage | Applicable PPI Type | HTS Suitability |
---|---|---|---|---|
FP | Simple operation High throughput Real-time and homogeneity Friendly to experimenters | Interference from light scattering, quenching, and auto-fluorescence. | All types | Yes |
Pull Down | Direct protein–protein interactions can be verified Conjugated beads with strong affinity and high elution purity | Cannot fully reflect the true state of intracellular protein interaction, and the fusion expressed GST tag may change the original folding of the target protein structure. | All types | No |
Co-IP | Reflect the real interaction of target PPI in intact cells | Low affinity and transient protein–protein interactions may not be detected. May not reflect direct interaction, a third party may act as a bridge in between. | All types | No |
ELISA | Highly sensitivity | Time-consuming Weak interactions are hard to detect | All types | No |
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Wu, X.; Zhang, Q.; Guo, Y.; Zhang, H.; Guo, X.; You, Q.; Wang, L. Methods for the Discovery and Identification of Small Molecules Targeting Oxidative Stress-Related Protein–Protein Interactions: An Update. Antioxidants 2022, 11, 619. https://doi.org/10.3390/antiox11040619
Wu X, Zhang Q, Guo Y, Zhang H, Guo X, You Q, Wang L. Methods for the Discovery and Identification of Small Molecules Targeting Oxidative Stress-Related Protein–Protein Interactions: An Update. Antioxidants. 2022; 11(4):619. https://doi.org/10.3390/antiox11040619
Chicago/Turabian StyleWu, Xuexuan, Qiuyue Zhang, Yuqi Guo, Hengheng Zhang, **aoke Guo, Qidong You, and Lei Wang. 2022. "Methods for the Discovery and Identification of Small Molecules Targeting Oxidative Stress-Related Protein–Protein Interactions: An Update" Antioxidants 11, no. 4: 619. https://doi.org/10.3390/antiox11040619