Overview of Proteolysis Targeting Chimeras (PROTACs)
PROTACs represent a new protein degradation approach that works by using the body's ubiquitin-proteasome system to target and degrade specific proteins. PROTACs consist of three components: A PROTAC molecule consists of three fundamental parts which include a targeting ligand specific to the protein of interest known as a POI ligand along with an E3 ubiquitin ligase ligand and an intermediate linker. These molecules form a bifunctional small molecule complex through chemical linking. Through their simultaneous binding to both the target protein and E3 ubiquitin ligase PROTACs trigger ternary complex formation which flags the target protein for ubiquitination enabling proteasomal recognition and degradation.
Fig. 1 The degradation mechanism of PROTACs.
Importance of E3 ligase ligands in PROTACs
Recent scientific research has started the integration of fluorescent labeling into E3 ubiquitin ligase ligands to improve both visualization and functional study techniques for PROTACs. Researchers can monitor PROTAC interactions with target proteins and E3 ligases in real-time using fluorescently labeled E3 ligands which provide insights into PROTAC mechanisms.
The use of fluorescent E3 ligands introduces new methods to improve PROTACs. Scientific evaluation of PROTACs' potency and specificity becomes more intuitive through fluorescent signal observation which directs subsequent chemical modifications and optimizations. The technique enables researchers to identify new E3 ligase ligands which expands opportunities for PROTAC development.
PROTACs represent a groundbreaking targeted protein degradation technology that is reshaping the drug development field. The critical role of E3 ubiquitin ligase ligands in this process enables fluorescently labeled E3 ligands to create fresh research paths and applications for PROTACs.
Fig. 2 First reported Protac-1 (1) and the most common E3 ligase ligands.
Advantages of Fluorescent E3 Ligase Ligands
Enhanced Detection and Monitoring Capabilities
Using fluorescence imaging techniques researchers can observe protein degradation live with fluorescently labeled E3 ligands. Fluorescence polarization and time-resolved fluorescence resonance energy transfer (TR-FRET) technology enable detection of E3 ligands binding to target proteins or PROTACs to monitor PROTAC activity.
Real-Time Imaging and Quantification of Protein Degradation
Fluorescent labeling of ligands gives researchers real-time visual feedback to track protein degradation processes. Luciferase-labeled E3 ligands demonstrate improved fluorescence when lysine residues undergo mutation which aids in the enhanced monitoring of protein degradation processes.
Synthesis of Fluorescent E3 Ligase Ligands
Incorporating fluorescent tags into E3 ligase ligands primarily involves these approaches.
1. Chemical Modification
Chemical synthesis enables direct incorporation of fluorophores into E3 ligand structures. The CRBN ligand binds with a sulfonate ester precursor which results in a fluorescent covalently modified compound.
2. Molecular Design
The process of molecular modeling combined with optimization design allows researchers to identify appropriate linkage sites and introduce fluorescent groups. The optimization of fluorescently labeled ligands can be achieved through structure-based drug design (SBDD) and flexible drug design (FBDD) approaches which utilize crystallographic data.
3. Biosynthesis
Scientists use genetic engineering to incorporate fluorescent genes into the E3 ligand's coding sequence which results in fluorescently labeled proteins expression.
Applications of Fluorescent E3 Ligase Ligands
In Vitro Studies
- Application in Cell Experiments
Fluorescent E3 ubiquitin ligase ligands serve as essential components in PROTAC research. The application of fluorescent labeling allows scientists to observe PROTAC activity and protein degradation processes as they happen in real-time. A fluorescently tagged E3 ligand attaches to a target protein to establish a ternary complex which leads to target protein ubiquitination followed by proteasomal degradation. Fluorescence labeling technology enables researchers to assess both PROTAC molecule binding to E3 ligases and their degradation efficiency inside cells.
Fluorescently labeled ligands provide substantial benefits over non-fluorescent ligands for visualization purposes and real-time tracking capabilities. Fluorescence-based experimental methods enable researchers to measure target protein degradation rates with greater time resolution precision.
- Comparison with Non-Fluorescent Ligands
Non-fluorescent PROTAC molecules function effectively in select scenarios but require more elaborate detection and validation procedures. Fluorescently labeled PROTAC molecules allow direct cellular visualization through basic fluorescence imaging techniques which leads to substantial improvements in research productivity.
In Vivo Studies
- Potential for In Vivo Imaging and Monitoring of PROTAC Efficacy
The potential of fluorescent E3 ubiquitin ligase ligands emerges strongly from in vivo research studies. Scientists can track how PROTAC molecules distribute and degrade targets inside living organisms in real-time through imaging technologies when they add fluorescent tags to PROTACs. Research has proven that fluorescent tagging of PROTAC molecules allows for in vivo protein degradation which also demonstrates their therapeutic success via fluorescence signal alterations.
- Challenges and Future Directions
Fluorescently labeled PROTAC molecules provide substantial benefits for in vivo research but researchers must still address existing challenges. Biological background noise creates interference that complicates the interpretation of fluorescence signals. Fluorescent labeling of PROTAC molecules may alter their pharmacokinetic properties including how they metabolize and distribute in tissues.
Case Studies and Examples
VHL Ligands
- Development of Fluorescent VHL Ligands
VH298 represents a VH032 derivative that serves as a fluorescent probe in the HTRF VHL-Red Ligand system for analyzing VHL protein interactions. The fluorescently tagged ligand demonstrates important utility in PROTAC drug development by enabling scientists to explore how VHL ligands connect with their target proteins. VH298 serves as the recommended permeability control compound for VHL detection tests aimed at assessing PROTAC series intracellular bioavailability.
Researchers commonly use VH032 along with its derivatives VH032-cyclopropane-F and VH298 to create VHL ligands for PROTACs. VHL E3 ligase recruitment by these ligands triggers target protein ubiquitination followed by proteasomal degradation. Researchers used VH032-based PROTACs to analyze the breakdown of several proteins associated with cancer.
Fig. 3 Von Hippel-Lindau (VHL) E3 ligase in PROTACs design
CRBN Ligands
- Examples of Fluorescent CRBN Ligands
Studies demonstrate that thalidomide along with its derivatives like lenalidomide serve as typical CRBN ligands. The CRBN ligands fulfill dual roles by recruiting the CRBN E3 ligase and functioning as molecular glues which modify the CRBN surface to bind new substrates. The CRBN ligands exhibit lower molecular weights with preferable physicochemical traits including reduced HBD and HBA numbers and diminished lipophilicity which makes them more suitable for drug development.
Clinical trials have extensively utilized CRBN-based PROTACs. Thalidomide-based ligands that target CRBN have shown effectiveness in the treatment of multiple myeloma and colorectal cancer. Clinical preclinical studies indicate that CRBN ligand-based PROTACs deliver strong anti-tumor effects and their reduced molecular weight paired with positive pharmacokinetic traits makes them ideal candidates for therapeutic applications.
References
- Xi, Jia-Yue, et al. Bioorganic Chemistry 125 (2022): 105848.
- Espinoza-Chávez, Rocío Marisol, et al. "Targeted protein degradation for infectious diseases: from basic biology to drug discovery." ACS bio & med Chem Au 3.1 (2022): 32-45.
- Setia, Nisha, Haider Thaer Abdulhameed Almuqdadi, and Mohammad Abid. European Journal of Medicinal Chemistry 265 (2024): 116041.
