Background: Targeted protein degradation relies on inducing proximity between an E3 ubiquitin ligase and a target protein, and subsequent proteasomal degradation of the latter. Biophysical methods allow the measurement of the ternary complex formation by recombinant target and E3 ligase proteins in the presence of molecular glues and bifunctional degraders. The development of new chemotypes of degraders mediating ternary complex formation of unknown dimensions and geometries requires the use of different biophysical approaches.
Methods: The TR-FRET and AlphaLISA platforms have been applied to study molecular glues and bifunctional degraders. The performance of the label-based proximity assays was compared with the BLI method, which is a label-free, sensor-based approach.
Results: We present and compare two commonly used assays to monitor proximity induction, AlphaLISA and TR-FRET. The LinkScape system consisting of the CaptorBait peptide and the CaptorPrey protein is a novel method of protein labeling compatible with TR-FRET assay.
Conclusions: The TR-FRET and AlphaLISA proximity assays enable detection of ternary complexes formed between an E3 Ligase, a target protein and a small molecule degrader. Experiments with different chemotypes of GSPT1 degraders showed that ALphaLISA was more susceptible to chemotype-dependent interference than TR-FRET assay.
General Significance: The discovery and optimization of small-molecule inducers of ternary complexes is greatly accelerated by using biophysical assays. The LinkScape-based TR-FRET assay is an alternative to antibody-based proximity assays due to the CaptorPrey's subnanomolar affinity to the CaptorBait-tagged protein target, and the 10-fold lower molecular weight of the CaptorPrey protein compared to the antibody.
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http://dx.doi.org/10.1016/j.bbagen.2023.130398 | DOI Listing |
Sci Rep
January 2025
Geophysics Department, Faculty of Science, Cairo University, Giza, 12613, Egypt.
The Hammam Faraun, Matulla, and Nubia formations in the Ashrafi oil field, in the southern Gulf of Suez, Egypt, are key hydrocarbon reservoirs with significant economic importance. These formations, characterized by their favorable reservoir properties and structural settings, play a crucial role in oil and gas accumulation. Their study provides valuable insights into regional petroleum systems and guides exploration and production activities.
View Article and Find Full Text PDFCell Commun Signal
January 2025
Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, 100191, China.
Background: Ovarian cancer (OC), particularly high-grade serous ovarian carcinoma (HGSOC), is the leading cause of mortality from gynecological malignancies worldwide. Despite the initial effectiveness of treatment, acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) represents a major challenge for the clinical management of HGSOC, highlighting the necessity for the development of novel therapeutic strategies. This study investigated the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a pivotal regulator of glycolysis, in PARPi resistance and explored its potential as a therapeutic target to overcome PARPi resistance.
View Article and Find Full Text PDFBMJ Open
January 2025
University Research Clinic for Cancer Screening, Randers Regional Hospital, Randers, Denmark.
Objective: This study explored and compared stakeholder perspectives on enhancements to cervical cancer screening for vulnerable women across seven European countries.
Design: In a series of Collaborative User Boards, stakeholders were invited to collaborate on identifying facilitators to improve cervical cancer screening.
Setting: This study was part of the CBIG-SCREEN project which is funded by the European Union and targets disparities in cervical cancer screening for vulnerable women (www.
J Immunother Cancer
January 2025
Section of Nephrology, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
Immune checkpoint inhibitor (ICI) therapy is a cornerstone treatment for many cancers, but it can induce severe immunotoxicity, including acute interstitial nephritis (AIN). Currently, kidney biopsy is required to differentiate ICI-AIN from other causes of acute kidney injury (AKI). However, this invasive approach can lead to morbidity, delayed glucocorticoid treatment for patients with AIN, and unnecessarily prolonged suspension of ICI therapy in non-AIN patients.
View Article and Find Full Text PDFJ Allergy Clin Immunol
January 2025
Departments of Animal Science, Integrative Biology and Physiology, University of Minnesota,St. Paul, MN, 55108. Electronic address:
Background: Environmental allergens induce the release of danger signals from the airway epithelium that trigger type 2 immune responses and promote airway inflammation.
Objective: To investigate the role of allergen-stimulated P2Y receptor activation in regulating ATP, IL-33 and DNA release by human bronchial epithelial (hBE) cells and mouse airways.
Methods: hBE cells were exposed to Alternaria alternata extract and secretion of ATP, IL-33 and DNA were studied in vitro.
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