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A Structure-guided Active Site Affinity Ligand Unmasks a Stress-Sensitizing Role for BLVRB in the Endoplasmic
Rahul Raghavan Thekke Veedu1,2, Jawaad Sheriff1, Natasha M Nesbitt1
1Blood Cell Technologies, JLABS@NYC, 101 Sixth Avenue, New York, New York 10013, United States.
Biliverdin IXb reductase (BLVRB) activity was measured in real-time using a novel nanoBRET platform. This technology allows for precise analysis of BLVRB inhibitors and their role in cellular stress responses.
Area of Science:
- Biochemistry
- Cellular Biology
- Pharmacology
Background:
- Biliverdin IXb reductase (BLVRB) is an oxidoreductase involved in hematopoiesis and cellular stress.
- Understanding BLVRB active site engagement is crucial but currently undefined.
Purpose of the Study:
- To develop a real-time platform for measuring BLVRB target engagement.
- To characterize BLVRB inhibitors and their cellular effects.
Main Methods:
- Development of a nanoBRET platform for real-time target engagement assays.
- Structure-guided design and synthesis of BODIPY-labeled BLVRB inhibitors.
- In vitro and cellular assays, including nanoBRET, enzymatic inhibition, and crystallographic studies.
- Live-cell imaging to determine inhibitor localization and cellular impact.
Main Results:
- The nanoBRET platform successfully enabled real-time measurement of BLVRB target engagement.
- BODIPY-labeled inhibitors showed high-affinity binding and validated BLVRB active site engagement.
- NanoBRET results strongly correlated with enzymatic inhibition data and crystallographic findings.
- Live-cell imaging revealed ER localization of BLVRB and transient suppression of GRP78/BiP without inducing a canonical unfolded protein response.
Conclusions:
- The nanoBRET platform is a valuable tool for studying BLVRB activity and inhibitor interactions.
- BLVRB active site engagement acts as a stress sensitizer, modulating ER proteostasis.
- These findings provide insights into a redox-regulated mechanism influencing cellular stress responses.
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