2. Academic Validation
  3. Direct observation of small molecule activator binding to single PR65 protein

Direct observation of small molecule activator binding to single PR65 protein

  • NPJ Biosens. 2025;2(1):2. doi: 10.1038/s44328-024-00018-7.
Annie Yang-Schulz # 1,  Maria Zacharopoulou # 2,  Sema Zeynep Yilmaz 3,  Anupam Banerjee 4,  Satyaki Saha 4 5,  Daniel Nietlispach 6,  Michael Ohlmeyer 7,  Mert Gur 8,  Laura S Itzhaki 2,  Ivet Bahar 4 5,  Reuven Gordon 1
Affiliations

Affiliations

  • 1 Department of Electrical Engineering, University of Victoria, Victoria, BC V8W 3P6 Canada.
  • 2 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD UK.
  • 3 Department of Mechanical Engineering, Istanbul Technical University, Istanbul, 34437 Turkey.
  • 4 Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794 USA.
  • 5 Department of Biochemistry and Cell Biology, School of Medicine, Stony Brook University, Stony Brook, NY 11794 USA.
  • 6 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW UK.
  • 7 Atux Iskay LLC, Plainsboro, New Jersey, NJ 08536 USA.
  • 8 Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213 USA.
  • # Contributed equally.
PMID: 39830999 DOI: 10.1038/s44328-024-00018-7
Abstract

The reactivation of heterotrimeric protein Phosphatase 2A (PP2A) through small molecule activators is of interest to therapeutic intervention due to its dysregulation, which is linked to chronic conditions. This study focuses on the PP2A scaffold subunit PR65 and a small molecule activator, ATUX-8385, designed to bind directly to this subunit. Using a label-free single-molecule approach with nanoaperture optical tweezers (NOT), we quantify its binding, obtaining a dissociation constant of 13.6 ± 2.5 μM, consistent with ensemble fluorescence anisotropy results but challenging to achieve with Other methods due to low affinity. Single-molecule NOT measurements reveal that binding increases optical scattering, indicating PR65 elongation. This interpretation is supported by all-atom molecular dynamics simulations showing PR65 adopts more extended conformations upon binding. This work highlights NOT's utility in quantifying binding kinetics and structural impact, offering insights valuable for drug discovery.

Keywords

Cancer; Pharmacology; Techniques and instrumentation.

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