Fragment-based drug discovery for transthyretin kinetic stabilisers using a novel capillary zone electrophoresis method
Wenjie Chen
Abstract
A Capillary Zone Electrophoresis (CZE) fragment screening methodology was developed and applied to the human plasma protein Transthyretin (TTR), normally soluble, but could misfold and aggregate, causing amyloidosis. Termed Free Probe Peak Height Restoration (FPPHR), it monitors changes in the level of free ligand known to bind TTR (the Probe Ligand) in the presence of competing fragments. 129 fragments were screened, 12 of the 16 initial hits (12.4% hit rate) were co-crystallised with TTR, 11 were found at the binding site (92% confirmation rate).
Introduction
With seven approved fragment-derived drugs and many others currently in clinical trials [1], Fragment-Based Drug Discovery (FBDD) is a widely accepted strategy alongside High-Throughput Screening (HTS) in the field of early-stage drug discovery. As well as the need of a reliable system for obtaining ligand-protein structural data, another prerequisite for the success of FBDD is the ability to detect weakly binding fragments.
Materials and method
Chemicals, reagents & protein
Tafamidis and Compound SEL101928 were synthesised by Selcia Ltd. (Ongar, Essex, UK). Thyroxine T4 and 8-Anilino-1-naphthalenesulfonic acid ammonium salt (8-ANS) were purchased from Sigma (Poole, Dorset, UK). All other tested compounds were either from the chemical store of Selcia Ltd., the Selcia Fragment Library (SFL) or purchased from ChemBridge Corporation (San Diego, CA, USA), or Sigma (St. Louis, MO, USA).
Results
Development of TTR CZE FPPHR fragment screening assay
Effect of TTR binding on free 8-ANS UV absorbance peak height and area.
The migration time, peak height and area of free 8-ANS (30 µ M) were averaged around 2.3 minutes, 1640 and 10274 Arbitrary Units (AU) respectively with excellent replicability (Fig 2, Trace A & B,). When 8-ANS (30 μM) was mixed with TTR (20 μM) in a different vial of Inject Buffer and separated again, two peaks appeared (Fig 2, Trace C & D).
Discussion
In a CE separation apparatus, the user has full control on the contents of Running Buffer, Inject Buffer and filling of the capillary. This assay setup flexibility has facilitated development of different modes of separation and numerous variants of ACE methods including the patented CEfrag™ method. FPPHR materially differs from them by not filling the capillary with protein-containing solution and not mixing test compounds with the protein.
Acknowledgments
The research work here formed part of a Ph.D. project (2010–2014) co-supervised by Professor Stephen P. Wood and Dr. Carol Austin. All capillary electrophoresis experiments were performed in Selcia Ltd (now part of Eurofins). The author thanks the UCL Wolfson Drug Discovery Unit for the provision of reagents (including recombinant TTR S52P mutant) and use of equipment.
Citation: Chen W (2025) Fragment-based drug discovery for transthyretin kinetic stabilisers using a novel capillary zone electrophoresis method. PLoS One 20(5): e0323816. https://doi.org/10.1371/journal.pone.0323816
Editor: Junzheng Yang, Guangdong Nephrotic Drug Engineering Technology Research Center, Institute of Consun Co. for Chinese Medicine in Kidney Diseases, CHINA
Received: November 25, 2024; Accepted: April 14, 2025; Published: May 14, 2025
Copyright: © 2025 Wenjie Chen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. Crystal structure coordinates and their structure factors have been deposited in the Protein Data Bank (PDB), www.pdb.org PDB IDs: 9H7A, 9H7B, 9H7C, 9H7D, 9H7E, 9H7F, 9H7G, 9H7H, 9H7I, 9H7J, 9H7K, 9H7L, 9H7M, 9H7N, 9H7O, 9H7P
Funding: Wenjie Chen was a recipient of an IMPACT PhD studentship grant code HN9A/BXTX, which is sponsored by Selcia Ltd. and University College London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The author has declared that no competing interests exist
