A novel small molecule screening assay using normal human chondrocytes toward osteoarthritis drug discovery
Philip R. Coryell, Paul B. Hardy, Susan Chubinskaya, Kenneth H. Pearce, Richard F. Loeser
Abstract
Osteoarthritis (OA) is the most common form of arthritis and a leading cause of pain and disability in adults. A central feature is progressive cartilage degradation and matrix fragment formation driven by the excessive production of matrix metalloproteinases (MMPs), such as MMP-13, by articular chondrocytes. Inflammatory factors, including interleukin 6 (IL-6), are secreted into the joint by synovial fibroblasts, and can contribute to pain and inflammation. No therapeutic exists that addresses the underlying loss of joint tissue in OA. To address this, we developed and utilized a cell-based high-throughput OA drug discovery platform using normal human chondrocytes treated with a recombinant fragment of the matrix protein fibronectin (FN-f) as a catabolic stimulus relevant to OA pathogenesis and a readout using a fluorescent MMP-13 responsive probe.
Introduction
Osteoarthritis (OA) is a disease of the articular joints characterized by the destruction of cartilage, synovial inflammation, and abnormal bone remodeling. It typically occurs in the knees, hips, hands, and spine, which can lead to loss of mobility and debilitating pain [1, 2]. Over 500 million individuals globally are affected by symptomatic OA, including 32 million in the U.S., which is expected to rise to 67 million by 2030 [3–5]. OA treatment costs the U.S. healthcare system over $27 billion annually [6] and even more in lost economic productivity due to OA disability.
Materials and method
Antibodies
Primary antibodies used were anti-MMP-13 (Millipore Sigma, MAB3321) at 1:1000 dilution, anti-IL-6 (Millipore Signal, MABF41) at 1:1000 dilution, and anti-β-Tubulin (Cell Signaling, #2146) at 1:1000 dilution. Secondary antibodies used were anti-rabbit IgG, HRP-linked antibody (Cell Signaling, #7074) and anti-mouse IgG, HRP-linked antibody (Cell Signaling, #7076) at 1:2000 dilutions.
Results
Screen design and MMP-13 probe validation
To find small molecule inhibitors of OA catabolic signaling, we developed a high-throughput compound screening protocol that used primary human chondrocytes treated with FN-f as a stimulus and MMP-13 as a readout (Fig 1). To measure MMP-13 production using an HTS platform, we developed a fluorogenic probe that measured MMP-13 activity in cell culture media via Förster resonance energy transfer (FRET).
Acknowledgments
We wish to acknowledge The Gift of Hope Organ and Tissue Donor Network and thank the donor families for providing donor tissue and the University of North Carolina Department of Orthopedic Surgery for assistance obtaining OA tissue. We also wish to acknowledge Stephanie Haro for her assistance with tissue isolation, cell culture, and preparing samples for immunoblot.
Citation: Coryell PR, Hardy PB, Chubinskaya S, Pearce KH, Loeser RF (2024) A novel small molecule screening assay using normal human chondrocytes toward osteoarthritis drug discovery. PLoS ONE 19(11): e0308647. https://doi.org/10.1371/journal.pone.0308647
Editor: Pierre Bobé, Universite Paris-Saclay, FRANCE
Received: April 9, 2024; Accepted: July 26, 2024; Published: November 1, 2024
Copyright: © 2024 Coryell et al. 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: The data are all contained within the manuscript and the Supporting information files.
Funding: Rheumatology Research Foundation National Institutes of Health grant UL1TR002489. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0308647#ack
