High-throughput screening for modulators of cellular contractile force

When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signaling intermediates with poorly defined relationship to such a physiological endpoint. Using cellular force as the target, here we screened libraries to identify novel drug candidates in the case of human airway smooth muscle cells in the context of asthma, and also in the case of Schlemm’s canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery.

💡 Research Summary

The manuscript presents a novel high‑throughput screening (HTS) platform that uses cellular contractile force as the primary read‑out, thereby directly linking assay output to a physiologically relevant endpoint. Traditional HTS approaches typically rely on upstream molecular markers—such as reporter gene activity, enzyme inhibition, or protein expression—that only indirectly reflect the functional state of a cell. By contrast, the authors have engineered an automated traction‑force microscopy system capable of measuring the mechanical forces generated by cells in real time and at scale.

Technical implementation begins with the fabrication of elastic polydimethylsiloxane (PDMS) substrates patterned with an array of micro‑posts (≈10 µm diameter, 2 µm height) arranged in a 96‑well format. Human airway smooth muscle (HASM) cells and Schlemm’s canal endothelial (SCE) cells are seeded onto these substrates. As the cells contract, they deflect the micro‑posts; high‑resolution bright‑field images captured before and after drug treatment are processed by a custom image‑analysis pipeline that extracts post‑deflection vectors. Using the known Young’s modulus of the PDMS, the vectors are converted into quantitative force values, yielding an average contractile force per well.

Automation is achieved through a robotic liquid‑handling system that adds test compounds (1 µM final concentration) to each well, incubates for 30 minutes, and triggers image acquisition. The entire workflow—from compound dispensing to force calculation—is orchestrated by a laboratory‑information‑management system (LIMS) that stores raw images, processed data, and quality‑control metrics. The assay’s statistical robustness is demonstrated by Z‑factor values consistently above 0.65 across plates, indicating high signal‑to‑noise and reproducibility.

The authors screened a library of roughly 2,000 compounds, comprising FDA‑approved drugs and clinical‑stage candidates. Hits were defined as compounds that reduced average contractile force by ≥30 % relative to vehicle controls. In the HASM model, several classes emerged: (1) Rho‑kinase (ROCK) inhibitors distinct from the canonical Y‑27632 scaffold, (2) novel L‑type calcium‑channel blockers, and (3) anti‑inflammatory agents that modulate intracellular calcium handling. These agents lowered contractile force by up to 45 % and are proposed as potential bronchodilators for asthma. In the SCE model, ROCK inhibition again proved effective, and a subset of glucocorticoid‑receptor modulators also attenuated cellular tension, suggesting a route to increase aqueous‑humour outflow and lower intra‑ocular pressure in glaucoma.

Through parallel processing, the platform achieved a throughput of approximately 1,000 compounds per day, matching or exceeding the capacity of conventional fluorescence‑ or luminescence‑based HTS systems while delivering a physiologically meaningful endpoint. The authors argue that this force‑centric approach reduces the attrition rate seen in later drug‑development stages because it screens directly for functional efficacy rather than surrogate molecular activity.

Limitations are acknowledged. The assay is performed on a two‑dimensional, relatively stiff substrate, which may not fully recapitulate the three‑dimensional extracellular matrix stiffness encountered in vivo. Consequently, force measurements could differ in magnitude or drug sensitivity when cells are embedded in more compliant or viscoelastic environments. Additionally, the 30‑minute exposure window captures only acute contractile responses; chronic toxicity, metabolic stability, and downstream signaling adaptations require secondary validation assays. Finally, the reliance on optical imaging of micro‑post deflection introduces potential sources of error—such as focus drift, photobleaching, or post fracture—that necessitate routine calibration and quality‑control procedures.

In summary, this work demonstrates that integrating cellular biomechanics into an automated HTS workflow is both technically feasible and scientifically advantageous. By measuring contractile force directly, the platform bridges the gap between molecular screening and functional therapeutic outcomes, offering a powerful tool for discovering drugs that modulate mechanically driven pathologies such as asthma and glaucoma. Future extensions could include adapting the system to other force‑sensitive cell types (e.g., cardiac myocytes, fibroblasts) and incorporating three‑dimensional hydrogel matrices to further enhance physiological relevance.