Resolving the ambiguous binding site of quercetin at the calcineurin subunit junction using funnel metadynamics with deep learning collective variables

Clinical Snapshot

Key Findings

• P Value: Not reported in abstract

• Effect Size: Free energy of binding -8.36 ± 0.60 kcal/mol

• Primary Outcome: Successful identification of quercetin binding site at calcineurin subunit junction

• Nnt Or Sensitivity: Correlation coefficient r² = 0.85 with experimental FRET measurements

• Confidence Interval: ±0.60 kcal/mol for binding energy

Clinical Application

Provides computational framework applicable to other challenging protein-ligand systems Relevant to Australian pharmaceutical research institutions and TGA drug development pathways for novel immunosuppressants Drug discovery researchers developing calcineurin inhibitors for immunosuppression

Abstract

Calcineurin represents a prominent target for immunosuppressive drugs, where conventional macrocyclic inhibitors utilize an immunophilin-dependent mechanism for their inhibition but are consequently hindered by adverse effects and variable pharmacokinetics. The flavonoid quercetin has been shown to inhibit calcineurin in a non-competitive, immunophilin-independent manner, but its exact binding site remains ambiguous at the junction between calcineurin subunits A and B, with the open solvent-exposed nature of this region proving challenging to model. In this study, we employ funnel metadynamics with collective variables derived from a deep learning model to identify the binding site of quercetin. A selective strategy using multiple simulations with stricter funnel definitions and a smaller number of carefully chosen descriptors for model training proved more effective than a broad-based approach. These simulations were able to effectively distinguish the binding site of quercetin from three experimentally suggested sites, with a calculated free energy of binding of -8.36 ± 0.60 kcal/mol showing excellent agreement with experiment. Ligand-tryptophan distances similarly corroborated measurements from FRET assays with a r2 of 0.85. The corresponding binding pose showed that quercetin inserts itself into a channel between Arg122, Gly123, Tyr124 on one side and Phe160, Thr161, and Asn345 on the other, stabilizing its binding through a network of hydrogen bonds. The findings of this study provide insights into the modelling of challenging binding sites and ligands using deep learning driven metadynamics simulations and provide the foundations for rational development of immunophilin-independent inhibitors of calcineurin.

References

1. 1.Loo, J. S. E., & Yau, M. Q. (2026). Resolving the ambiguous binding site of quercetin at the calcineurin subunit junction using funnel metadynamics with deep learning collective variables. Journal of Computer-Aided Molecular Design. https://doi.org/10.1073/pnas.1303186110