Vince Bart Cardenas
| Beyond the 1:1 Ligand-Protein Paradigm: An In Silico Assay for Competitive Ligand Binding |
| Cardenas Vince Bart 1, Stefano Raniolo 1, Paolo Conflitti 1, and Vittorio Limongelli 1 1Euler Institute, Faculty of Biomedical Sciences, Università della Svizzera italiana, Lugano CH-6900, Switzerland |
| Abstract Competitive binding assays (CBAs) are central to pharmacology and drug discovery, providing metrics of ligand affinity and potency [1]. Yet they only provide limited mechanistic insight into the molecular events underlying ligand competition. How multiple ligands dynamically explore receptor surfaces, compete for access, and influence one another’s dynamics remain experimentally difficult to resolve. All-atom molecular dynamics (AAMD) simulations potentially bridge this gap, but atomistic simulations of true competitive binding environments remain computationally prohibitive as they require multiple freely diffusing ligands, long timescales, and extensive sampling [2]. Here, we present a computational framework of CBA-like conditions using fully unbiased, multiligand coarse-grained molecular dynamics (CGMD) simulations [3], applied to ligands of opposing efficacy, the full agonist NECA and inverse agonist ZM241385, at the adenosine A2A receptor, a prototypical GPCR and key pharmacological target [4]. By simulating varying ligand ratios, a concentration-dependent competition is emulated, while allowing both ligands to diffuse freely, explore the receptor and membrane, and interact dynamically amongst themselves. The protocol captures hallmark features of CBA, including spontaneous binding, unbinding, and direct competition across an aggregate millisecond simulation timescale, enabling structural, mechanistic, thermodynamic, and kinetic characterization inaccessible to conventional experiments. Without imposing prior binding poses, both ligands found their experimental binding modes and reproduced their experimental relative binding energies. Moreover, the simulations revealed an extracellular vestibular region that hosts transient ligand-receptor binding, accommodate ligand-ligand interactions, and modulate ligand access to the orthosteric site. Occupancy by NECA in this region facilitated ZM241385 pocket entry and prolonged its residence time, revealing a cooperative mechanism in an otherwise competitive process. These findings provide a molecular perspective on ligand competition and highlight CGMD’s promising potential for probing multiligand dynamics beyond the reach of conventional assays. More broadly, it opens new avenues towards understanding how competitive-cooperative processes can shape binding pathways, kinetics, and outcomes in drug discovery. |
| References [1] Campbell, I.B; et al. Medicinal chemistry in drug discovery in big pharma: the past, present, and future. Drug Discovery Today 2018, 23, 219-234. DOI: 10.1016/j.drudis.2017.10.007. [2] Hollingsworth, S.A.; Dror, R.O. Molecular dynamics simulations for all. Neuron 2018, 99, 1129-1143. DOI: 10.1016/j.neuron.2018.08.011. [3] Ingolfsson, H.I; et al. The power of coarse graining in biomolecular simulations. WIREs Computational Molecular Science 2014, 4, 225-248. DOI: 10.1002/wcms.1169. |