A Systematic Benchmark Study of Free Energy Methods for Quantifying Light-Responsive Binding Affinities of...

Molecular photoswitches enable spatiotemporal photocontrol of protein function, but their design requires high target selectivity and large light-dependent changes in binding affinity and/or efficacy. These properties are especially difficult to optimize in membrane receptors due to membrane-protein interactions. Computational design remains challenging because few benchmarks rigorously compare free-energy methods against experiment. Here, we establish such a benchmark for photoswitchable antagonists of β-adrenergic receptors, exemplifying most successful designs in the photopharmacology of class A G protein-coupled receptors (GPCRs) to date. We evaluated widely used free-energy methods for predicting how substituents and chirality affect light-responsive binding and subtype selectivity. Thermodynamic integration shows the best agreement with experiment, followed by umbrella sampling, whereas metadynamics and end-point methods perform poorly. Our simulations reveal interactions stabilizing cis OP2 in β₂-AR and the key role of PHE289 in isomer-specific binding, consistent with mutagenesis data. Overall, this work provides a robust computational framework for GPCR photopharmacology.