Relative Binding Free-Energy Calculations at Lipid-Exposed Sites: Deciphering Hot Spots

Relative binding free-energy (RBFE)
calculations are experiencing
resurgence in the computer-aided drug design of novel small molecules
due to performance gains allowed by cutting-edge molecular mechanic
force fields and computer hardware. Application of RBFE to soluble
proteins is becoming a routine, while recent studies outline necessary
steps to successfully apply RBFE at the orthosteric site of membrane-embedded
G-protein-coupled receptors (GPCRs). In this work, we apply RBFE to
a congeneric series of antagonists that bind to a lipid-exposed, extra-helical
site of the P2Y1 receptor. We find promising performance of RBFE,
such that it may be applied in a predictive manner on drug discovery
programs targeting lipid-exposed sites. Further, by the application
of the microkinetic model, binding at a lipid-exposed site can be
split into (1) membrane partitioning of the drug molecule followed
by (2) binding at the extra-helical site. We find that RBFE can be
applied to calculate the free energy of each step, allowing the uncoupling
of observed binding free energy from the influence of membrane affinity.
This protocol may be used to identify binding hot spots at extra-helical
sites and guide drug discovery programs toward optimizing intrinsic
activity at the target.