SAR-Guided Scoring Function and Mutational Validation Reveal the Binding Mode of CGS-8216 at the α1+/γ2– Benzodiazepine Site

The
structural resolution of a bound ligand–receptor complex
is a key asset to efficiently drive lead optimization in drug design.
However, structural resolution of many drug targets still remains
a challenging endeavor. In the absence of structural knowledge, scientists
resort to structure–activity relationships (SARs) to promote
compound development. In this study, we incorporated ligand-based
knowledge to formulate a docking scoring function that evaluates binding
poses for their agreement with a known SAR. We showcased this protocol
by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216
at the benzodiazepine binding site of the GABAA receptor.
Further evaluation of the final pose by molecular dynamics and free
energy simulations revealed a close proximity between the pendent
phenyl ring of the PQ and γ2D56, congruent with the low potency
of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A
mutation and in fact observed a 10-fold potency increase in the carboxyphenyl
analogue, providing experimental evidence in favor of our binding
hypothesis.