Deconstruction of Dual-Site Tankyrase Inhibitors Provides Insights into Binding Energetics and Suggests Critical Hotspots for Ligand Optimization

Designing inhibitors is a complex task that requires
a deep understanding
of protein–ligand interactions and their dynamics. Ligands
often interact with multiple binding subsites, with noncovalent interactions
affecting binding affinity. Conformational changes and plasticity
of both, the ligand and the protein influence binding energetics.
We investigated the tankyrase ADP-ribosyltransferase as a promising
drug target regulating many cellular pathways. Despite the existence
of diverse tankyrase inhibitors, their binding energetics and contributions
of flexible cryptic subpockets to binding affinity remain elusive.
To examine these aspects, we deconstructed inhibitors to key fragments,
dissected their energetic contribution to the affinity, and determined
their binding mode by X-ray crystallography. Varying ligand efficiencies
of the deconstructed, pocket-binding fragments revealed the cryptic
nature of subpockets. These insights enabled us to redesign inhibitors
with novel linkers, the observed key area for optimization, increasing
the potency in enzymatic and cell-based assays by 7.5-fold and 6.2-fold
compared to the parent ligand.