SAR and molecular mechanics reveal the importance of ring entropy in the biosynthesis and activity of a natural product

摘要

Macrocycles are appealing drug candidates due to their high-affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally-encoded family of natural products that exhibits potent antimicrobial effects against gram-positive bacteria. Since thiocillin is derived from a genetically-encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogs. To understand thiocillin’s structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin’s macrocyclic ring. We report the identification of eight unique compounds more potent than WT thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin’s macrocyclic structure revealed a striking requirement for a low entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high potency binding.