Discovery of Novel Macrocyclic Ligands for Difficult Targets : Applications to Natural Product Derived Keap1 Inhibitors

Abstract: The development of small-molecule ligands for biological targets that possess large, featureless or highly polar binding sites is a challenging task. This thesis is focused on a novel lead generation strategy to identify macrocyclic ligands for difficult-to-drug targets, as well as on the relationship between cell permeability and the conformations of macrocycles.A database of natural products was investigated to compile a set of macrocyclic cores to be used for in silico screening on difficult-to-drug targets. Docking of this set on Keap1, a target considered challenging due to its large and polar binding site, identified the core of the natural product cyclothialidine as a starting point for lead generation. Synthesis and evaluation of a small number of analogues provided a novel macrocyclic Keap1 inhibitor with potency in the low micromolar range that displayed cellular activity. Investigation of the structure-activity relationship of the lead inhibitor identified two positions amenable for optimization. In silico libraries were constructed at both positions using structure-based design to improve the affinity for Keap1. Subsequent synthesis of approximately 100 compounds led to an optimized lead series with potency in the low nanomolar range, providing a 100-fold improvement from the starting point. Additionally, the difference in passive cell permeability for a pair of diastereoisomeric macrocycles was rationalized on the basis of differences in their solution-phase conformations, that were determined by NMR spectroscopy. Moreover, for two sets of moderately flexible isomeric macrocycles, it was shown that the molecular descriptors predicted from conformational sampling correlated with cell permeability. This method may find use for prioritization of macrocycles prior to embarking on demanding synthetic routes.