Definitive Metabolite Identification Coupled with Automated Ligand Identification System (ALIS) Technology: A Novel Approach to Uncover Structure–Activity Relationships and Guide Drug Design in a Factor IXa Inhibitor Program

A potent
and selective Factor IXa (FIXa) inhibitor was subjected
to a series of liver microsomal incubations, which generated a number
of metabolites. Using automated ligand identification system-affinity
selection (ALIS-AS) methodology, metabolites in the incubation mixture
were prioritized by their binding affinities to the FIXa protein.
Microgram quantities of the metabolites of interest were then isolated
through microisolation analytical capabilities, and structurally characterized
using MicroCryoProbe heteronuclear 2D NMR techniques. The isolated
metabolites recovered from the NMR experiments were then submitted
directly to an in vitro FIXa enzymatic assay. The
order of the metabolites’ binding affinity to the Factor IXa
protein from the ALIS assay was completely consistent with the enzymatic
assay results. This work showcases an innovative and efficient approach
to uncover structure–activity relationships (SARs) and guide
drug design via microisolation–structural characterization
and ALIS capabilities.