Maurizio Pellecchia’s lab at the Burnham Institute has been one of the most active academic groups using fragment-based ligand discovery, and their recent paper in J. Med. Chem. describes an NMR-based approach to discover small-molecule mimetics of SMAC. The four N-terminal amino acids of SMAC bind to the protein XIAP, thereby blocking its interaction with caspase-9 and allowing apoptosis to proceed. A small-molecule mimic of SMAC could thus be useful as a chemical probe to better understand the biology of apoptosis, and, ultimately, could be useful as a cancer therapeutic.
The researchers started with the alanine “fragment” of the tetrapeptide Ala-Val-Pro-Ile (AVPI) and generated a virtual library of nearly 1400 alanine-containing derivatives. Molecular modeling narrowed this down to 15 which were then actually synthesized and tested by NMR to assess their binding affinity to a domain of XIAP; BI-75A1 was found to be a weak binder. Molecular modeling suggested that this new fragment (with a molecular weight just under 300) could in turn be “grown” to improve affinity, and after roughly 900 compounds were docked, 28 were then synthesized and tested. Of these, the most potent turned out to be BI-75D2, with a low micromolar dissociation constant in both NMR and isothermal titration calorimetry assays.
BI-75D2 exhibited improved stability in human plasma and S9 fraction compared to the starting peptide AVPI, as well as increased permeability. BI-75D2 also showed modest (16 micromolar) activity in a cell-based apoptosis induction assay, in contrast to the (inactive) AVPI peptide. Further biological experiments support the hypothesis that the small molecule induces apoptosis by binding to XIAP.
From a drug perspective, BI-75D2 still has a long way to go: it is a relatively weak binder, has a molecular weight greater than 500 Daltons, and contains several structural features that make a medicinal chemist squirm and a toxicologist squeal. Moreover, BI-75D2 has a fairly low ligand efficiency (LE), and this actually got worse as the affinity was improved. Nonetheless, as a chemical probe it may have value. It is also a demonstration of how fragment-inspired techniques can be used to attain novel molecules in an academic setting.