Blind Man’s Bluff – Elaboration of Fragment Hits in the Absence of Structure for the Development of Antitrypsin Deficiency Inhibitors

Abstract

The three pillars of rational drug design from a fragment library are an efficient screen, a robust assay, and atomic-resolution structures of the protein–ligand complexes. However, not all targets are amenable to structure determination by X-ray crystallography or NMR spectroscopy. In particular, targets involved in diseases of protein misfolding are inherently intractable. In the absence of structures, we are blind. However, the lack of structural information need not preclude the use of fragment-based approaches. The use of appropriate NMR techniques can enable us to detect the effects of protein binding on ligand resonances. In our efforts to identify compounds that affect the kinetics of α1-antitrypsin misfolding, we have used saturation transfer difference NMR spectroscopy to detect hits from mixtures of compounds in a fragment library. In the absence of structures, the initial challenge is three-fold: to (1) distinguish between binding sites; (2) evaluate the relative affinities of hits; and (3) advance them to the stage where activity can be detected in biological assays. We largely achieved these aims by the use of Carr–Purcell–Meiboom–Gill NMR competition experiments that detect differential relaxation of the ligand on protein binding.