Abstract
The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation.
Highlights
The desire to modulate protein function with small molecules that can be administered as drugs has led to a plethora of studies attempting to define and calculate the ‘‘druggability’’ of sites on a protein [1,2,3,4,5]
In this study we demonstrate how the approach of employing multiple protein conformers at the selection-of-pocket stage can be combined with predictions of druggability, to aid the identification of transient, novel druggable pockets often missed in single conformer approaches
Druggable pockets on the surface of proteins can be missed by in silico screens to identify the most promising target site on a protein, commonly based upon a single structural snapshot. Such pockets are of particular interest in cases where the protein target undergoes large conformational variations, as in the archetypal serpin A1AT
Summary
The desire to modulate protein function with small molecules that can be administered as drugs has led to a plethora of studies attempting to define and calculate the ‘‘druggability’’ of sites on a protein [1,2,3,4,5]. These include protein-protein interactions and proteins belonging to large homologous superfamilies e.g. kinases. In the former, the interfaces are larger and flatter [6]. In the latter, inhibiting the common active site risks serious cross-class side effects. Both these issues may be addressed by targeting clefts that are not necessarily associated directly with the protein’s biochemical function. The idea is that binding of small molecules to such clefts may be more favourable and could still allosterically modulate protein function, e.g. via preferential stabilization of a particular state within the conformational landscape of the protein in solution
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