Crystal structures of the σ₂receptor template large-library docking for selective chemotypes activein vivo

Abstract

The σ2 receptor is a poorly understood transmembrane receptor that has attracted intense interest in many areas of biology including cancer imaging, Alzheimer's disease, schizophrenia, and neuropathic pain. However, little is known regarding the molecular details of the receptor, and few highly selective ligands are available. Here, we report the crystal structure of the σ2 receptor in complex with the clinical drug candidate roluperidone and the probe compound PB28. These structures, in turn, templated a large-scale docking screen of 490 million make-on-demand molecules. Of these, 484 compounds were synthesized and tested, prioritizing not only high-ranking docked molecules, but also those with mediocre and poor scores. Overall, 127 compounds with binding affinities superior to 1 μM were identified, all in new chemotypes, 31 of which had affinities superior to 50 nM. Intriguingly, hit rate fell smoothly and monotonically with docking score. Seeking to develop selective and biologically active probe molecules, we optimized three of the original docking hits for potency and for selectivity, achieving affinities in the 3 to 48 nM range and to up to 250-fold selectivity vs. the σ1 receptor. Crystal structures of the newly discovered ligands bound to the σ2 receptor were subsequently determined, confirming the docked poses. To investigate the contribution of the σ2 receptor in pain processing, and to distinguish it from the contribution of the σ1 receptor, two potent σ2-selective and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands demonstrated time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model, supporting a role for the σ2 receptor in nociception, and a possible role for σ1/σ2 polypharmacology. This study illustrates the opportunities for rapid discovery of in vivo active and selective probes to study under-explored areas of biology using structure-based screens of diverse, ultra-large libraries following the elucidation of protein structures.

A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes

“…Convincingly, while this manuscript was in revision, the first crystal structure of Tmem97 in complex with synthetic σ2 ligands was reported, evidencing a ligand binding site with striking resemblance to our homology-based model. 46 As in EBP and other sterol-binding proteins, an array of hydrophobic and aromatic residues line a central Tmem97 ligand-binding cavity with a helical “cap” at the top. In our docked 20( S )-OHC:Tmem97 model, the C3 hydroxyl group of 20( S )-OHC forms a hydrogen bond to the imidazole side chain of H106, while the iso-octyl tail is accommodated by a hydrophobic pocket that includes F66, P113, V146, Y147, and Y150.…”

A practical guide to large-scale docking

“…At the end of this protocol, a receptor will have been converted into a dock-readable binding pocket, the system optimized on retrospective control calculations, and the system screened prospectively against a large chemical library. While this protocol has been specific to a single target that yielded successful results 13 , we have equally applied this protocol to a number of targets with general success demonstrating its broad applicability 12,83,118 . We have attempted to curate all of the relevant steps, though expert intuition brought by each user for their target will result in a different selection of final hits for purchase.…”

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

“…While the strengths of this study were the identification of multiple new MPro inhibitor scaffolds, with subsequent crystal structures supporting the docking predictions, the work also revealed liabilities of docking screens. In contrast to campaigns against G protein-coupled receptors [38][39][40][41] and other integral membrane receptors 42,43 , hit rates against MPro were in the 7 to 15% range, rather than the 25 to 60% range. Meanwhile, the activities of the better MPro docking hits were in the 20 to 100 µM range, not the lowto mid-nM range found against the integral membrane proteins.…”