The σ receptor is an enigmatic protein that has attracted significant attention because of its involvement in diseases as diverse as cancer and neurological disorders. Unlike virtually all other receptors of medical interest, it has eluded molecular cloning since its discovery, and the gene that codes for the receptor remains unknown, precluding the use of modern biological methods to study its function. Using a chemical biology approach, we purified the σ receptor from tissue, revealing its identity as TMEM97, an endoplasmic reticulum-resident transmembrane protein that regulates the sterol transporter NPC1. We show that TMEM97 possesses the full suite of molecular properties that define the σ receptor, and we identify Asp29 and Asp56 as essential for ligand recognition. Cloning the σ receptor resolves a longstanding mystery and will enable therapeutic targeting of this potential drug target.

With over 60,000 protein structures available in the Protein Data Bank, it is frequently possible use one of them to obtain starting phase information and to solve new crystal structures. Molecular replacement1–4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5–8, have allowed the rapid solution of large numbers of protein structures. Despite extensive work9–14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modeling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modeling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction datasets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction datasets of better than 3.2Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

Repurposing drugs as treatments for COVID-19 has drawn much attention. Beginning with sigma receptor ligands, and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.

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