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

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Structure-Based Design of a Chemical Probe Set for the 5-HT5A Serotonin Receptor.

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-µM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR.

Colloidal Aggregators in Biochemical SARS-CoV-2 Repurposing Screens.

To fight COVID-19, much effort has been directed toward in vitro drug repurposing. Here, we investigate the impact of colloidal aggregation, a common screening artifact, in these repurposing campaigns. We tested 56 drugs reported as active in biochemical assays for aggregation by dynamic light scattering and by detergent-based enzyme counter screening; 19 formed colloids at concentrations similar to their literature IC50's, and another 14 were problematic. From a common repurposing library, we further selected another 15 drugs that had physical properties resembling known aggregators, finding that six aggregated at micromolar concentrations. This study suggests not only that many of the drugs repurposed for SARS-CoV-2 in biochemical assays are artifacts but that, more generally, at screening-relevant concentrations, even drugs can act artifactually via colloidal aggregation. Rapid detection of these artifacts will allow the community to focus on those molecules that genuinely have potential for treating COVID-19.

Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2.

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 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 we 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.

Phospholipidosis is a shared mechanism underlying the in vitro antiviral activity of many repurposed drugs against SARS-CoV-2.

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.

A Crowding Barrier to Protein Inhibition in Colloidal Aggregates.

Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.