Colloidal Aggregation Confounds Cell-Based Covid-19 Antiviral Screens.

Colloidal aggregation is one of the largest contributors to false positives in early drug discovery. Here, we consider aggregation's role in cell-based infectivity assays in Covid-19 drug repurposing. We investigated the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate the impact of aggregation on antiviral efficacy in cells, we presaturated the colloidal drug suspensions with BSA or spun them down by centrifugation and measured the effects on spike pseudovirus infectivity. Antiviral potencies diminished by at least 10-fold following both BSA and centrifugation treatments, supporting a colloid-based mechanism. Aggregates induced puncta of the labeled spike protein in fluorescence microscopy, consistent with sequestration of the protein on the colloidal particles. These observations suggest that colloidal aggregation is common among cell-based antiviral drug repurposing and offers rapid counter-screens to detect and eliminate these artifacts.

AlphaFold2 structures guide prospective ligand discovery.

AlphaFold2 (AF2) models have had wide impact but mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and serotonin 2A (5-HT2A) receptors, testing hundreds of new molecules and comparing results with those obtained from docking against the experimental structures. Hit rates were high and similar for the experimental and AF2 structures, as were affinities. Success in docking against the AF2 models was achieved despite differences between orthosteric residue conformations in the AF2 models and the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5-HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based drug design.

Large library docking for cannabinoid-1 receptor agonists with reduced side effects.

Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

Small vs. Large Library Docking for Positive Allosteric Modulators of the Calcium Sensing Receptor.

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism.

Colloidal aggregation confounds cell-based Covid-19 antiviral screens.

Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on pure-protein screens; here we consider aggregations role in cell-based infectivity assays in Covid-19 drug repurposing. We began by investigating the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal-particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate antiviral efficacy of the drugs in cells we used spike pseudotyped lentivirus and pre-saturation of the colloids with BSA. The antiviral potency of the aggregators was diminished by at least 10-fold and often entirely eliminated in the presence of BSA, suggesting antiviral activity can be attributed to the non-specific nature of the colloids. In confocal microscopy, the aggregates induced fluorescent puncta of labeled spike protein, consistent with sequestration of the protein on the colloidal particles. Addition of either non-ionic detergent or of BSA disrupted these puncta. These observations suggest that colloidal aggregation is common among cell-based anti-viral drug repurposing, and perhaps cell-based assays more broadly, and offers rapid counter-screens to detect and eliminate these artifacts, allowing the community invest resources in compounds with true potential as a Covid-19 therapeutic.

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 Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase.

An under-explored target for SARS-CoV-2 is the S-adenosyl methionine (SAM)-dependent methyltransferase Nsp14, which methylates the N7-guanosine of viral RNA at the 5'-end, allowing the virus to evade host immune response. We sought new Nsp14 inhibitors with three large library docking strategies. First, up to 1.1 billion lead-like molecules were docked against the enzyme's SAM site, leading to three inhibitors with IC50 values from 6 to 50 µM. Second, docking a library of 16 million fragments revealed 9 new inhibitors with IC50 values from 12 to 341 µM. Third, docking a library of 25 million electrophiles to covalently modify Cys387 revealed 7 inhibitors with IC50 values from 3.5 to 39 µM. Overall, 32 inhibitors encompassing 11 chemotypes had IC50 values < 50 µM and 5 inhibitors in 4 chemotypes had IC50 values < 10 µM. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing starting points for future optimization.