Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

Enantioselective Syntheses of Wickerols A and B.

The evolution of a successful strategy for the synthesis of the strained, cage-like antiviral diterpenoids wickerols A and B is described. Initial attempts to access the carbocyclic core were surprisingly challenging and in retrospect, presaged the many detours needed to ultimately arrive at the fully adorned wickerol architecture. In most cases, conditions to trigger desired outcomes with respect to both reactivity and stereochemistry were hard-won. The successful synthesis ultimately leveraged alkenes in virtually all productive bond-forming events. A series of conjugate addition reactions generated the fused tricyclic core, a Claisen rearrangement was used to install an otherwise unmanageable methyl-bearing stereogenic center, and a Prins cyclization closed the strained bridging ring. This final reaction proved enormously interesting because the strain of the ring system permitted diversion of the presumed initial Prins product into several different scaffolds.

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the Mpro. Starting from crystal structures of the Mpro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the Mpro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC50 of 13 nM versus the recombinant Mpro, and similar potency was observed for its P1' N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 Mpro inhibition, we also explored the activity of MG-78 against the Mpro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 Mpro), moderate (1.45 µM, Coxsackievirus 3Cpro), and relatively poor (6.7 µM, enterovirus A71 3Cpro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus Mpros but further optimization would be needed to target enterovirus 3Cpros efficiently.

Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine from Octopus cyanea.

The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1), 2-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and auramine (3) have been isolated from the sea anemone Heteractis aurora. Both indole alkaloids were synthesized for the confirmation of the structures. Homarine (4), along with uracil (5), hypoxanthine (6), and inosine (7) have been obtained from Octopus cyanea.

Detection of a new piperideine alkaloid in the pygidial glands of some Stenus beetles.

Rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) in their pygidial glands to protect themselves from predation and microorganismic infestation. The biosynthesis of stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) was previously investigated in Stenus bimaculatus, Stenus similis, and Stenus solutus, respectively. The piperideine alkaloid cicindeloine (5) occurs also as a major compound in the pygidial gland secretion of Stenus cicindeloides. The three metabolites follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from L-lysine and the side chain from L-isoleucine. The different alkaloids are finally obtained by few modifications of shared precursor molecules, such as 2,3,4,5-tetrahydro-5-(2-methylbutylidene)pyridine (1). This piperideine alkaloid was synthesized and detected by GC/MS and GC at a chiral phase in the pygidial glands of Stenus similis, Stenus tarsalis, and Stenus cicindeloides.

Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (Mpro, 3CLpro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. Mpro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 Mpro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC50 of 120 nM against the Mpro and EC50 values of 0.8-3.4 µM for antiviral activity in different cell types. Crystal structures have been elucidated for the Mpro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.

Identification of Adenosine-to-Inosine RNA Editing with Acrylonitrile Reagents.

New chemical probes have been designed to facilitate the identification of adenosine-to-inosine (A-to-I) edited RNAs. These reagents combine a conjugate acceptor for selective inosine covalent modification with functional groups for bioorthogonal biotinylation. The resulting biotinylated RNA was enriched and verified with RT-qPCR. This powerful chemical approach provides new opportunities to identify and quantify A-to-I editing sites.

Ceropegia sandersonii Mimics Attacked Honeybees to Attract Kleptoparasitic Flies for Pollination.

Four to six percent of plants, distributed over different angiosperm families, entice pollinators by deception [1]. In these systems, chemical mimicry is often used as an efficient way to exploit the olfactory preferences of animals for the purpose of attracting them as pollinators [2,3]. Here, we report a very specific type of chemical mimicry of a food source. Ceropegia sandersonii (Apocynaceae), a deceptive South African plant with pitfall flowers, mimics attacked honeybees. We identified kleptoparasitic Desmometopa flies (Milichiidae) as the main pollinators of C. sandersonii. These flies are well known to feed on honeybees that are eaten by spiders, which we thus predicted as the model chemically mimicked by the plant. Indeed, we found that the floral scent of C. sandersonii is comparable to volatiles released from honeybees when under simulated attack. Moreover, many of these shared compounds elicited physiological responses in antennae of pollinating Desmometopa flies. A mixture of four compounds-geraniol, 2-heptanone, 2-nonanol, and (E)-2-octen-1-yl acetate-was highly attractive to the flies. We conclude that C. sandersonii is specialized on kleptoparasitic fly pollinators by deploying volatiles linked to the flies' food source, i.e., attacked and/or freshly killed honeybees. The blend of compounds emitted by C. sandersonii is unusual among flowering plants and lures kleptoparasitic flies into the trap flowers. This study describes a new example of how a plant can achieve pollination through chemical mimicry of the food sources of adult carnivorous animals.