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Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (M pro ). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC 50 values of 15 ± 4 and 4.2 ± 0.7 μM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with M pro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead M pro inhibitor for the development of therapeutics for SARS-CoV-2 infection. Here, using in vitro assays and structural analysis, the authors characterize the anti-SARS-CoV-2 properties of two small molcules, showing these to bind and target the virus main protease (M pro ), and to exhibit a synergistic antiviral effect when combined with remdesivir in vitro.

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1H-NMR, 13C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC50 value of 160 nM. Compound 13b exhibited an enzyme IC50 value of 4.9 µM. However, it exhibited a potent antiviral EC50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL pro ) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CL pro from PEDV. Analysis of the PEDV 3CL pro structure and comparison to other coronaviral 3CL pro ’s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL pro ’s are conserved, with the exception of a loop that comprises the protease S 2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL pro , ( R )- 16 , to have inhibitor activity against PEDV 3CL pro , despite that SARS-3CL pro and PEDV 3CL pro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in ( R )- 16 binding to SARS-3CL pro are conserved in PEDV-3CL pro ; however, the sequence variation and positional difference in the loop forming the S 2 pocket may account for large observed difference in IC 50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CL pro architecture and contributes to the broader structural knowledge of coronaviral 3CL pro ’s.

Abstract COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment. Competing Interest Statement A.D.M has a sponsored program contract with Pfizer to test compounds for inhibition of coronavirus proteases. JW has a sponsored research agreement with Pfizer to test compounds for inhibition of coronavirus proteases. The Frieman Laboratory was funded by Pfizer for the work in this manuscript. Footnotes * One Sentence Summary: PF-07304814, a novel phosphate prodrug is disclosed as an investigational novel intravenous small molecule 3CL protease inhibitor for the potential treatment of COVID-19 and other coronavirus infections. * One small typo in the abstract. The compound ID number should be PF-00835231 and in the abstract the number is PF-00835321 in one place. This has been corrected in the abstract and in the manuscript. Version 3: Added in vivo efficacy data, section Activity of PF-00835231 in a mouse model of SARS-CoV-1 infection and Figure 4.

The World Health Organization highlighted the potential protective role of exercise against cognitive decline, all-cause dementia, Alzheimer's disease (AD), and vascular dementia in healthy individuals. We have previously shown that exercise is particularly beneficial for older, cognitively unimpaired apolipoprotein E4 ( ε4) carriers. A key unanswered question is whether a long-term, high-intensity aerobic exercise intervention initiated in a cohort of previously inactive older individuals at genetic risk for AD has neuroprotective properties. CYCLE-AD is a randomized, single-blind, single-center, controlled trial of a home-based, high-intensity exercise intervention involving 150 older ε4 carriers (ages 65-80 years) who are healthy, cognitively unimpaired, and physically inactive. Participants are randomized into two groups: indoor cycling (IC) or usual and customary care (UCC) (target of 75 each). IC participants exercise 3×/week on an upright stationary cycle ergometer at a moderate-vigorous intensity for 18 months. Those in the UCC group are expected to maintain enrollment levels of activity. Comparison of IC and UCC groups on change in primary and secondary outcomes over baseline, 9-month, and 18-month evaluations. Primary outcomes are VO (Fitness), 5-trial total recall on the Rey Auditory Verbal List Learning Test (Episodic Memory), and total hippocampal volume derived from structural MRI (Brain Atrophy). Secondary outcomes include comprehensive neurocognitive and physical function test batteries, MRI scans including structural and functional connectivity measures, and blood-based biomarkers. Over an 18-month interval, physically inactive ε4 carriers who engage in high-intensity aerobic exercise will demonstrate less cognitive decline and hippocampal atrophy than physically inactive ε4 carriers who did not participate in a formal exercise program. Successful demonstration of a scalable, home-based, high-intensity aerobic exercise intervention in altering the trajectory of AD pathophysiology and its effects on cognitive functioning will transform AD treatment, improve patient outcomes and quality of life, and reduce healthcare costs.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems. Here, the authors show structural, biochemical, and functional insights into the discovery of epichaperome‐ directed chemical probes for use in central nervous system diseases. Probes emerging from this work have translated to human clinical studies in Alzheimer’s disease and cancer.

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs. Cryo-EM structure and inhibition assays reveal a non-nucleoside inhibitor of the respiratory syncytial virus polymerase complex that acts by binding to an induced-fit pocket in the capping domain.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9-megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99.99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.