Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.

The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.

A Novel Ambroxol-Derived Tetrahydroquinazoline with a Potency against SARS-CoV-2 Proteins.

We report synthesis of a novel 1,2,3,4-tetrahydroquinazoline derivative, named 2-(6,8-dibromo-3-(4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazolin-2-yl)phenol (1), which was obtained from the hydrochloride of 4-((2-amino-3,5-dibromobenzyl)amino)cyclohexan-1-ol (ambroxol hydrochloride) and salicylaldehyde in EtOH. The resulting compound was produced in the form of colorless crystals of the composition 1∙0.5EtOH. The formation of the single product was confirmed by the IR and 1H spectroscopy, single-crystal and powder X-ray diffraction, and elemental analysis. The molecule of 1 contains a chiral tertiary carbon of the 1,2,3,4-tetrahydropyrimidine fragment and the crystal structure of 1∙0.5EtOH is a racemate. Optical properties of 1∙0.5EtOH were revealed by UV-vis spectroscopy in MeOH and it was established that the compound absorbs exclusively in the UV region up to about 350 nm. 1∙0.5EtOH in MeOH exhibits dual emission and the emission spectra contains bands at about 340 and 446 nm upon excitation at 300 and 360 nm, respectively. The DFT calculations were performed to verify the structure as well as electronic and optical properties of 1. ADMET properties of the R-isomer of 1 were evaluated using the SwissADME, BOILED-Egg, and ProTox-II tools. As evidenced from the blue dot position in the BOILED-Egg plot, both human blood-brain barrier penetration and gastrointestinal absorption properties are positive with the positive PGP effect on the molecule. Molecular docking was applied to examine the influence of the structures of both R-isomer and S-isomer of 1 on a series of the SARS-CoV-2 proteins. According to the docking analysis results, both isomers of 1 were found to be active against all the applied SARS-CoV-2 proteins with the best binding affinities with Papain-like protease (PLpro) and nonstructural protein 3 (Nsp3_range 207-379-AMP). Ligand efficiency scores for both isomers of 1 inside the binding sites of the applied proteins were also revealed and compared with the initial ligands. Molecular dynamics simulations were also applied to evaluate the stability of complexes of both isomers with Papain-like protease (PLpro) and nonstructural protein 3 (Nsp3_range 207-379-AMP). The complex of the S-isomer with Papain-like protease (PLpro) was found to be highly unstable, while the other complexes are stable.

An Unexpected Enzyme in Vascular Smooth Muscle Cells: Angiotensin II Upregulates Cholesterol-25-Hydroxylase Gene Expression.

Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.

Rhenium(V) Complexes as Cysteine-Targeting Coordinate Covalent Warheads.

Interest in covalent enzyme inhibitors as therapeutic agents has seen a recent resurgence. Covalent enzyme inhibitors typically possess an organic functional group that reacts with a key feature of the target enzyme, often a nucleophilic cysteine residue. Herein, the application of small, modular ReV complexes as inorganic cysteine-targeting warheads is described. These metal complexes were found to react with cysteine residues rapidly and selectively. To demonstrate the utility of these ReV complexes, their reactivity with SARS-CoV-2-associated cysteine proteases is presented, including the SARS-CoV-2 main protease and papain-like protease and human enzymes cathepsin B and L. As all of these proteins are cysteine proteases, these enzymes were found to be inhibited by the ReV complexes through the formation of adducts. These findings suggest that these ReV complexes could be used as a new class of warheads for targeting surface accessible cysteine residues in disease-relevant target proteins.

RNA dependent RNA polymerase (RdRp) as a drug target for SARS-CoV2.

RNA-dependent RNA polymerase (RdRp), also called nsp12, is considered a promising but challenging drug target for inhibiting replication and hence, the growth of various RNA-viruses. In this report, a computational study is performed to offer insights on the binding of Remdesivir and Galidesivir with SARS-CoV2 RdRp with natural substrate, ATP, as the control. It was observed that Remdesivir and Galidesivir exhibited similar binding energies for their best docked poses, -6.6 kcal/mole and -6.2 kcal/mole, respectively. ATP also displayed comparative and strong binding free energy of -6.3 kcal/mole in the catalytic site of RdRp. However, their binding locations within the active site are distinct. Further, the interaction of catalytic site residues (Asp760, Asp761, and Asp618) with Remdesivir and Galidesivir is comprehensively examined. Conformational changes of RdRp and bound molecules are demonstrated using 100 ns explicit solvent simulation of the protein-ligand complex. Simulation suggests that Galidesivir binds at the non-catalytic location and its binding strength is relatively weaker than ATP and Remdesivir. Remdesivir also binds at the catalytic site and showed high potency to inhibit the function of RdRp. Binding of co-factor units nsp7 and nsp8 with RdRp (nsp12) complexed with Remdesivir and Galidesivir was also examined. MMPBSA binding energy for all three complexes has been computed across the 100 ns simulation trajectory. Overall, this study suggests, Remdesivir has anti-RdRp activity via binding at a catalytic site. In contrast, Galidesivir may not have direct anti-RdRp activity but it can induce a conformational change in the RNA polymerase.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. M^pro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of M^pro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against M^pro with IC₅₀ of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 M^pro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of M^pro, and ACF has been previously reported as a PL^pro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Computer-Aided Screening for Potential Coronavirus 3-Chymotrypsin-like Protease (3CLpro) Inhibitory Peptides from Putative Hemp Seed Trypsinized Peptidome.

To control the COVID-19 pandemic, antivirals that specifically target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently required. The 3-chymotrypsin-like protease (3CLpro) is a promising drug target since it functions as a catalytic dyad in hydrolyzing polyprotein during the viral life cycle. Bioactive peptides, especially food-derived peptides, have a variety of functional activities, including antiviral activity, and also have a potential therapeutic effect against COVID-19. In this study, the hemp seed trypsinized peptidome was subjected to computer-aided screening against the 3CLpro of SARS-CoV-2. Using predictive trypsinized products of the five major proteins in hemp seed (i.e., edestin 1, edestin 2, edestin 3, albumin, and vicilin), the putative hydrolyzed peptidome was established and used as the input dataset. To select the Cannabis sativa antiviral peptides (csAVPs), a predictive bioinformatic analysis was performed by three webserver screening programs: iAMPpred, AVPpred, and Meta-iAVP. The amino acid composition profile comparison was performed by COPid to screen for the non-toxic and non-allergenic candidates, ToxinPred and AllerTOP and AllergenFP, respectively. GalaxyPepDock and HPEPDOCK were employed to perform the molecular docking of all selected csAVPs to the 3CLpro of SARS-CoV-2. Only the top docking-scored candidate (csAVP4) was further analyzed by molecular dynamics simulation for 150 nanoseconds. Molecular docking and molecular dynamics revealed the potential ability and stability of csAVP4 to inhibit the 3CLpro catalytic domain with hydrogen bond formation in domain 2 with short bonding distances. In addition, these top ten candidate bioactive peptides contained hydrophilic amino acid residues and exhibited a positive net charge. We hope that our results may guide the future development of alternative therapeutics against COVID-19.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

The potential effects of DPP-4 inhibitors on cardiovascular system in COVID-19 patients.

With the outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the public healthcare systems are facing great challenges. Coronavirus disease 2019 (COVID-19) could develop into severe pneumonia, acute respiratory distress syndrome and multi-organ failure. Remarkably, in addition to the respiratory symptoms, some COVID-19 patients also suffer from cardiovascular injuries. Dipeptidyl peptidase-4 (DPP-4) is a ubiquitous glycoprotein which could act both as a cell membrane-bound protein and a soluble enzymatic protein after cleavage and release into the circulation. Despite angiotensin-converting enzyme 2 (ACE2), the recently recognized receptor of SARS-CoV and SARS-CoV-2, which facilitated their entries into the host, DPP-4 has been identified as the receptor of middle east respiratory syndrome coronavirus (MERS-CoV). In the current review, we discussed the potential roles of DPP-4 in COVID-19 and the possible effects of DPP-4 inhibitors on cardiovascular system in patients with COVID-19.

Mutation Effects on Structure and Dynamics: Adaptive Evolution of the SARS-CoV-2 Main Protease.

The main protease of SARS-CoV-2 (Mpro) plays a critical role in viral replication; although it is relatively conserved, Mpro has nevertheless evolved over the course of the COVID-19 pandemic. Here, we examine phenotypic changes in clinically observed variants of Mpro, relative to the originally reported wild-type enzyme. Using atomistic molecular dynamics simulations, we examine effects of mutation on protein structure and dynamics. In addition to basic structural properties such as variation in surface area and torsion angles, we use protein structure networks and active site networks to evaluate functionally relevant characters related to global cohesion and active site constraint. Substitution analysis shows a continuing trend toward more hydrophobic residues that are dependent on the location of the residue in primary, secondary, tertiary, and quaternary structures. Phylogenetic analysis provides additional evidence for the impact of selective pressure on mutation of Mpro. Overall, these analyses suggest evolutionary adaptation of Mpro toward more hydrophobicity and a less-constrained active site in response to the selective pressures of a novel host environment.

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro enzyme through in silico approach.

A new SARS coronavirus (SARS-CoV-2) belonging to the genus Betacoronavirus has caused a pandemic known as COVID-19. Among coronaviruses, the main protease (Mpro) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral RNA and recognizes specific cleavage sites. There are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. Therefore, targeting the SARS-CoV-2 Mpro enzyme with small molecules can block viral replication. The present study is aimed at the identification of promising lead molecules for SARS-CoV-2 Mpro enzyme through virtual screening of antiviral compounds from plants. The binding affinity of selected small drug-like molecules to SARS-CoV-2 Mpro, SARS-CoV Mpro and MERS-CoV Mpro were studied using molecular docking. Bonducellpin D was identified as the best lead molecule which shows higher binding affinity (-9.28 kcal/mol) as compared to the control (-8.24 kcal/mol). The molecular binding was stabilized through four hydrogen bonds with Glu166 and Thr190 as well as hydrophobic interactions via eight residues. The SARS-CoV-2 Mpro shows identities of 96.08% and 50.65% to that of SARS-CoV Mpro and MERS-CoV Mpro respectively at the sequence level. At the structural level, the root mean square deviation (RMSD) between SARS-CoV-2 Mpro and SARS-CoV Mpro was found to be 0.517 Å and 0.817 Å between SARS-CoV-2 Mpro and MERS-CoV Mpro. Bonducellpin D exhibited broad-spectrum inhibition potential against SARS-CoV Mpro and MERS-CoV Mpro and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions.

Replication of the 30-kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared with single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16-30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high-affinity RNA-protein interaction in a unidirectional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

Genetically Encoded Detection of Biosynthetic Protease Inhibitors.

Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds. The detection scheme relies on a bacterial two-hybrid (B2H) system that links protease inactivation to the transcription of a swappable reporter gene. This system, which can accomodate multiple biochemical outputs (i.e., luminescence and antibiotic resistance), permitted the facile incorporation of four disease-relevant proteases. A B2H designed to detect the inactivation of the main protease of severe acute respiratory syndrome coronavirus 2 enabled the identification of a terpenoid inhibitor of modest potency. An analysis of multiple pathways that make this terpenoid, however, suggested that its production was necessary but not sufficient to confer a survival advantage in growth-coupled assays. This finding highlights an important challenge associated with the use of genetic selection to search for inhibitors─notably, the influence of pathway toxicity─and underlines the value of including multiple pathways with overlapping product profiles in pathway screens. This study provides a detailed experimental framework for using microbes to screen libraries of biosynthetic pathways for targeted protease inhibitors.

Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease.

In this study, we described an easy-to-perform nano-luciferase (nLuc) sensor for the rapid detection of 3-chymotrypsin-like protease (3CLpro) encoded by SARS-CoV-2. The technology is based on the cleavage reaction of recombinant-nLuc via 3CLpro. The nLuc-based assay is a general, one-step method and is naturally specific in detection. The stability, sensitivity, detection range, and response time are fully characterized. The application of 3CLpro detection in artificial and human saliva as well as antiviral drug screening demonstrates that the method can quantify 3CLpro with high sensitivity in one step. With its unique features, the nLuc-based assay may find broad applications in the auxiliary diagnosis of SARS-CoV-2, as well as other types of coronavirus infection.