A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CL^Pro of coronaviruses, so that the luminescent biosensor is turned on upon 3CL^Pro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.     

Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (M^pro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 M^pro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 M^pro at 2.5 µM, which is more potent than against SAR-CoV-1 M^pro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 M^pro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.     

Scutellaria barbata D. Don Inhibits the Main Proteases (Mpro and TMPRSS2) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic emerged to severely impact the global population, creating an unprecedented need for effective treatments. This study aims to investigate the potential of Scutellaria barbata D. Don (SB) as a treatment for SARS-CoV-2 infection through the inhibition of the proteases playing important functions in the infection by SARS-CoV-2. FRET assay was applied to investigate the inhibitory effects of SB on the two proteases involved in SARS-CoV-2 infection, M^pro and TMPRSS2. Additionally, to measure the potential effectiveness of SB treatment on infection inhibition, cellular models based on the Calu3 and VeroE6 cells and their TMPRSS2- expressing derivatives were assessed by viral pseudoparticles (Vpp) infection assays. The experimental approaches were conjugated with LC/MS analyses of the aqueous extracts of SB to identify the major constituent compounds, followed by a literature review to determine the potential active components of the inhibitory effects on protease activities. Our results showed that SB extracts inhibited the enzyme activities of M^pro and TMPRSS2. Furthermore, SB extracts effectively inhibited SARS-CoV-2 Vpp infection through a TMPRSS2-dependent mechanism. The aqueous extract analysis identified six major constituent compounds present in SB. Some of them have been known associated with inhibitory activities of TMPRSS2 or M^pro. Thus, SB may effectively prevent SARS-CoV-2 infection and replication through inhibiting M^pro and TMPRSS2 protease activities.     

Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules

We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3C^pro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2A^pro, 3A, 3C^pro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2A^pro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3C^pro, whereas G3BP2 is not cleaved by 3C^pro or 2A^pro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2A^pro and 3C^pro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2A^pro and heat shock protein (Hsp)90 inhibition but not 3C^pro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant.     

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression

High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87–91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC₅₀ = 7 ± 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.     

Structure-Based Identification of Natural Products as SARS-CoV-2 Mpro Antagonist from Echinacea angustifolia Using Computational Approaches

Coronavirus disease-19 (COVID-19) pandemic, caused by the novel SARS-CoV-2 virus, continues to be a global threat. The number of cases and deaths will remain escalating due to the lack of effective therapeutic agents. Several studies have established the importance of the viral main protease (M^pro) in the replication of SARS-CoV-2 which makes it an attractive target for antiviral drug development, including pharmaceutical repurposing and other medicinal chemistry approaches. Identification of natural products with considerable inhibitory potential against SARS-CoV-2 could be beneficial as a rapid and potent alternative with drug-likeness by comparison to de novo antiviral drug discovery approaches. Thereof, we carried out the structure-based screening of natural products from Echinacea-angustifolia, commonly used to prevent cold and other microbial respiratory infections, targeting SARS-CoV-2 M^pro. Four natural products namely, Echinacoside, Quercetagetin 7-glucoside, Levan N, Inulin from chicory, and 1,3-Dicaffeoylquinic acid, revealed significant docking energy (>−10 kcal/mol) in the SARS-CoV-2 M^pro catalytic pocket via substantial intermolecular contacts formation against co-crystallized ligand (<−4 kcal/mol). Furthermore, the docked poses of SARS-CoV-2 M^pro with selected natural products showed conformational stability through molecular dynamics. Exploring the end-point net binding energy exhibited substantial contribution of Coulomb and van der Waals interactions to the stability of respective docked conformations. These results advocated the natural products from Echinacea angustifolia for further experimental studies with an elevated probability to discover the potent SARS-CoV-2 M^pro antagonist with higher affinity and drug-likeness.     

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration–approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (M^pro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K_i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of M^pro (K_i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K_i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

On March 11, 2020, the World Health Organization recognized the COVID-19 outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a pandemic. Although vaccines are being approved for emergency use to fight COVID-19 (https://www.cdc.gov/vaccines/covid-19/index.html), there is an urgent need for effective new drug treatments to reduce the morbidity and mortality for patients who have already contracted COVID-19, not yet received the vaccine, are infected with SARS-CoV-2 variants that are more resistant to the vaccines, and for future coronavirus pandemics. The conventional process of drug discovery is based on automated high-throughput screening (HTS) or structure-based drug design. HTS requires a complex infrastructure, the development a miniaturized assay tailored to the individual target, and generally the need for extensive medicinal chemistry to optimize modestly potent hits that arise from the screen. The development of such screening methods is therefore sluggish and not well-suited to meet the present public health demands imposed by the COVID-19 pandemic. As an HTS alternative, structure-based approaches are advantageous when available since the rational synthesis of analogs can accelerate the generation of potent compounds. Prior to our work, other investigations have relied on structure-based methods and computational approaches to optimize moderately potent M^pro inhibitors (1, 2). However, the need for a high-resolution X-ray structure to initiate a drug discovery campaign can be extremely rate-limiting, if not altogether prohibitive. Finally, drug repurposing is often utilized as an efficient discovery strategy since it can bypass issues related to safety; however, the application of a compound to a target for which it was not optimized may never achieve maximal therapeutic benefit. A screening process which circumvents these challenges would significantly accelerate the pace for identification of clinical candidate compounds. DNA-encoded chemistry technology (DEC-Tec) is an increasingly attractive strategy to explore chemical space to identify small molecules and high-affinity binders for a multitude of protein targets (3–7). DEC-Tec involves the creation of libraries of drug-like molecules covalently attached to a unique DNA barcode that enables identification of binders for a target in a pool of millions to billions of compounds. DEC-Tec addresses these pitfalls related to HTS by screening of billions of DNA-tagged small molecules as a single mixture using an affinity selection assay (4, 8–13). This expanded number of drug-like small molecules allows for the identification of high-affinity ligands using a technologically practical format. For example, the modest protein requirement and low price of DNA sequencing both contribute to the inexpensive cost of screening small-molecule DNA-encoded chemical libraries. Additionally, the various steps involved in the selection process, which include the affinity selection to the protein, DNA sample preparation, sequencing, and data analysis, can each be performed in several days, providing for a highly expeditious screening process. Finally, the large number of compounds employed in DEC-Tec is leveraged to provide binders in the absence of preexisting structural information related to the target or its ligand-binding preferences. We believe that these features of DEC-Tec provided clear advantages for discovering novel small-molecule agents against SARS-CoV-2 targets.With our collection of over 55 DEC-Tec libraries (4 billion unique molecules) including protease-biased libraries (6) and various libraries utilizing DNA-compatible reactions developed in-house (14–22), the Center for Drug Discovery at Baylor College of Medicine has the operational capacity to support emergent action at the onset of pandemics. The genome of SARS-CoV-2 comprises six major open reading frames including two polyproteins that undergo extensive proteolytic processing to create functional proteins that perform tasks essential for viral propagation (23). This processing is largely achieved by SARS-CoV-2 main protease (M^pro or 3CL^pro) (24), a cysteine protease enzyme indispensable for the virus lifecycle and a key therapeutic target. Our hypothesis is that SARS-CoV-2 M^pro screening of billions of DNA-encoded molecules, generation of small-molecule-M^pro cocrystals, and minimal medicinal chemistry follow-up would generate drug-like inhibitors of M^pro for emergent use in patients infected with SARS-CoV-2, related variants, and related coronaviruses.To screen our DEC-Tec collection, we first constructed the pSUMO-SARS-CoV-2-M^pro plasmid carrying the SUMO-M^pro-His6 tag (SI Appendix, Fig. S1). The M^pro open reading frame sequence was flanked on the N terminus by its endogenous cleavage site (SAVLQ↓SGFRK) and on its C terminus by a PreScission cleavage site (SGVTFQ↓GP). The SUMO-M^pro-His6 recombinant fusion protein was expressed from Escherichia coli BL21(DE3), and the M^pro enzyme with its authentic N terminus and the His6 tag was purified as described in Methods and SI Appendix, Fig. S2. Because our nickel magnetic screen capture test requires a His tag, the M^pro-His6 protein was used for DEC-Tec library screening. For crystallography and enzymatic assays, the M^pro-His6 protein was treated with PreScission enzyme to remove the C-terminal 6-His tag, and the M^pro was further purified by gel filtration chromatography. Using a fluorescent peptide, Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH₂, as a reporter substrate, we first confirmed that the purified recombinant M^pro-His6 and the M^pro (“native”) lacking the His6 tag were very active and demonstrated nearly identical bioactivity in vitro.Forty unique DNA-encoded chemical libraries (DECLs) cumulatively containing 3.987 billion drug-like compounds were pooled together for the screen of M^pro-His6 binding compounds. Each library was prequantitated by qPCR, and library pooling was conducted to have 1 million copies of each compound present in the pool. Our selection for M^pro-His6 binders comprised a three-round affinity selection with an M^pro-His6 protein concentration of 1 µM. An independent affinity selection was performed in parallel without protein to serve as a no-target control to identify any nonprotein specific enrichment. Our Illumina next-generation sequencing identified a chemical series consistently enriched with excellent structure–enrichment relationships (SER) from qDOS28_1, one of our DEC-Tec libraries (Fig. 1 and SI Appendix, Scheme S1). The process of modern HTS screening has the capability to interrogate the activity of ∼100,000 small molecules per day in a specialized well-based assay (25, 26). Here, DEC-Tec was used to evaluate 4 billion small molecules as a single mixture in 1 d against M^pro. Thus DEC-Tec, through its breakneck rate of screening, offers a tremendous advantage to infectious disease targets of pathogens that pose both present and imminent public health threats.Open in a separate windowFig. 1.Enrichment profile of qDOS28_1 against SARS-CoV-2 M^pro at 1 μM. The selection data have shown the enrichment of the same BB2 (in red) and BB3 (in black) with various BB1 (in blue), where the BB1 features di-substituted amine. The methyl amide (in black) represents the DNA attachment point. The number of counts in the box represents the number of observed library members.The DNA sequences resulting from the M^pro selection were analyzed (27) to determine structural features that were enriched to prioritize compounds to be synthesized without the DNA barcode (off DNA). Compounds within DECLs are generally constructed from the union of three building blocks (trisynthons). Compounds enriched from the selection can be analyzed for those sharing one or two building blocks (monosynthons vs. disynthons) in common, leading to the identification of critical structure–enrichment relationships. The consideration of these SER leads to potent compounds in an efficient manner, supplanting the need for many rounds of laborious medicinal chemistry. Based on the M^pro selection analysis, the trisynthon CDD-1714 (“hit” molecule) and its smaller disynthons CDD-1712 and CDD-1713 (Fig. 2) were synthesized off DNA in two to four steps from commercially available materials (SI Appendix). To characterize the potency of selected compounds toward M^pro, we utilized the above described fluorescent peptide reporter assay. For initial compound screening, 25 µM of compound was incubated with M^pro and only compounds which inhibited M^pro proteolytic activity >90% were considered as candidates. The inhibition constant (K_i) values of these compounds were determined with concentrations ranging from 4 nM to 4,000 nM (SI Appendix, Fig. S3 and Table S1). Since M^pro-His6 was used in library screening, we performed parallel enzyme inhibition assays using either M^pro-His6 or M^pro to evaluate the potency of compounds toward M^pro proteolytic activity in the presence and absence of His tag. Using this protease inhibition assay, we found that CDD-1713 and CDD-1714 inhibited M^pro with K_i values of 45 nM and 20 nM, respectively (Fig. 2 and SI Appendix, Fig. S3). These results indicated that building block 1 (BB1), closest to the DNA attachment site, was less critical for binding and inhibition; CDD-1713 was chosen for optimization efforts based on its low molecular weight (353.3 g/mol) and good cLogP (2.01). CDD-1713 contains a reactive aldehyde functional group capable of forging covalent bonds with proteins, and thus we initially turned our attention toward examining the importance of the aldehyde moiety. Deleting the aldehyde (CDD-1793) or replacing with hydroxymethyl (CDD-1776) completely abolished the activity, while replacing the aldehyde with hydroxymethyl ketone (CDD-1886) drastically decreased M^pro inhibition by greater than 100-fold (Fig. 2). We therefore concluded that the aldehyde was required for activity and next proceeded to further probe its electrophilic nature. Generation of the more electron-deficient des-methoxy analog (CDD-1976) showed better inhibition compared to CDD-1713. Aldehydes have the propensity to react nonselectively; however, CDD-1712 did not show any activity, and additionally we synthesized CDD-1847 which incorporates an N-methylindazole and found that it completely lost activity. Given this remote substitution to the aldehyde, it is likely that the M^pro inhibitory activity of CDD-1713 and related analogs results from a specific binding interaction with M^pro. While aldehydes may pose a liability in drugs, compounds that contain aldehydes have been evaluated in humans, demonstrating good selectivity and other drug-like properties (28–33). To confirm that the potent inhibitors bound tightly to M^pro, we performed a protein thermal shift stability assay (thermofluor assay). CDD-1713, CDD-1714, and CDD-1976 cause a concentration-dependent stabilization of M^pro, with CDD-1976 showing the most pronounced temperature shift at all three protein concentrations (SI Appendix, Fig. S4). Taken altogether, the DEC-Tec process involving affinity selection of 4 billion DNA-encoded compounds against M^pro, analysis of SER, and synthesis of the exemplary compounds (including with different electrophiles) off DNA (SI Appendix, Fig. S5 and Table S1) yielded potent and selective inhibitors of M^pro. We note that CDD-1713, which was inferred directly from the selection, was synthesized and validated in rapid fashion (less than 10 wk from start to finish; SI Appendix, Table S2), highlighting DEC-Tec’s ability to produce potent compounds without extensive synthetic optimization.Open in a separate windowFig. 2.SARS-CoV-2 M^pro hits, analogs and examples of enzymatic inhibitory activity of these key molecules. (A) Small molecules synthesized off DNA are shown. Numbers indicate K_i values determined as described in Methods. Inactive = compounds that inhibited M^pro activity by less than 90% with 25 µM compound were considered inactive. (B) Inhibition K_i value determination against M^pro-His6. Concentration-dependent inhibition curve of CDD-1714, CDD-1713, and CDD-1976.Using the M^pro sequence, BLAST search analysis of the reference proteins encoded by the human genome shows no significant similarity. To confirm that our M^pro inhibitors do not show any potential off-target inhibition of major proteases in humans, we tested their effects on four important proteases representing four classes of human protease enzymes. As might be expected from our BLAST search, none of our inhibitors block the enzymatic activity of cathepsin B (a cysteine protease like M^pro), thrombin (a serine protease), renin (an aspartic protease), or matrix metallopeptidase-1 (MMP-1; SI Appendix, Fig. S6). Thus, our potent molecules are anticipated to have specific effects on inhibition of viral protein processing in vivo without altering human cellular activity.To understand the interactions of CDD-1713 with M^pro we determined the X-ray crystal structure of the enzyme in complex with the inhibitor. For this purpose, the purified enzyme was cocrystallized with CDD-1713. The structure was determined in space group C121 at 1.8-Å resolution with a single monomer in the asymmetric unit (Fig. 3 and SI Appendix, Table S3). The biological dimer is formed by the monomer and its symmetry-related monomer across the crystallographic two-fold axis, as seen previously in M^pro structures (1, 34).Open in a separate windowFig. 3.Crystal structure of M^pro in complex with CDD-1713. (A) Structure of M^pro (tan) with CDD-1713 (cyan). The F_o – F_c density map is shown for the inhibitor with contouring level at 3σ. The catalytic site is located within the square. (B) Magnified view of the catalytic center. Carbon atoms of the inhibitor are cyan, nitrogen atoms are blue, and oxygen atoms are red. The M^pro amino acid residues involved in CDD-1713 binding are shown as stick models and labeled. M^pro residues that form hydrogen bonds (dashed black lines) and van der Waals interactions with CDD-1713 are colored in orange and green, respectively. One water molecule involved in hydrogen bond was colored red. The site chain of Asn142 is not shown to avoid obstruction of the view of hydrogen bonds on the aldehyde group. (C) F_o − F_c omit maps showing electron density of the covalent bond formed between Cys145 and CDD-1713 contoured at 3s. Continuous electron density is present between C¹³ on the aldehyde group of CDD-1713 and thiol on the side chain of Cys145. (D) Two-dimensional diagram of the M^pro interaction with CDD-1713 generated by Ligplot⁺. Carbon atoms are shown in black, nitrogen atoms are blue, and oxygen atoms are red. Ligands are colored cyan and hydrogen bonds are represented as black dashed lines, hydrogen bond contacts are colored orange, and hydrophobic contacts are represented as green spoke arcs. Water molecule was presented as red sphere. The length of each hydrogen bond is labeled.Examination of the structure reveals CDD-1713 is positioned in the active site of M^pro with the electron density clearly showing a 1.7-Å covalent bond from the aldehyde of CDD-1713 to Sγ of the catalytic residue Cys145 (Fig. 3 B and C). The carbonyl oxygen of the aldehyde forms hydrogen bonds with the main chain nitrogens of Gly143 and Cys145 that form the oxyanion hole of the enzyme. Similar interactions have been reported for bicyclopropane-containing inhibitors of M^pro with an aldehyde warhead (1). The active site of M^pro contains four subsites (S1′, S1, S2, and S3) that accommodate the amino acids of the peptide substrate or peptidomimetic inhibitors (P1′, P1, P2, and P3) (35, 36). M^pro has a stringent requirement for a P1 glutamine occupying the S1 subsite (35). The indazole ring of CDD-1713 inserts into the S1 pocket (Fig. 3). The NH of the indazole group forms hydrogen bonds with the side-chain Oε of Glu166 and the main-chain O of Phe140, while the N of the indazole forms a hydrogen bond with the Nε2 of His163 (Fig. 3). In addition, the indazole group makes hydrophobic interactions with Phe140, Leu141, Asn142, and Glu166 (Fig. 3). The extensive interactions of the indazole with residues in the S1 pocket is of note in that the residues in the S1 pocket are largely conserved among coronavirus M^pro enzymes, suggesting CDD-1713 may exhibit broad M^pro specificity (37). The central phenyl ring of CDD-1713 makes hydrophobic interactions with Asn142 and positions the aldehyde group for interaction with Cys143. The O-alkyl chain on the central phenyl ring occupies a region between the S2 and S1′ subsites. The terminal methyl groups of the dimethylamide make hydrophobic interactions with His41, Cys44, Thr45, Ser46, and Met49 (Fig. 3D). Note that the dimethylamide is partially buried in the structure, suggesting the BB1 group in CDD-1714 and the DNA-attachment site may sterically clash with the Thr45-Met49 region. However, the rotatable bonds in the O-alkyl chain may allow positioning consistent with binding of the BB1 group and solvent exposure of the DNA-attachment point (Fig. 3B). Further, the methoxy group attached to the central phenyl ring is solvent-exposed and does not interact with M^pro. The design of M^pro inhibitors has mostly centered on templates having multiple amide bonds to mimic the enzyme’s natural peptide substrates (1, 34, 38–41). CDD-1713 differs significantly from the amide-based inhibitors, allowing it to forge different interactions within the same M^pro active site (SI Appendix, Fig. S3E). Thus, the DEC-Tec approach supports the elucidation of novel pharmacophores to enzymes without specific design principles or prior knowledge of substrate preferences.Using mouse and human liver microsomes, CDD-1713 was found to be metabolically labile in both mouse and human assays, while CDD-1976 is more stable in human liver microsomes, but not mouse (SI Appendix, Table S4). CDD-1713 and CDD-1976 displayed moderate cell permeability in an uptake assay of HepG2 cells (SI Appendix, Fig. S7A) and no obvious cytotoxicity in HepG2 cells was observed for both compounds at 100 μM (SI Appendix, Fig. S7B). Both CDD-1713 and CDD-1976 are relatively stable in human and mouse plasma; 80% of compounds remained in plasma after 2-h incubation (SI Appendix, Fig. S7C).The viral inhibitory capacity of compounds CDD-1713 and CDD-1976 was demonstrated using a real-time cell analysis (RTCA) assay. To evaluate viral inhibition, different concentrations of the M^pro inhibitors were added to VERO E6 cells incubated with a live strain of the SARS-CoV-2 virus. Subsequently, the growth kinetics of the cells were followed over 75 h. In the positive control group, SARS-CoV-2 effectively caused cell death, which was observed as a decrease in the normalized cell index compared to the negative control group (no virus). CDD-1713 and CDD-1976 successfully reduced cell death in a dose-dependent manner, indicating that virus replication was stopped (Fig. 4). CDD-1976 was found to be most effective, with a calculated half-maximal inhibitory concentration (IC₅₀) of 2.50 µM, followed by CDD-1713 with an IC₅₀ of 5.19 µM. CDD-1847, a closely related analog that abolishes binding to M^pro, was unable to prevent cell death by the SARS-CoV-2 virus.Open in a separate windowFig. 4.Normalized cell index plotted versus concentration (log) of M^pro drug compounds measured using the xCELLigence RTCA. Average data points from duplicate measurements. A sigmoidal dose–response curve was fitted to determine IC₅₀ values for each M^pro drug compound (lines).In summary, using a DEC-Tec–based strategy we identified CDD-1713 and CDD-1976 as a class of potent and selective inhibitors of SARS-CoV-2 M^pro that block viral reproduction in a short span of time. X-ray crystallography was further deployed to elucidate the structural details of M^pro inhibition by CDD-1713, and this information should enable further development of drug-like M^pro inhibitors. These studies support DEC-Tec as an expedient and effective paradigm for generating therapeutics against critical targets within the SARS-CoV-2 genome.     

Glycyrrhizin Effectively Inhibits SARS-CoV-2 Replication by Inhibiting the Viral Main Protease

The outbreak of SARS-CoV-2 developed into a global pandemic affecting millions of people worldwide. Despite one year of intensive research, the current treatment options for SARS-CoV-2 infected people are still limited. Clearly, novel antiviral compounds for the treatment of SARS-CoV-2 infected patients are still urgently needed. Complementary medicine is used along with standard medical treatment and accessible to a vast majority of people worldwide. Natural products with antiviral activity may contribute to improve the overall condition of SARS-CoV-2 infected individuals. In the present study, we investigated the antiviral activity of glycyrrhizin, the primary active ingredient of the licorice root, against SARS-CoV-2. We demonstrated that glycyrrhizin potently inhibits SARS-CoV-2 replication in vitro. Furthermore, we uncovered the underlying mechanism and showed that glycyrrhizin blocks the viral replication by inhibiting the viral main protease M^pro that is essential for viral replication. Our data indicate that the consumption of glycyrrhizin-containing products such as licorice root tea of black licorice may be of great benefit for SARS-CoV-2 infected people. Furthermore, glycyrrhizin is a good candidate for further investigation for clinical use to treat COVID-19 patients.     

Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study

Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CL^pro provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally.     

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2

Background: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. Methods: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4⁺ and CD8⁺ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. Results: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4⁺CD45RA⁻CCR7⁻ phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. Conclusion: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8⁺, but not CD4⁺, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.     

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affinity complex with RTA, against ricin-induced toxicity in cell- based luciferase translation and cell cytotoxicity assays were evaluated. METHODS： To test the therapeutic potential of anti- RTA aptamers in Chinese hamster ovary （CliO） AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured us- ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet- razolium compound [3-（4,5-dimethylthiazol-2-yl）- 5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium]. RESULTS： Inhibition of protein synthesis by ricin in CliO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly- cosylated RTA （dgA） neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CliO AA8 cells. CONCLUSION： We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell- based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.     

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 Å cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K_D = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the “off-target” effect of a small molecule is mediated by an MAI mechanism.     

Functional cleavage of the common cytokine receptor γ chain (γc) by calpain

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γ_c) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type γ_c, but not γ_c containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γ_c in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γ_c. Moreover, in single positive CD4⁺ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γ_c blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γ_c represents a mechanism by which γ_c-dependent signaling can be controlled.     

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.     

Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents. METHODS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems. RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a. CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.     

Host cell factor requirement for hepatitis C virus enzyme maturation

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus. The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2/3. Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone/folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.