Discovery of Peptidic Ligands against the SARS-CoV-2 Spike Protein and Their Use in the Development of a Highly Sensitive Personal Use Colorimetric COVID-19 Biosensor.

In addition to efficacious vaccines and antiviral therapeutics, reliable and flexible in-home personal use diagnostics for the detection of viral antigens are needed for effective control of the COVID-19 pandemic. Despite the approval of several PCR-based and affinity-based in-home COVID-19 testing kits, many of them suffer from problems such as a high false-negative rate, long waiting time, and short storage period. Using the enabling one-bead-one-compound (OBOC) combinatorial technology, several peptidic ligands with a nanomolar binding affinity toward the SARS-CoV-2 spike protein (S-protein) were successfully discovered. Taking advantage of the high surface area of porous nanofibers, immobilization of these ligands on nanofibrous membranes allows the development of personal use sensors that can achieve low nanomolar sensitivity in the detection of the S-protein in saliva. This simple biosensor employing naked-eye reading exhibits detection sensitivity comparable to some of the current FDA-approved home detection kits. Furthermore, the ligand used in the biosensor was found to detect the S-protein derived from both the original strain and the Delta variant. The workflow reported here may enable us to rapidly respond to the development of home-based biosensors against future viral outbreaks.

Natural Product-Based Screening for Lead Compounds Targeting SARS CoV-2 Mpro.

Drugs that cure COVID-19 have been marketed; however, this disease continues to ravage the world without becoming extinct, and thus, drug discoveries are still relevant. Since Mpro has known advantages as a drug target, such as the conserved nature of the active site and the absence of homologous proteins in the body, it receives the attention of many researchers. Meanwhile, the role of traditional Chinese medicine (TCM) in the control of epidemics in China has also led to a focus on natural products, with the hope of finding some promising lead molecules through screening. In this study, we selected a commercial library of 2526 natural products from plants, animals and microorganisms with known biological activity for drug discovery, which had previously been reported for compound screening of the SARS CoV-2 S protein, but had not been tested on Mpro. This library contains compounds from a variety of Chinese herbs, including Lonicerae Japonicae Flos, Forsythiae Fructus and Scutellariae Radix, which are derived from traditional Chinese medicine prescriptions that have been shown to be effective against COVID-19. We used the conventional FRET method for the initial screening. After two rounds of selection, the remaining 86 compounds were divided into flavonoids, lipids, phenylpropanoids, phenols, quinones, alkaloids, terpenoids and steroids according to the skeleton structures, with inhibition rates greater than 70%. The top compounds in each group were selected to test the effective concentration ranges; the IC50 values were as follows: (-)-gallocatechin gallate (1.522 ± 0.126 µM), ginkgolic acid C15:1 (9.352 ± 0.531 µM), hematoxylin (1.025 ± 0.042 µM), fraxetin (2.486 ± 0.178 µM), wedelolactone (1.003 ± 0.238 µM), hydroxytyrosol acetate (3.850 ± 0.576 µM), vanitiolide (2.837 ± 0.225 µM), ß,ß-dimethylacrylalkannin (2.731 ± 0.308 µM), melanin (7.373 ± 0.368 µM) and cholesteryl sodium sulfate (2.741 ± 0.234µM). In the next step, we employed two biophysical techniques, SPR and nanoDSF, to obtain KD/Kobs values: hematoxylin (0.7 µM), (-)-gallocatechin gallate (126 µM), ginkgolic acid C15:1 (227 µM), wedelolactone (0.9770 µM), ß,ß-dimethylacrylalkannin (1.9004 µM,), cholesteryl sodium sulfate (7.5950 µM) and melanin (11.5667 µM), which allowed better assessments of the binding levels. Here, seven compounds were the winners. Then, molecular docking experiments were specially performed by AutoDock Vina to analyze the mode of interactions within Mpro and ligands. We finally formulated the present in silico study to predict pharmacokinetic parameters as well as drug-like properties, which is presumably the step that tells humans whether the compounds are drug-like or not. Moreover, hematoxylin, melanin, wedelolactone, ß,ß-dimethylacrylalkannin and cholesteryl sodium sulfate are in full compliance with the "Lipinski" principle and possess reasonable ADME/T properties, they have a greater potential of being lead compounds. The proposed five compounds are also the first to be found to have potential inhibitory effects on SARS CoV-2 Mpro. We hope that the results in this manuscript may serve as benchmarks for the above potentials.

Identification of small molecules capable of enhancing viral membrane fusion.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

Ultra High-Throughput Screening Assays for Inhibitors of TMPRSS2 and SPIKE Interaction

SARS-CoV-2, the coronavirus that causes the disease COVID- 19, was identified over three years ago, yet current small molecule therapies have limited usefulness and resistance to therapies and vaccines is inevitable. Ultra high-throughput screening (uHTS) assays for novel and repurposed inhibitors of a protein-protein interaction in the viral life cycle could be used to screen a vast number of compounds with a specific target of action. In particular, the interaction between viral SPIKE protein and human TMPRSS2 is an understudied, yet critical step in viral entry. Thus, we aim to create uHTS assays to rapidly and affordably identify inhibitors of the TMPRSS2 and SPIKE interaction for further biochemical studies and therapeutic development for SARS-CoV-2.We first sought to create a Time Resolved-Forster/Fluorescence Energy Transfer (TR-FRET) assay which uses lysates of cells with overexpressed SPIKE and TMPRSS2 and fluorescently labeled antibodies to detect interactions between these proteins. Initially, we developed and optimized this TR-FRET assay in a 384-well plate then miniaturized to a 1536-well plate. We conducted a pilot screen of compounds with known biological activity to test this assay's screening capabilities. To further narrow the hits from this TR-FRET screen, we developed an orthogonal uHTS Nanoluciferase Binary Technology (NanoBiT) assay to detect the interaction between tagged SPIKE and TMPRSS2 in live cells.With these two assays in hand, we expanded our TR-FRET screen to over 100 000 compounds and identified several that were also positive in the orthogonal NanoBiT assay. Four of these compounds were found to potentially interact with either SPIKE or TMPRSS2 from thermal shift experiments, providing support for their action as SPIKE and TMPRSS2 interaction inhibitors. Thus, we have developed TR-FRET and NanoBiT orthogonal uHTS assays which have allowed for the discovery of several possible repurposed and novel inhibitors of the SPIKE/ TMPRSS2 interaction. These uHTS assays can be employed as a model for future drug discovery efforts and the compounds identified may be used as exciting starting points for development of inhibitors of SARS-CoV-2. This research was supported in part by The Emory School of Medicine COVID Catalyst-I3 award, the NCI Emory Lung Cancer SPORE (SR, HF;P50CA217691) Career Enhancement Program (AI, P50CA217691), Emory initiative on Biological Discovery through Chemical Innovation (AI) and R01AI167356 (SS).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

A molecular toolbox for ADP-ribosyl binding proteins.

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Antiviral Activity of Acetylsalicylic Acid against Bunyamwera Virus in Cell Culture.

The Bunyavirales order is a large group of RNA viruses that includes important pathogens for humans, animals and plants. With high-throughput screening of clinically tested compounds we have looked for potential inhibitors of the endonuclease domain of a bunyavirus RNA polymerase. From a list of fifteen top candidates, five compounds were selected and their antiviral properties studied with Bunyamwera virus (BUNV), a prototypic bunyavirus widely used for studies about the biology of this group of viruses and to test antivirals. Four compounds (silibinin A, myricetin, L-phenylalanine and p-aminohippuric acid) showed no antiviral activity in BUNV-infected Vero cells. On the contrary, acetylsalicylic acid (ASA) efficiently inhibited BUNV infection with a half maximal inhibitory concentration (IC50) of 2.02 mM. In cell culture supernatants, ASA reduced viral titer up to three logarithmic units. A significant dose-dependent reduction of the expression levels of Gc and N viral proteins was also measured. Immunofluorescence and confocal microscopy showed that ASA protects the Golgi complex from the characteristic BUNV-induced fragmentation in Vero cells. Electron microscopy showed that ASA inhibits the assembly of Golgi-associated BUNV spherules that are the replication organelles of bunyaviruses. As a consequence, the assembly of new viral particles is also significantly reduced. Considering its availability and low cost, the potential usability of ASA to treat bunyavirus infections deserves further investigation.

High-throughput microfluidic droplets in biomolecular analytical system: A review.

Droplet microfluidic technology has revolutionized biomolecular analytical research, as it has the capability to reserve the genotype-to-phenotype linkage and assist for revealing the heterogeneity. Massive and uniform picolitre droplets feature dividing solution to the level that single cell and single molecule in each droplet can be visualized, barcoded, and analyzed. Then, the droplet assays can unfold intensive genomic data, offer high sensitivity, and screen and sort from a large number of combinations or phenotypes. Based on these unique advantages, this review focuses on up-to-date research concerning diverse screening applications utilizing droplet microfluidic technology. The emerging progress of droplet microfluidic technology is first introduced, including efficient and scaling-up in droplets encapsulation, and prevalent batch operations. Then the new implementations of droplet-based digital detection assays and single-cell muti-omics sequencing are briefly examined, along with related applications such as drug susceptibility testing, multiplexing for cancer subtype identification, interactions of virus-to-host, and multimodal and spatiotemporal analysis. Meanwhile, we specialize in droplet-based large-scale combinational screening regarding desired phenotypes, with an emphasis on sorting for immune cells, antibodies, enzymatic properties, and proteins produced by directed evolution methods. Finally, some challenges, deployment and future perspective of droplet microfluidics technology in practice are also discussed.

How to Discover Antiviral Drugs Quickly

Molecular-dynamics simulations together with virtual high-throughput screening provide a means of quick evaluation of existing drugs for antiviral activity. The authors explain how these methods serve in the quest for drugs to treat Covid-19.

In silico and in vitro assays reveal potential inhibitors against 3CLpro main protease of SARS-CoV-2.

The COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not showing any sign of slowing down even after the ongoing efforts of vaccination. The threats of new strains are concerning, as some of them are more infectious than the original one. A therapeutic against the disease is, therefore, of urgent need. Here, we use the DrugBank database to screen for potential inhibitors against the 3CLpro main protease of SARS-CoV-2. Instead of using the traditional approach of computational screening by docking, we developed a kernel ridge regressor (using a part of the docking data) to predict the binding energy of ligands. We used this model to screen the DrugBank database and shortlist two lead candidates (bromocriptine and avoralstat) for in vitro enzymatic study. Our results show that the 3CLpro enzyme activity in presence of 100 µM concentration of bromocriptine and avoralstat is 9.9% and 15.9%, respectively. Remarkably, bromocriptine exhibited submicromolar IC50 of 130 nM (0.13 µM). Avoralstat showed an IC50 of 2.16 µM. Further, the interactions of both drugs with 3CLpro were analyzed using molecular dynamics simulations of 100 ns. Results indicate that both ligands are stable in the binding pocket of the 3CLpro receptor. In addition, the MM-PBSA analysis revealed that bromocriptine (-29.37 kcal/mol) has a lower binding free energy compared to avoralstat (-6.91 kcal/mol). Further, hydrogen bond analysis also showed that bromocriptine interacts with the two catalytic residues, His41 and Cys145, more frequently than avoralstat.Communicated by Ramaswamy H. Sarma.

High-Throughput Screening for the Potential Inhibitors of SARS-CoV-2 with Essential Dynamic Behavior.

Global health security has been challenged by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. Due to the lengthy process of generating vaccinations, it is vital to reposition currently available drugs in order to relieve anti-epidemic tensions and accelerate the development of therapies for Coronavirus Disease 2019 (COVID-19), the public threat caused by SARS-CoV-2. High throughput screening techniques have established their roles in the evaluation of already available medications and the search for novel potential agents with desirable chemical space and more cost-effectiveness. Here, we present the architectural aspects of highthroughput screening for SARS-CoV-2 inhibitors, especially three generations of virtual screening methodologies with structural dynamics: ligand-based screening, receptor-based screening, and machine learning (ML)-based scoring functions (SFs). By outlining the benefits and drawbacks, we hope that researchers will be motivated to adopt these methods in the development of novel anti- SARS-CoV-2 agents.

SARS-CoV-2 RNA-dependent RNA polymerase as a target for high-throughput drug screening.

The ongoing COVID-19 pandemic caused by the SARS-CoV-2 has necessitated rapid development of drug screening tools. RNA-dependent RNA polymerase (RdRp) is a promising target due to its essential functions in replication and transcription of viral genome. To date, through minimal RNA synthesizing machinery established from cryo-electron microscopy structural data, there has been development of high-throughput screening assays for directly screening inhibitors that target the SARS-CoV-2 RdRp. Here, we analyze and present verified techniques that could be used to discover potential anti-RdRp agents or repurposing of approved drugs to target the SARS-CoV-2 RdRp. In addition, we highlight the characteristics and application value of cell-free or cell-based assays in drug discovery.

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past two years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here we report the development of a Time-Resolved Fluorescence/Förster Resonance Energy Transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well plate format for high-throughput screening with robust assay performance. To enable large scale compound screening, we further miniaturized the assay into a 1536-well ultra-high throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

Liposome-based high-throughput and point-of-care assays toward the quick, simple, and sensitive detection of neutralizing antibodies against SARS-CoV-2 in patient sera.

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in 2019 caused an increased interest in neutralizing antibody tests to determine the immune status of the population. Standard live-virus-based neutralization assays such as plaque-reduction assays or pseudovirus neutralization tests cannot be adapted to the point-of-care (POC). Accordingly, tests quantifying competitive binding inhibition of the angiotensin-converting enzyme 2 (ACE2) receptor to the receptor-binding domain (RBD) of SARS-CoV-2 by neutralizing antibodies have been developed. Here, we present a new platform using sulforhodamine B encapsulating liposomes decorated with RBD as foundation for the development of both a fluorescent, highly feasible high-throughput (HTS) and a POC-ready neutralizing antibody assay. RBD-conjugated liposomes are incubated with serum and subsequently immobilized in an ACE2-coated plate or mixed with biotinylated ACE2 and used in test strip with streptavidin test line, respectively. Polyclonal neutralizing human antibodies were shown to cause complete binding inhibition, while S309 and CR3022 human monoclonal antibodies only caused partial inhibition, proving the functionality of the assay. Both formats, the HTS and POC assay, were then tested using 20 sera containing varying titers of neutralizing antibodies, and a control panel of sera including prepandemic sera and reconvalescent sera from respiratory infections other than SARS-CoV-2. Both assays correlated well with a standard pseudovirus neutralization test (r = 0.847 for HTS and r = 0.614 for POC format). Furthermore, excellent correlation (r = 0.868) between HTS and POC formats was observed. The flexibility afforded by liposomes as signaling agents using different dyes and sizes can hence be utilized in the future for a broad range of multianalyte neutralizing antibody diagnostics.

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.

Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery.

Massive efforts on both vaccine development and antiviral research were launched to combat the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We contributed, amongst others, by the development of a high-throughput screening (HTS) antiviral assay against SARS-CoV-2 using a fully automated, high-containment robot system. Here, we describe the development of this novel, convenient and phenotypic dual-reporter virus-cell-based high-content imaging assay using the A549+hACE2+TMPRSS2_mCherry reporter lung carcinoma cell line and an ancestral SARS-CoV-2_Wuhan_mNeonGreen reporter virus. Briefly, by means of clonal selection, a host cell subclone was selected that (i) efficiently supports replication of the reporter virus with high expression, upon infection, of the NeonGreen fluorescent reporter protein, (ii) that is not affected by virus-induced cytopathogenic effects and, (iii) that expresses a strong fluorescent mCherry signal in the nucleus. The selected clone matched these criteria with an infection rate on average of 75% with limited cell death. The average (R)Z'-factors of the assay plates were all >0.8, which indicates a robust assay suitable for HTS purposes. A selection of reference compounds that inhibits SARS-CoV-2 replication in vitro were used to validate this novel dual-reporter assay and confirms the data reported in the literature. This assay is a convenient and powerful tool for HTS of large compound libraries against SARS-CoV-2.

Cell-based high throughput screening to identify FDA/ EMA approved drugs that inhibit the endothelial pro-thrombotic switch during severe COVID19 infection

Background: A major complication of COVID19 is severe endothelial injury with micro-and macro-thrombotic disease in the lung and other organs. Several studies have identified high levels of inflammatory cytokines ( cytokine storm ), powerful activators of the endothelium, in plasma of severe COVID19 patients;indeed, COVID19 plasma was shown to activate endothelial cells (EC) in vitro. A consequence of EC activation is loss of anti-coagulant function, with release of pro-thrombotic Von Willebrand Factor (VWF). High levels of plasma VWF in severe COVID19 patients indicate systemic endothelial activation and increased risk of thrombosis. Aim(s): To identify drugs that decrease endothelial activation and VWF release, which may have a therapeutic impact in COVID19 patients. Method(s): We established an in vitro model of endothelial activation driven by 6 cytokines selected because of their high levels in COVID19 plasma. Cells were treated with the 6-cytokine cocktail for 24 hr;endothelial activation was confirmed by a panel of markers including ICAM1, measured by RT-qPCR and immunofluorescence (IF). Result(s): The treatment induced release of VWF and increased VWF-platelet string formation in a platelet flow-based assay. To identify drugs that blocked cytokine-induced VWF release, a high-throughput screening was carried out in human umbilical vein EC (HUVEC);VWF and ICAM1 expression were detected by IF;DAPI was used as nuclear stain. High content imaging screen of 3049 drugs from FDA/EMA-approved drug libraries identified drugs able to decrease VWF release following cytokine treatment. Top hits from several therapeutic classes including anti-inflammatory, anti-viral and hormones were taken forward for validation. Two hits were confirmed to inhibit cytokine-induced VWF release and VWF-platelet string formation. Selected findings were validated in lung microvascular EC. Conclusion(s): This study identified candidate drugs that reduce the enhanced VWF release caused by the cytokine storm typical of severe COVID19;these may be beneficial in the treatment of the pro-thrombotic risk in COVID19 patients.

A high-throughput SARS-CoV-2 pseudovirus multiplex neutralization assay.

Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus (VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples, and run-to-run variability. For complete details on the use and execution of this protocol, please refer to Sievers et al. (2022).1.