A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 µM) and GS441524 (IC50 15.2 µM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

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.

Protocol for high-throughput screening of SARS-CoV-2 main protease inhibitors using a robust fluorescence polarization assay.

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors.

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

Development of Integrated Systems for On-Site Infection Detection.

The modern healthcare system faces an unrelenting threat from microorganisms, as evidenced by global outbreaks of new viral diseases, emerging antimicrobial resistance, and the rising incidence of healthcare-associated infections (HAIs). An effective response to these threats requires rapid and accurate diagnostic tests that can identify causative pathogens at the point of care (POC). Such tests could eliminate diagnostic uncertainties, facilitating patient triaging, minimizing the empiric use of antimicrobial drugs, and enabling targeted treatments. Current standard methods, however, often fail to meet the needs of rapid diagnosis in POC settings. Culture-based assays entail long processing times and require specialized laboratory infrastructure; nucleic acid (NA) tests are often limited to centralized hospitals due to assay complexity and high costs. Here we discuss two new POC tests developed in our groups to enable the rapid diagnosis of infection. The first is nanoPCR that takes advantages of core-shell magnetoplasmonic nanoparticles (MPNs): (i) Au shell significantly accelerates thermocycling via volumetric, plasmonic light-to-heat conversion and (ii) a magnetic core enables sensitive in situ fluorescent detection via magnetic clearing. By adopting a Ferris wheel module, the system expedites multisamples in parallel with a minimal setup. When applied to COVID-19 diagnosis, nanoPCR detected SARS-CoV-2 RNA down to 3.2 copy/µL within 17 min. In particular, nanoPCR diagnostics accurately identified COVID-19 cases in clinical samples (n = 150), validating its clinical applicability. The second is a polarization anisotropy diagnostic (PAD) system that exploits the principle of fluorescence polarization (FP) as a detection modality. Fluorescent probes were designed to alter their molecular weight upon recognizing target NAs. This event modulates the probes' tumbling rate (Brownian motion), which leads to changes in FP. The approach is robust against environmental noise and benefits from the ratiometric nature of the signal readout. We applied PAD to detect clinically relevant HAI bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus). The PAD assay demonstrated detection sensitivity down to the single bacterium level and determined both drug resistance and virulence status. In summary, these new tests have the potential to become powerful tools for rapid diagnosis in the infectious disease space. They do not require highly skilled personnel or labor-intensive analyses, and the assays are quick and cost-effective. These attributes will make nanoPCR and PAD well-aligned with a POC workflow to aid physicians to initiate prompt and informed patient treatment.

A robust high-throughput fluorescent polarization assay for the evaluation and screening of SARS-CoV-2 fusion inhibitors.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal-C) were measured to be 6.1 nM, 2.5 nM, and 8.9 µM respectively. In addition, we found Sal-A has a slightly lower IC50 (3.9 µM) than Sal-C. Interestingly, simple caffeic acid can also disrupt the formation of 6-HB with a sub-mM concentration. Pilot high throughput screening (HTS) of a small marine natural product library validates the assay with a Z' factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitors and assess their binding affinity.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19.

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.