Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos.

Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.

Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

Efficient and rapid fluorescent protein knock-in with universal donors in mouse embryonic stem cells.

Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.

Tissue-specific and endogenous protein labeling with split fluorescent proteins.

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

Bright individuals: Applications of fluorescent protein-based reporter systems in single-cell cellular microbiology.

Activation and function of virulence functions of bacterial pathogens are highly dynamic in time and space, and can show considerable heterogeneity between individual cells in pathogen populations. To investigate the complex events in host-pathogen interactions, single cell analyses are required. Fluorescent proteins (FPs) are excellent tools to follow the fate of individual bacterial cells during infection, and can also be deployed to use the pathogen as a sensor for its specific environment in host cells or host organisms. This Resources describes design and applications of dual fluorescence reporters (DFR) in cellular microbiology. DFR feature constitutively expressed FPs for detection of bacterial cells, and FPs expressed by an environmentally regulated promoter for interrogation of niche-specific cues or nutritional parameters. Variations of the basic design allow the generation of DFR that can be used to analyze, on single cell level, bacterial proliferation during infection, subcellular localization of intracellular bacteria, stress response, or persister state. We describe basic considerations for DFR design and review recent applications of DFR in cellular microbiology.

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

mBeRFP: a versatile fluorescent tool to enhance multichannel live imaging and its applications.

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins offers interesting new perspectives to bypass this limitation. Here, we provide a detailed characterization of mBeRFP for in vivo live imaging and its applications in Drosophila. Briefly, we show that a single illumination source is sufficient to stimulate mBeRFP and GFP simultaneously. We demonstrate that mBeRFP can be easily combined with classical green and red fluorescent proteins without any crosstalk. We also show that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications, such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano-biologists in their current research.

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

A Genetically Encoded Fluorescent Heme Sensor Detects Free Heme in Plants.

Heme is produced in plants via a plastid-localized metabolic pathway and is subsequently distributed to all cellular compartments. In addition to covalently and non-covalently bound heme, a comparatively small amount of free heme that is not associated with protein is available for incorporation into heme-dependent proteins in all subcellular compartments and for regulatory purposes. This "labile" fraction may also be toxic. To date, the distribution of the free heme pool in plant cells remains poorly understood. Several fluorescence-based methods for the quantification of intracellular free heme have been described. For this study, we used the previously described genetically encoded heme sensor 1 (HS1) to measure the relative amounts of heme in different plant subcellular compartments. In a proof of concept, we manipulated heme content using a range of biochemical and genetic approaches and verified the utility of HS1 in different cellular compartments of Arabidopsis (Arabidopsis thaliana) and tobacco (N. tabacum and N. benthamiana) plants transformed either transiently or stably with HS1 and HS1(M7A), a variant with lower affinity for heme. This approach makes it possible to trace the distribution and dynamics of free heme and provides relevant information about its mobilization. The application of these heme sensors will create opportunities to explore and validate the importance of free heme in plant cells and to identify mutants that alter the subcellular allocation of free heme.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats.

BACKGROUND: Acellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI-NCSC-SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. METHODS: The effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI-NCSC-SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. RESULTS: (1) EPI-NCSC-SCLCs had good compatibility with ANAs in vitro. (2) In the ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI-NCSC-SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). CONCLUSIONS: EPI-NCSC-SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI-NCSC-SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.

Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle.

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

The key role of glutamine for protein expression and isotopic labeling in insect cells.

Nuclear magnetic resonance studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotope-labeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N nuclear magnetic resonance spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the tricarboxylic acid cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-α,ß,ß-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled ß1-adrenergic receptor using the synthesized 15N2-glutamine-α,ß,ß-d3.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection.

Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated.

Efficient generation of endogenous protein reporters for mouse development.

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals, yet the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. Here, we present two strategies for higher throughput production of endogenous protein reporters in mice, focusing on the blastocyst model of development. Our first strategy makes use of a split fluorescent protein, mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest, was highly efficient in embryos, potentially obviating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. Our second strategy achieves in-frame knock-in of a relatively small protein tag, which provides high efficiency and higher sensitivity protein reporting. Together, these two approaches provide complementary advantages and enable broad downstream applications.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

pH sensitivity of YFPs is reduced upon AlphaRep binding: proof of concept in vitro and in living cells.

Yellow fluorescent proteins (YFPs) are commonly used in biology to track cellular processes, particularly as acceptors in experiments using the Förster Resonant Energy Transfer (FRET) phenomenon. However, their fluorescence intensity is strongly pH-dependent, limiting their utility in acidic environments. Here, we explore the pH sensitivity of YFPs upon binding with an artificial repeat protein (αRep) both in vitro and in living cells. We show that αRep binds to Citrine, with high affinity in the nanomolar range at physiological and acidic pHs, leading to increased thermal stability of the complex. Moreover, αRep binding reduces Citrine's pKa by 0.75 pH units, leading to a decreased sensitivity to pH fluctuations. This effect can be generalized to other YFPs as Venus and EYFP in vitro. An efficient binding of αRep to Citrine has also been observed in living cells both at pH 7.4 and pH 6. This interaction leads to reduced variations of Citrine fluorescence intensity in response to pH variations in cells. Overall, the study highlights the potential of αReps as a tool to modulate the pH sensitivity of YFPs, paving the way for future exploration of biological events in acidic environments by FRET in combination with a pH-insensitive cyan donor.

Directed evolution of the fluorescent protein CGP with in situ biosynthesized noncanonical amino acids.

The incorporation of noncanonical amino acids (ncAAs) into proteins can enhance their function beyond the abilities of canonical amino acids and even generate new functions. However, the ncAAs used for such research are usually chemically synthesized, which is expensive and hinders their application on large industrial scales. We believe that the biosynthesis of ncAAs using metabolic engineering and their employment in situ in target protein engineering with genetic code expansion could overcome these limitations. As a proof of principle, we biosynthesized four ncAAs, O-L-methyltyrosine, 3,4-dihydroxy-L-phenylalanine, 5-hydroxytryptophan, and 5-chloro-L-tryptophan using metabolic engineering and directly evolved the fluorescent consensus green protein (CGP) by combination with nine other exogenous ncAAs in Escherichia coli. After screening a TAG scanning library expressing 13 ncAAs, several variants with enhanced fluorescence and stability were identified. The variants CGPV3pMeoF/K190pMeoF and CGPG20pMeoF/K190pMeoF expressed with biosynthetic O-L-methyltyrosine showed an approximately 1.4-fold improvement in fluorescence compared to the original level, and a 2.5-fold improvement in residual fluorescence after heat treatment. Our results demonstrated the feasibility of integrating metabolic engineering, genetic code expansion, and directed evolution in engineered cells to employ biosynthetic ncAAs in protein engineering. These results could further promote the application of ncAAs in protein engineering and enzyme evolution. IMPORTANCE: Noncanonical amino acids (ncAAs) have shown great potential in protein engineering and enzyme evolution through genetic code expansion. However, in most cases, ncAAs must be provided exogenously during protein expression, which hinders their application, especially when they are expensive or have poor cell membrane penetration. Engineering cells with artificial metabolic pathways to biosynthesize ncAAs and employing them in situ for protein engineering and enzyme evolution could facilitate their application and reduce costs. Here, we attempted to evolve the fluorescent consensus green protein (CGP) with biosynthesized ncAAs. Our results demonstrated the feasibility of using biosynthesized ncAAs in protein engineering, which could further stimulate the application of ncAAs in bioengineering and biomedicine.

Improved fluorescence-based assay for rapid screening and evaluation of SARS-CoV-2 main protease inhibitors.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health. In parallel with vaccines, efficacious antivirals are urgently needed. SARS-CoV-2 main protease (Mpro) is an attractive drug target for antiviral development owing to its key roles in virus replication and host immune evasion. Due to the limitations of currently available methods, the development of novel high-throughput screening assays is of the highest importance for the discovery of Mpro inhibitors. In this study, we first developed an improved fluorescence-based assay for rapid screening of Mpro inhibitors from an anti-infection compound library using a versatile dimerization-dependent red fluorescent protein (ddRFP) biosensor. Utilizing this assay, we identified MG-101 as a competitive Mpro inhibitor in vitro. Moreover, our results revealed that ensitrelvir is a potent Mpro inhibitor, but baicalein, chloroquine, ebselen, echinatin, and silibinin are not. Therefore, this robust ddRFP assay provides a faithful avenue for rapid screening and evaluation of Mpro inhibitors to fight against COVID-19.