Studying SARS-CoV-2 with Fluorescence Microscopy

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus–cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy—which in our opinion can provide important information about the molecular biology of SARS-CoV-2—are discussed.     

SARS-CoV-2 Papain-Like Protease Potential Inhibitors—In Silico Quantitative Assessment

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC₅₀ values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC₅₀ values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.     

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PL^Pro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PL^Pro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PL^Pro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PL^Pro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC₅₀ value below 40 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC₅₀ value below 10 μM. Among four inhibitors with an IC₅₀ value below 10 μM, SJB2-043 is the most unique in that it does not fully inhibit PL^Pro but has a noteworthy IC₅₀ value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PL^Pro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PL^Pro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.     

Atomistic-Level Description of the Covalent Inhibition of SARS-CoV-2 Papain-like Protease

Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys¹¹¹), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys¹¹¹ deprotonation of the thiol group by a Brønsted–Lowry base; (iii) Cys¹¹¹-S⁻ addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys¹¹¹, that is, prior to the Cys¹¹¹ deprotonation. For the second step, MD simulations revealed that His²⁷² would deprotonate Cys¹¹¹ after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys¹¹¹ deprotonation, whose equilibrium is largely shifted toward the neutral Cys¹¹¹ form. Although thermodynamically disfavored, if Cys¹¹¹ is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys¹¹¹-S⁻ negative charge redistribution over the bound ligand, proton transfer from the initially protonated His²⁷² is favored, finally leading to an irreversibly modified Cys¹¹¹ and a restored His²⁷². These results elucidate the selectivity of Cys¹¹¹ to enable formation of a covalent bond, even if a weak proton acceptor is available, as His²⁷².     

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors**

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M^Pro) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M^Pro), we have designed and synthesized a series of SC2M^Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M^Pro active-site cysteine C145. All inhibitors display high potency with K_i values at or below 100 nM. The most potent compound, MPI3, has as a K_i value of 8.3 nM. Crystallographic analyses of SC2M^Pro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M^Pro inhibitors with ultra-high antiviral potency.     

Structure-Activity Studies Reveal Scope for Optimisation of Ebselen-Type Inhibition of SARS-CoV-2 Main Protease

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of M^pro, particularly with respect to improved selectivity.     

A Structural Comparison of SARS-CoV-2 Main Protease and Animal Coronaviral Main Protease Reveals Species-Specific Ligand Binding and Dimerization Mechanism

Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (M^pro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV M^pro and can thermodynamically stabilize its folding. The structure of CCoV M^pro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV M^pro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV M^pro and other animal CoV M^pros with SARS-CoV-2 M^pro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral M^pros, SARS-CoV-2 M^pro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46–49 in animal coronaviral M^pros has been replaced by a stable α-helix in SARS-CoV-2 M^pro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV M^pros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV M^pros among different species.     

The mRubyFT Protein,Genetically Encoded Blue-to-Red Fluorescent Timer

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.     

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.     

The Role of Antibodies in the Treatment of SARS-CoV-2 Virus Infection,and Evaluating Their Contribution to Antibody-Dependent Enhancement of Infection

Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helping create strong immunological memory. Antibody-dependent enhancement (ADE) occurs when non-neutralising antibodies recognise and bind to a pathogen, but are unable to prevent infection, and is widely known and is reported as occurring in infection caused by several viruses. This narrative review explores the ADE phenomenon, its occurrence in viral infections and evaluates its role in infection by SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19). As of yet, there is no clear evidence of ADE in SARS-CoV-2, though this area is still subject to further study.     

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca²⁺ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening.     

In silico allicin induced S-thioallylation of SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused due to new coronavirus infection with ³716075 deaths across the world as reported by the World Health Organization (WHO). SARS-CoV-2 main protease (M^pro) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of COVID-19. The preclinical drugs ebselen and PX-12 are potent inhibitors of SARS-CoV-2 M^pro and covalently modifies the active site Cys-145 residue of M^pro through selenosulfide/disulfide. In the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of SARS-CoV-2 M^pro using PX-12 as a benchmark reference compound. The results indicate that allicin induces dual S-thioallylation of Cys-145 and Cys-85/ Cys-156 residues of SARS-CoV-2 M^pro. Using density functional theory (DFT), Gibbs free energy change (DG) is calculated for the putative reactions between N-acetylcysteine amide thiol and allicin/allyl sulfenic acid. The overall reaction is exergonic and allyl disulfide of Cys-145 residue of M^pro is involved in a sulfur mediated hydrogen bond. The results indicate that allicin causes dual S-thioallylation of SARS-CoV-2 M^pro which may be of interest for treatment and attenuation of ongoing coronavirus infection.     

Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CL^pro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CL^pro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CL^pro–PF-07321332 and 3CL^pro–α-ketoamide complexes remained stable compared with 3CL^pro–ritonavir and 3CL^pro–lopinavir. Investigating the dynamic behavior of ligand–protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41–Cys145 of 3CL^pro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CL^pro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.     

LSSmScarlet,dCyRFP2s,dCyOFP2s and CRISPRed2s,Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.     

In Silico Study of piRNA Interactions with the SARS-CoV-2 Genome

A prolonged pandemic with numerous human casualties requires a rapid search for means to control the various strains of SARS-CoV-2. Since only part of the human population is affected by coronaviruses, there are probably endogenous compounds preventing the spread of these viral pathogens. It has been shown that piRNA (PIWI-interacting RNAs) interact with the mRNA of human genes and can block protein synthesis at the stage of translation. Estimated the effects of piRNA on SARS-CoV-2 genomic RNA (gRNA) in silico. A cluster of 13 piRNA binding sites (BS) in the SARS-CoV-2 gRNA region encoding the oligopeptide was identified. The second cluster of BSs 39 piRNAs also encodes the oligopeptide. The third cluster of 24 piRNA BS encodes the oligopeptide. Twelve piRNAs were identified that strongly interact with the gRNA. Based on the identified functionally important endogenous piRNAs, synthetic piRNAs (spiRNAs) are proposed that will suppress the multiplication of the coronavirus even more strongly. These spiRNAs and selected endogenous piRNAs have little effect on human 17494 protein-coding genes, indicating a low probability of side effects. The piRNA and spiRNA selection methodology created for the control of SARS-CoV-2 ({"type":"entrez-nucleotide","attrs":{"text":"NC_045512.2","term_id":"1798174254","term_text":"NC_045512.2"}}NC_045512.2) can be used to control all strains of SARS-CoV-2.     

Action of Dipeptidyl Peptidase-4 Inhibitors on SARS-CoV-2 Main Protease

In a recent publication, Eleftheriou et al. proposed that inhibitors of dipeptidyl peptidase-4 (DPP-4) are functional inhibitors of the main protease (M^pro) of SARS-CoV-2. Their predictions prompted the authors to suggest linagliptin, a DPP-4 inhibitor and approved anti-diabetes drug, as a repurposed drug candidate against the ongoing COVID-19 pandemic. We used an enzymatic assay measuring the inhibition of M^pro catalytic activity in the presence of four different commercially available gliptins (linagliptin, sitagliptin, alogliptin and saxagliptin) and several structural analogues of linagliptin to study the binding of DPP-4 inhibitors to M^pro and their functional activity. We show here that DPP-4 inhibitors like linagliptin, other gliptins and structural analogues are inactive against M^pro.     

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca²⁺ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.     

FRCaMP,a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with K_d of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.     

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His₆-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.