Biology subjects

Zeng, J.

Publications and source records attributed to Zeng, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Effective infection with dengue virus in experimental neonate and adult mice through intranasal route

As a member of the family Flaviviridae, the dengue virus is transmitted to humans through mosquito vectors. The virus causes dengue fever with flu-like symptoms, life-threatening dengue hemorrhagic fever, and dengue shock syndrome that affects four billion people in 128 countries. It can also be transmitted through atypical routes, such as needle stick injury, vertical transmission, and the receipt of contaminated blood or organs. Although sporadic cases cannot be classified as atypical transmission routes and raise respiratory exposure concerns, the risks remain unclear. Here, we analyzed the respiratory infectivity of the dengue virus in BALB/c suckling and A6 adult mice using the dengue virus serotype 2 and intranasal inoculation. The results showed that the clinical symptoms of intranasally infected mice included excitement, emaciation, malaise, and death, and histopathological changes and viremia dynamics were observed in multiple tissues, including the brain, liver, and spleen. Notably, the DENV serotype 2 showed clear brain tropism after intranasal infection, and viral loads peaked at seven days post-inoculation. Furthermore, the virus was isolated from mouse brains, and the sequence homology between the viral stock and dengue virus isolates was 99.99%. Similar results were observed in adult IFN-/{beta} receptor-deficient mice. Taken together, our results suggest that the DENV serotype 2 can infect suckling mice and adult immune-deficient mice via the nasal route. The results of our study broaden our perception of atypical dengue transmission routes and may help explain dengue virus infections that occur without the presence of mosquito vectors. ImportanceDengue is transmitted mainly via mosquito vectors. Flu-like dengue fever or life-threatening severe dengue threatens four billion people in 128 countries. Non-vector dengue cases have occurred occasionally since 1990. These cases cannot be classified as atypical transmission routes and are suspected to have arisen via sexual or respiratory transmission. Recently, the sexual transmission of dengue has been confirmed in humans. The respiratory infectivity of other members of the Flaviviridae family, such as Zika and Japanese encephalitis viruses, has been determined under experimental conditions. The potential for respiratory infection with the dengue virus has not yet been confirmed. In this study, we analyzed the respiratory infectivity of the dengue virus in suckling mice and adult immunodeficient mice via intranasal inoculation, thereby exploring the potential for respiratory transmission in cases with non-vector dengue infection, especially for patients who were exposed to dengue virus contaminants.

microbiology↗

bioRxiv2022

Optimization of NLS Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells

Type V CRISPR-Cas12a systems are an attractive alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells. Here we describe optimization to the nuclear localization signal composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in mammalian cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. A 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not decrease the specificity of editing in HEK293T and CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

molecular biology↗

bioRxiv2022

Impact of various vaccine boosters on neutralization against Omicron following prime vaccinations with inactivated or adenovirus-vectored vaccine

Since the first report on November 24, 2021, the Omicron SARS-CoV-2 variant is now overwhelmingly spreading across the world. Two SARS-CoV-2 inactivated vaccines (IAVs), one recombinant protein subunit vaccine (PRV), and one adenovirus-vectored vaccine (AdV) have been widely administrated in many countries including China to pursue herd immunity. Here we investigated cross-neutralizing activities in 341 human serum specimens elicited by full-course vaccinations with IAV, PRV and AdV, and by various vaccine boosters following prime IAV and AdV vaccinations. We found that all types of vaccines induced significantly lower neutralizing antibody titers against the Omicron variant than against the prototype strain. For prime vaccinations with IAV and AdV, heterologous boosters with AdV and PRV, respectively, elevated serum Omicron-neutralizing activities to the highest degrees. In a mouse model, we further demonstrated that among a series of variant-derived RBD-encoding mRNA vaccine boosters, it is only the Omicron booster that significantly enhanced Omicron neutralizing antibody titers compared with the prototype booster following a prime immunization with a prototype S-encoding mRNA vaccine candidate. In summary, our systematical investigations of various vaccine boosters inform potential booster administrations in the future to combat the Omicron variant.

immunology↗

bioRxiv2022

Delineate clonal dynamics of ovarian cancer initiating cells during pre-malignant progression using a mouse genetic mosaic system

Different cellular compartments within a tissue present distinct cancer-initiating capacities. Current approaches to delineate cancer-initiating heterogeneity require a well-understood lineage hierarchy and cell-specific genetic tools, which are lacking for many tissues. Here, we circumvented this hurdle and investigated the capacity of fallopian tube cells in initiating serous ovarian cancer, utilizing a mouse genetic system that generates scattered GFP-labeled mutant cells. Through quantitative tracing of clonal expansion of individual mutant cells, we revealed that only a rare primitive subset enriched in the distal fallopian tube is capable of clonal expansion whereas others stall immediately upon acquiring oncogenic mutations. Expanded clones from this subset then encountered further attrition: many stalled shortly after while others sustained small cluster of proliferative cells and biased differentiation toward primitive fate. Taken together, our study showcases quantitative clonal tracing as a powerful approach to investigate cancer-initiating heterogeneity and clonal trajectory in tissues with limited prior knowledge.

cancer biology↗

bioRxiv2021

Extend Mixed Models to Multi-layer Neural Networks for Genomic Prediction Including Intermediate Omics Data

With the growing amount and diversity of intermediate omics data complementary to genomics (e.g., DNA methylation, gene expression, and protein abundance), there is a need to develop methods to incorporate intermediate omics data into conventional genomic evaluation. The omics data helps decode the multiple layers of regulation from genotypes to phenotypes, thus forms a connected multi-layer network naturally. We developed a new method named NN-LMM to model the multiple layers of regulation from genotypes to intermediate omics features, then to phenotypes, by extending conventional linear mixed models ("LMM") to multi-layer artificial neural networks ("NN"). NN-LMM incorporates intermediate omics features by adding middle layers between genotypes and phenotypes. Linear mixed models (e.g., pedigree-based BLUP, GBLUP, Bayesian Alphabet, single-step GBLUP, or single-step Bayesian Alphabet) can be used to sample marker effects or genetic values on intermediate omics features, and activation functions in neural networks are used to capture the nonlinear relationships between intermediate omics features and phenotypes. NN-LMM had significantly better prediction performance than the recently proposed single-step approach for genomic prediction with intermediate omics data. Compared to the single-step approach, NN-LMM can handle various patterns of missing omics measures, and allows nonlinear relationships between intermediate omics features and phenotypes. NN-LMM has been implemented in an open-source package called "JWAS".

genetics↗

bioRxiv2021

FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by targeting a glycosyl hydrolase coding gene at transcriptional level

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence and hypersensitivity to hydrogen peroxide (H2O2). These results indicate that milRNA biosynthesis is crucial for Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly downregulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR analysis. Through deletion and overexpression of milR-87 in the wild-type Foc strain, functions of milR-87 were studied. The results showed that milR-87 is crucial for Foc virulence in infection process. We furthermore identified a glycosyl hydrolase-coding gene, FOIG_15013, as the direct target of milR-87. The FOIG_15013 deletion mutant displayed a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases. Author summaryThe fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. As a typical representative of F. oxysporum species complex, the pathogen has been widely concerned. However, pathogenesis of Foc is not fully elucidated. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, one Argonaute coding gene, and two Dicer coding genes FoDCL1 and FoDCL2, which are involved in milRNA biosynthesis, are significantly induced during the early infection stage of Foc. The results suggested that the milRNAs biosynthesis mediated by these genes may play an active role in the infection process of Foc. Based on this assumption, we subsequently found a FoQDE2-dependent milRNA (milR-87) and identified its target gene. Functional analysis showed that FoQDE2, miR-87 and its target gene were involved in the pathogenicity of Foc in different degree. The studies help us gain insight into the pathogenesis with FoQDE2, milR-87, and its target gene as central axis in Foc. The identified pathogenicity-involved milRNA provides an active target for developing novel and efficient biocontrol agents against Panama disease.

microbiology↗

bioRxiv2021

SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury

Robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) accounts for high viral transmissibility, yet whether neutralizing IgA antibodies can control it remains unknown. Here, we evaluated receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1 and B8-dIgA2 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparably potent neutralization against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viruses in lungs, pre-exposure intranasal B8-dIgA1 or B8-dIgA2 led to 81-fold more infectious viruses and severer damage in NT than placebo. Virus-bound B8-dIgA1 and B8-dIgA2 could engage CD209 as an alternative receptor for entry into ACE2-negative cells and allowed viral cell-to-cell transmission. Cryo-EM revealed B8 as a class II neutralizing antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Therefore, RBD-specific neutralizing dIgA engages an unexpected action for enhanced SARS-CoV-2 nasal infection and injury in Syrian hamsters.

microbiology↗

bioRxiv2021

Isolation, identification and molecular characterization of Aeromonas spp. from farmed loach (Misgurnus anguillicaudatus)

Gram-negative Aeromonas bacteria is known to contaminate aquaculture products and retains the ability to infect wide range of host including fishes, shrimps and humans. This study is designed to evaluate the presence of Aeromonas in healthy loaches (Misgurnus anguillicaudatus) collected from the markets in Hainan and Guangdong provinces of China. Based on the molecular sequencing and phylogenetic analysis with single 16S rRNA and concatenated genes (gyrA and ropD), 104 isolates were identified as Aeromonas species followed by further classification to A. veronii (83.65%), A. jandaei (9.62%), A. hydrophila (3.85%) and A. allosaccharophila (2.88%). More than half of the isolates displayed hemolytic activity of 87.5% and were able to form moderate biofilm (78.26%). Fourteen antibiotics from ten representative classes were screened that demonstrated complete resistance to ampicillin, 89.4% and 68.3% to lincomycin and nalidixic acid. Moreover, a notable detection and prevalence was found in screening of ten virulence-related and nine resistance genes. To our knowledge, it is the first report of its kind demonstrating potential threat of the commercial loaches carrying Aeromonas to human health. These findings will assist professionals working in clinical settings to optimize their prescription accordingly and researchers to access the impacts of Aeromonas resistance on human health. ImportanceThis study demonstrates high infection rate of loaches by Aeromonas spp. in Hainan and Guangdong provinces of China. The most prevalent species was Aeromonas veronii. Most of the isolates displayed hemolytic activity and formed moderate biofilm. Multiple virulence genes, resistance genes and multi-antibiotic resistance were detected. Aeromonads risks should be considered in loach industry.

microbiology↗

bioRxiv2021

Widely naturalized species are not more promiscuous in the use of different nitrogen forms, but benefit more from inorganic nitrogen

Nitrogen (N) is an essential mineral nutrient necessary for plant growth and has been considered a crucial factor influencing invasion success. Former studies mainly focused on responses of alien plants to different levels of N availability. However, in natural soils, N comes in different forms. Few experimental studies have explored responses of naturalized alien species to different N forms, and whether such responses are related to their naturalization success globally. We selected 22 common herbaceous species native to Germany that have all become naturalized and thus been introduced elsewhere in the world. We grew these species under six different N conditions that differed in the amount or form of N, and assessed their growth performance in a greenhouse experiment. We found that plants produced more biomass when grown under high N levels than under low N levels, and when N was provided in inorganic form than when provided in organic form. Neither biomass production nor promiscuity to different N forms was related to naturalization success of the species. However, the biomass response to inorganic N, relative to organic N, was stronger for the widely naturalized species than for the less widely naturalized ones. Our study shows that although the widely naturalized alien species were not more promiscuous than the less widely naturalized species, they took more advantage of the inorganic-N forms. This indicates that naturalization success might be partly driven by a species ability to take advantage of increased inorganic N levels.

ecology↗

bioRxiv2021

Atp13a5 Marker Reveals Pericytes of The Central Nervous System in Mice

Perivascular mural cells including vascular smooth cells (VSMCs) and pericytes are integral components of the vascular system. In the central nervous system (CNS), pericytes are also known as the guardian of the blood-brain barrier, blood-spinal cord barrier and blood-retinal barrier, and play key roles in maintaining cerebrovascular and neuronal functions. However, the functional difference between CNS and peripheral pericytes has not been resolved at the genetic and molecular levels. Hence, the generation of reliable CNS pericyte-specific models and genetic tools remains very challenging. Here, we report a new CNS pericyte marker in mice. This cation-transporting ATPase 13A5 (Atp13a5) marker is highly specific to the pericytes in brain, spinal cord and retina. We generated a transgenic model with a knock-in tdTomato reporter and Cre recombinase. The tdTomato reporter reliably labels the CNS pericytes, but not found in any other CNS cell types including closely related VSMCs, or in peripheral organs. More importantly, Atp13a5 is turned on at embryonic day E15, suggesting brain pericytes are shaped by the developing neural environment. We hope that the new tools will allow us to further explore the heterogeneity of pericytes and achieve a better understanding of CNS pericytes in health and diseases.

neuroscience↗

bioRxiv2021

Why are SNAREpins rod-shaped?

SNARE proteins are the core components of the cellular machineries that fuse membranes for neurotransmitter or hormone release and other fundamental processes. Fusion is accomplished when SNARE proteins hosted by apposing membranes form SNARE complexes called SNAREpins, but the mechanism of fusion remains unclear. Computational simulations of SNARE-mediated membrane fusion are challenging due to the millisecond timescales of physiological membrane fusion. Here we used ultra-coarse-grained (UCG) simulations to investigate the minimal requirements for a molecular intracellular fusogen, and to elucidate the mechanisms of SNARE-mediated fusion. We find fusion by simple body forces that push vesicles together is highly inefficient. Inter-vesicle fusogens with different aspect ratios can fuse vesicles only if they are rodlike, of sufficient length to clear the fusogens from the fusion site by entropic forces. Simulations with rod-shaped SNAREpin-like fusogens fused 50-nm vesicles on ms timescales, driven by entropic forces along a reproducible fusion pathway. SNARE-SNARE and SNARE-membrane entropic forces cleared the fusion site and pressed the vesicles into an extended contact zone (ECZ), drove stalk nucleation at the high curvature ECZ boundary, and expanded the stalk into a long-lived hemifusion diaphragm in which a simple pore completed fusion. Our results provide strong support for the entropic hypothesis of SNARE-mediated membrane fusion, and implicate the rodlike structure of the SNAREpin complex as a necessity for entropic force production and fusion.

biophysics↗

bioRxiv2021

Coupling of Ca2+-triggered unclamping and membrane fusion during neurotransmitter release

Neurotransmitter (NT) release is accomplished by a machinery that unclamps fusion in response to calcium and then fuses the synaptic vesicle and plasma membranes. These are often thought of as distinct tasks assigned to non-overlapping components. Vesicle release rates have a power law dependence on [Ca2+] with an exponent of 3-5, long taken to indicate that 3-5 Ca2+ ions bind the calcium sensor Synaptotagmin to trigger release. However, dependencies at low [Ca] are inconsistent with simple sequential binding to a single Ca2+ sensor followed by a final fusion step. Here we developed coarse-grained molecular dynamics simulations of the NT release machinery accounting for Synaptotagmin-mediated unclamping and SNARE-mediated fusion. Calcium-triggered unclamping and SNARE-mediated fusion emerged from simulations as contemporaneous, coupled processes. Increasing cytosolic [Ca2+], the instantaneous fusion rate increased as SNAREpins were progressively and reversibly released by dissociation of Synaptotagmin-SNAREpin complexes. Simulations reproduced the observed dependence of release rates on [Ca2+], but the power law was unrelated to the number of Ca2+ ions required. Action potential-evoked vesicle release probabilities depended on the number of transiently unclamped SNAREpins, explaining experimental dependencies of release probabilities on both unclamping and membrane-fusing machinery components. These results describe a highly cooperative NT release machinery with intrinsically inseparable unclamping and membrane-fusing functionalities.

neuroscience↗

bioRxiv2021

Associative learning drives longitudinally-graded presynaptic plasticity of neurotransmitter release along axonal compartments

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. Here we show that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the CaV2.1 calcium channel subunit cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor (IP3R), which was required to maintain responsivity to odors in untrained conditions. Downstream from the mushroom body, a set of MBONs that receive their input from the {gamma}3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which discriminative effects influence appetitive memory and decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.

neuroscience↗

bioRxiv2021

Human genetic diversity modifies therapeutic gene editing off-target potential

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to nominate and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and {beta}-thalassemia1. We find that the top candidate off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels ([~]9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. The CRISPRme tool highlights alternative allele-specific off-target editing as a prevalent risk of gRNAs considered for therapeutic gene editing. Our report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We suggest that variant-aware off-target assessment should be considered in therapeutic genome editing efforts going forward and provide a powerful approach for comprehensive off-target nomination.

genetics↗

bioRxiv2021

Large fluctuations of the fusion intermediate help SARS-CoV-2 capture host cell membranes

Cell entry by SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by capture of the host cell membrane and fusion with the viral envelope. Capture and fusion require the prefusion S2 to transit to its potent, fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are unavailable, and the mechanisms and timing of membrane capture and fusion are not established. Here, we constructed a full-length model of the CoV-2 FI by extrapolating from known CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the FI was remarkably flexible and executed large bending and extensional fluctuations due to three hinges in the C-terminal base. Simulations suggested a host cell membrane capture time of [~] 2 ms. Isolated fusion peptide simulations identified an N-terminal helix that directed and maintained binding to the membrane but grossly underestimated the binding time, showing that the fusion peptide environment is radically altered when attached to its host fusion protein. The large configurational fluctuations of the FI generated a substantial exploration volume that aided capture of the target membrane, and may set the waiting time for fluctuation-triggered refolding of the FI that draws the viral envelope and host cell membrane together for fusion. These results describe the FI as a machinery designed for efficient membrane capture and suggest novel potential drug targets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/439051v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1374b59org.highwire.dtl.DTLVardef@133d21forg.highwire.dtl.DTLVardef@127f2ddorg.highwire.dtl.DTLVardef@1b9ab8f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp13 Helicase

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterised pharmaceuticals for nsp13 inhibitors using a FRET-based high-throughput screening (HTS) approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

biochemistry↗