SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening

Gorshkov, Kirill; Vasquez, Desarey Morales; Chiem, Kevin; Ye, Chengjin; Tran, Bruce Nguyen; Torre, Juan Carlos de la; Moran, Thomas M.; Chen, Catherine Z.; Martínez-Sobrido, Luis; Zheng, Wei

doi:10.1021/acsptsci.1c00182

Cited by 7 publications

(3 citation statements)

References 38 publications

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“…[49] Some recent TR-FRETbased HTS campaigns involve screening for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compounds, estrogen receptor inhibitors, histone lysine demethylases inhibitors, dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors, and actin-myosin interaction modulators. [64,[66][67][68][69] Another fluorescence-based assay is fluorescence polarization (FP), which has been used in HTS for decades. [70] This technique detects PPIs based on the polarization of fluorophores.…”

Section: Fluorescence-and Bioluminescence-based Assaysmentioning

confidence: 99%

“…[ 49 ] Some recent TR‐FRET‐based HTS campaigns involve screening for anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) compounds, estrogen receptor inhibitors, histone lysine demethylases inhibitors, dual‐specificity tyrosine phosphorylation‐regulated kinase 1A (DYRK1A) inhibitors, and actin‐myosin interaction modulators. [ 64,66–69 ]…”

Section: Hts‐based Drug Discoverymentioning

confidence: 99%

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The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

Lim

2022

Advanced Biology

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An early example of HTS in drug development came from the identification of new antibiotics using 96-well microtiter plates, whereby fermentations of an established actinomycetes library were incubated with a panel of microorganisms, and after optimization, a screening capacity of 10 000 fermentation broths per week was achieved. [1] In the following decades, the rapid development of molecular biology techniques and advances in automated equipment have changed how new drugs are identified, and a variety of HTS-identified drugs have been approved to treat different diseases. [2] For a comprehensive list of HTS-identified drugs, please refer to the excellent review by Macarron et al. [2] High-throughput applications can also be combined with other unbiased "Omics" technologies used to study genomics, epigenomics, transcriptomics, proteomics, and metabolomics. [3] Diabetes is an incurable disease characterized by the inability of the body to maintain normoglycemia due to decreased sensitivity of various tissues to insulin and insufficient amounts of circulating insulin, a hormone produced by pancreatic β-cells. [4] Diabetes is associated with chronic complications, like macrovascular diseases (e.g., cardiovascular diseases) and microvascular diseases (e.g., damages in nerve, kidney, eye, and foot). [5,6] More than 537 million adults are currently living with diabetes, and this number is expected to increase to 693 million by 2045. In 2021, 6.7 million deaths worldwide were attributed to diabetes, [7] which made it the ninth leading cause of death. [8] Globally, the prevalence of diabetes also brings a heavy burden to healthcare systems, with an estimated expenditure of over 966 billion U.S. dollars per year. [7] Diabetes is generally categorized into two groups: Type I diabetes mellitus (T1DM) and Type II diabetes mellitus (T2DM), but there are still some cases that have different etiologies, such as gestational diabetes mellitus and monogenic diabetes. [9] Since the discovery of insulin in 1921, [10] significant progress has been made in diabetes pharmacotherapy and therapeutic technology; [11][12][13] however, since a permanent cure is unavailable, the discovery and development of anti-diabetic drugs is still at the forefront of diabetes research. With the rapid advancement in high-throughput omics technologies, combined with accumulated genome-wide association study (GWAS) data, a better understanding of diabetes pathophysiology may one day lead to the discovery of novel therapeutic targets. [14,15] This review will During the past decades, unprecedented progress in technologies has revolutionized traditional research methodologies. Among these, advances in highthroughput drug screening approaches have permitted the rapid identification of potential therapeutic agents from drug libraries that contain thousands or millions of molecules. Moreover, high-throughput-based therapeutic target discovery strategies can comprehensively interrogate relationships between biomolecules (e.g., gene, RNA, and protein) and diseas...

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Section: Fluorescence-and Bioluminescence-based Assaysmentioning

confidence: 99%

Section: Hts‐based Drug Discoverymentioning

confidence: 99%

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

Lim

2022

Advanced Biology

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“…In addition, numerous cell-based screening assays with SARS-CoV-2 have also been recently reported 12 , 15 – 18 . Unlike the S and E proteins, however, the structural nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 protein in infected cells, has not been fully exploited as a target for drug development against SARS-CoV-2 19 .…”

Section: Introductionmentioning

confidence: 99%

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

Mercaldi

Bezerra²,

Batista³

et al. 2022

Sci Rep

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The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

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Boosting Excited‐State Energy Transfer by Anchoring Dipole Orientation in Binary Thermally Activated Delayed Fluorescence/J‐Aggregate Assemblies

Ma,

Guo,

Gao

et al. 2024

Chemistry A European J

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Förster resonance energy transfer (FRET) has been widely applied in fluorescence imaging, sensing and so on, while developing useful strategy of boosting FRET efficiency becomes a key issue that limits the application. Except optimizing spectral properties, promoting orientation factor (κ2) has been well discussed but rarely utilized for boosting FRET. Herein, we constructed binary nano‐assembling of two thermally activated delayed fluorescence (TADF) emitters (2CzPN and DMAC‐DPS) with J‐type aggregate of cyanine dye (C8S4) as doping films by taking advantage of their electrostatic interactions. Time‐resolved spectroscopic measurements indicated that 2CzPN/Cy‐J films exhibit an order of magnitude higher kFRET than DMAC‐DPS/Cy‐J films. Further quantitative analysing on kFRET and kDET indicated higher orientation factor (κ2) in 2CzPN/Cy‐J films play a key role for achieving fast kFRET, which was subsequently confirmed by anisotropic measurements. Corresponding DFT/TDDFT calculation revealed strong “two‐point” electrostatic anchoring in 2CzPN/Cy‐J films that is responsible for highly orientated transitions. We provide a new strategy for boosting FRET in nano‐assemblies, which might be inspired for designing FRET‐based devices of sensing, imaging and information encryption.

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SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening

Cited by 7 publications

References 38 publications

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

Boosting Excited‐State Energy Transfer by Anchoring Dipole Orientation in Binary Thermally Activated Delayed Fluorescence/J‐Aggregate Assemblies

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