A review for cell-based screening methods in drug discovery

Fang, Wei; Wang, Sicen; Gou, Xin

doi:10.52601/bpr.2021.210042

Cited by 26 publications

(10 citation statements)

References 128 publications

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“…One of the most promising applications of fluorescent BER probes is high-throughput screening (HTS) to identify inhibitors and/or activators of BER enzymes. In particular, cell-based screening assays have the advantage of generating biologically relevant data that can better predict the complexity of a therapeutic response. , However, cell-based HTS remains inaccessible for most BER enzymes due to the lack of biostable probes amenable to intracellular analysis. Given the robust performance of our chimeric probe design in cells, we therefore tested whether FU-7 could be applicable to small molecule screening using the known h UNG inhibitor nifuroxazide as a model compound .…”

Section: Resultsmentioning

confidence: 99%

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

Zhong

Sczepanski

2023

J. Am. Chem. Soc.

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The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other offtarget interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric D/L-DNA architecture that exploits the bioorthogonal properties of mirror-image L-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

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Section: Resultsmentioning

confidence: 99%

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

Zhong

Sczepanski

2023

J. Am. Chem. Soc.

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“…Compared to conventional microfluidic droplet devices, these devices operate a smaller number of droplets (several up to tens), and the droplet volume is relatively larger (from microliters down to a hundred nanoliters). [ 212 ] However, the ability to configure and automate complex multistep operations makes these platforms a potential universal tool for drug screening.…”

Section: Final Remarks and Outlookmentioning

confidence: 99%

Recent Advances on Cell Culture Platforms for In Vitro Drug Screening and Cell Therapies: From Conventional to Microfluidic Strategies

Cardoso

Castanheira

Lanceros‐Méndez

et al. 2023

Adv Healthcare Materials

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The clinical translations of drugs and nanomedicines depend on coherent pharmaceutical research based on biologically accurate screening approaches. Since establishing the 2D in vitro cell culture method, the scientific community has improved cell-based drug screening assays and models. Those advances result in more informative biochemical assays and the development of 3D multicellular models to describe the biological complexity better and enhance the simulation of the in vivo microenvironment. Despite the overall dominance of conventional 2D and 3D cell macroscopic culture methods, they present physicochemical and operational challenges that impair the scale-up of drug screening by not allowing a high parallelization, multidrug combination, and high-throughput screening. Their combination and complementarity with microfluidic platforms enable the development of microfluidics-based cell culture platforms with unequivocal advantages in drug screening and cell therapies. Thus, this review presents an updated and consolidated view of cell culture miniaturization's physical, chemical, and operational considerations in the pharmaceutical research scenario. It clarifies advances in the field using gradient-based microfluidics, droplet-based microfluidics, printed-based microfluidics, digital-based microfluidics, SlipChip, and paper-based microfluidics. Finally, it presents a comparative analysis of the performance of cell-based methods in life research and development to achieve increased precision in the drug screening process.

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“…Prioritizing hit compounds identified from a primary compound screen is important for drug development [1][2][3][4][5][6] as follow-up secondary assays are resource-intensive, particularly cell-based assays of target protein function [7,8]. Cell-based assays monitor compound activity in a physiologically relevant environment, increasing confidence in results [7,[9][10][11] while retaining compatibility with higher-throughput screening formats [12]. The choice of secondary assay must therefore strike a balance between testing fewer, high-priority compounds in a higher-confidence assay, or more compounds in a lower-confidence assay, since resources not unlimited [13].…”

Section: Introductionmentioning

confidence: 99%

A novel heuristic of rigid docking scores positively correlates with full-length nuclear receptor LRH-1 regulation.

Haratipour,

Foutch,

Blind

2024

Preprint

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The nuclear receptor Liver Receptor Homolog-1 (LRH-1, NR5A2) is a ligand-regulated transcription factor and validated drug target for several human diseases. LRH-1 activation is regulated by small molecule ligands, which bind to the ligand binding domain (LBD) within the full-length LRH-1. We recently identified 57 compounds that bind LRH-1, and unexpectedly found these compounds regulated either the isolated LBD, or the full-length LRH-1 in cells, with little overlap. Here, we correlated compound binding energy from a single rigid-body scoring function with full-length LRH-1 activity in cells. Although docking scores of the 57 hit compounds did not correlate with LRH-1 regulation in wet lab assays, a subset of the compounds had large differences in binding energy docked to the isolated LBD vs. full-length LRH-1, which we used to empirically derive a new metric of the docking scores we call "ΔΔG". Initial regressions, correlations and contingency analyses all suggest compounds with high ΔΔG values more frequently regulated LRH-1 in wet lab assays. We then docked all 57 compounds to 18 crystal structures of LRH-1 to obtain averaged ΔΔG values, which robustly and reproducibly associated with full-length LRH-1 activity in cells. Network analyses on the 18 crystal structures of LRH-1 suggest unique communication paths exist between the subsets of LRH-1 crystal structures that produced high vs. low ΔΔG values, identifying a structural relationship between ΔΔG and the position of Helix 6, a previously established regulatory helix important for LRH-1 regulation. Together, these data suggest computational docking can be used to quickly calculate ΔΔG, which positively correlated with the ability of these 57 hit compounds to regulate full-length LRH-1 in cell-based assays. We propose ΔΔG as a novel computational tool that can be applied to LRH-1 drug screens to prioritize compounds for secondary screening.Although docking scores of the 57 hit compounds did not correlate with LRH-1 regulation in wet lab assays, a subset of the compounds had large differences in binding energy docked to the isolated LBD vs. full-length LRH-1, which we used to empirically derive a new metric of the docking scores we call "ΔΔG". Initial regressions, correlations and contingency analyses all suggest compounds with high ΔΔG values more frequently regulated LRH-1 in wet lab assays. We then docked all 57 compounds to 18 crystal structures of LRH-1 to obtain averaged ΔΔG values, which robustly and reproducibly associated with full-length LRH-1 activity in cells. Network analyses on the 18 crystal structures of LRH-1 suggest unique communication paths exist between the subsets of LRH-1 crystal structures that produced high vs. low ΔΔG values, identifying a structural relationship between ΔΔG and the position of Helix 6, a previously established regulatory helix important for LRH-1 regulation. Together, these data suggest computational docking can be used to quickly calculate ΔΔG, which positively correlated with the ability of these 57 hit compounds to regulate full-length LRH-1 in cell-based assays. We propose ΔΔG as a novel computational tool that can be applied to LRH-1 drug screens to prioritize compounds for secondary screening.

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A review for cell-based screening methods in drug discovery

Cited by 26 publications

References 128 publications

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

Recent Advances on Cell Culture Platforms for In Vitro Drug Screening and Cell Therapies: From Conventional to Microfluidic Strategies

A novel heuristic of rigid docking scores positively correlates with full-length nuclear receptor LRH-1 regulation.

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