High-Throughput Screening Using Mass Spectrometry within Drug Discovery

Rohman, Mattias; Wingfield, Jonathan

doi:10.1007/978-1-4939-3673-1_3

Cited by 23 publications

(20 citation statements)

References 12 publications

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“…Key to a successful discovery campaign of such chemical probes is the availability of reliable screening methods that could enable medium to high throughput screening with low false-positive and false-negative rates. Various assays including mass spectrometry 30–32 , fluorescence 33 , and radiometric assays 24 have been used for screening libraries of compounds. Mass spectrometrybased assays require more expensive instrumentation and expertise.…”

Section: Discussionmentioning

confidence: 99%

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

Devkota

Schapira

Perveen

et al. 2021

Preprint

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The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 ± 0.2 μM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

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Section: Discussionmentioning

confidence: 99%

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

Devkota

Schapira

Perveen

et al. 2021

Preprint

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“…To improve throughput, solidphase extraction (SPE) can be utilized instead of LC for desalting. The Agilent RapidFire system further automates sample aspiration, SPE desalting, and ESI MS injection steps to achieve a cycling time of~10 s [27,28].…”

Section: Basic Concepts In Ms and High-throughput Ms Approachesmentioning

confidence: 99%

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

Zhang

2020

BMC Energy

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Enzymes play indispensable roles in producing biofuels, a sustainable and renewable source of transportation fuels. Lacking rational design rules, the development of industrially relevant enzyme catalysts relies heavily on highthroughput screening. However, few universal methods exist to rapidly characterize large-scale enzyme libraries. Therefore, assay development is necessary on an ad hoc basis to link enzyme properties to spectrophotometric signals and often requires the use of surrogate, optically active substrates. On the other hand, mass spectrometry (MS) performs label-free enzyme assays that utilize native substrates and is therefore generally applicable. But the analytical speed of MS is considered rate limiting, mainly due to the use of time-consuming chromatographic separation in traditional MS analysis. Thanks to new instrumentation and sample preparation methods, direct analyte introduction into a mass spectrometer without a prior chromatographic step can be achieved by laser, microfluidics, and acoustics, so that each sample can be analyzed within seconds. Here we review recent advances in MS platforms that improve the throughput of enzyme library screening and discuss how these advances can potentially facilitate biofuel research by providing high sensitivity, selectivity and quantitation that are difficult to obtain using traditional assays. We also highlight the limitations of current MS assays in studying biofuel-related enzymes and propose possible solutions.

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“…( Paddon et al, 2013 )], and too consuming of time and resources for high-throughput screening. A rapid methodology for detecting improved production of oxidized products of amorphadiene would be needed such as rapid mass spectrometry ( Rohman and Wingfield, 2016 ) or surrogate assays based on spectrophotometric or fluorescent methods that could cut the time required to measure titers to 10 s or less per sample, albeit with the statistical reproducibility required to detect genuine improvements on artemisinic acid production from the background variability inherent in a high-throughput screen.…”

Section: Increasing Activity Of Heterologously Expressed Cyp71av1mentioning

confidence: 99%

Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin

et al. 2018

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The antimalarial drug artemisinin is a natural product produced by the plant Artemisia annua. Extracts of A. annua have been used in Chinese herbal medicine for over two millennia. Following the re-discovery of A. annua extract as an effective antimalarial, and the isolation and structural elucidation of artemisinin as the active agent, it was recommended as the first-line treatment for uncomplicated malaria in combination with another effective antimalarial drug (Artemisinin Combination Therapy) by the World Health Organization (WHO) in 2002. Following the WHO recommendation, the availability and price of artemisinin fluctuated greatly, ranging from supply shortfalls in some years to oversupply in others. To alleviate these supply and price issues, a second source of artemisinin was sought, resulting in an effort to produce artemisinic acid, a late-stage chemical precursor of artemisinin, by yeast fermentation, followed by chemical conversion to artemisinin (i.e., semi-synthesis). Engineering to enable production of artemisinic acid in yeast relied on the discovery of A. annua genes encoding artemisinic acid biosynthetic enzymes, and synthetic biology to engineer yeast metabolism. The progress of this effort, which resulted in semi-synthetic artemisinin entering commercial production in 2013, is reviewed with an emphasis on recent publications and opportunities for further development. Aspects of both the biology of artemisinin production in A. annua, and yeast strain engineering are discussed, as are recent developments in the chemical conversion of artemisinic acid to artemisinin.

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High-Throughput Screening Using Mass Spectrometry within Drug Discovery

Cited by 23 publications

References 12 publications

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin

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