High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

Forbes, Chris D.; Toth, Joshuaine; Özbal, Can C.; LaMarr, William A.; Pendleton, Jennifer A.; Rocks, Sandra; Gedrich, Richard; Osterman, David G.; Landro, James A.; Lumb, Kevin J.

doi:10.1177/1087057107301320

Cited by 39 publications

(30 citation statements)

References 19 publications

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“…From previous reports the fastest rate for electrospray or MALDI-based methods was ~5 to 10 sec per sample. 3,11,12 To test the limit of acquisition speed on the MALDI-QqQ system, an entire 384-well plate of control reactions was scanned at a standard rate of 1.2 sec per sample (<8 min per plate) and then again at the fastest rate of 0.3 sec per sample (<2 min per plate). As is demonstrated in Figure 3, even at the highest acquisition rate, the reproducibility of the substrate and product ratios are maintained with a robust Z′ value at 0.65.…”

Section: Resultsmentioning

confidence: 99%

Development of an Inhibitor Screening Platform via Mass Spectrometry

Rathore

Corr²,

Scott³

et al. 2008

SLAS Discovery

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Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC 50 values for several known kinase inhibitors against cyclic AMP-dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS-based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. INTRODUCTION HIGH-THROUGHPUT SCREENING (HTS) plays a central role in the drug discovery process starting at the early "hit" discovery stage then continuing through to lead optimization. As such, the screening community has continuously developed new technologies to meet the broadening assay needs and to expedite the work flow of drug discovery. Within the pharmaceutical and biotechnology enzyme screening community, fluorescent-and chemiluminescent-based detection methods continue to be the routine assay platforms of choice for isolated enzyme assays. These assays are desirable because they have been developed to be simple homogeneous assays that often offer universal methods to evaluate a variety of targets using the same reagents. However, 1 of the primary challenges for these approaches continues to be how to maintain the high speed of analysis while minimizing false-positive or false-negative readouts. Inherent to this challenge is the nature of fluorescence and chemiluminescence readout, which can lend itself to false readouts because of the properties of various compound classes that enhance or quench the signals. Thus a comparable approach in speed, robust readout, and cost that further minimizes the potential for false readouts would be a benefit to the field.One approach that offers promise in this arena is mass spectrometry. A variety of mass spectrometry (MS) techniques have been used to measure enzyme activity, enzyme kinetics, inhibition, and more recently for HTS applications (reviewed in the studies of Greis 1 and De Boer et...

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Section: Resultsmentioning

confidence: 99%

Development of an Inhibitor Screening Platform via Mass Spectrometry

Rathore

Corr²,

Scott³

et al. 2008

SLAS Discovery

View full text Add to dashboard Cite

show abstract

“…This enabling technology has previously been successfully used in lead discovery campaigns for challenging targets. [14][15][16][17] It overcomes the problem of time-consuming sample separation by using an in-line solid-phase extraction (SPE) cartridge system for sample clean up and analyte concentration. This delivers the sample directly to an electrospray ionization (ESI) triple quadrupole mass spectrometer at a rate of approximately 7 s per well.…”

Section: Introductionmentioning

confidence: 99%

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

et al. 2014

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Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z′ value 0.8) and provided several tractable hit series for further investigation.

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“…It offers a direct, label-free readout of product formation with reduced risk of interference compared with alternatives and no requirement for antibody generation. 16,17 However, the capacity of this system renders it a medium-throughput rather than a high-throughput technology. Fluorescent-based methods are therefore still employed for diversity screening of these targets.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously reported on the application of RapidFire (Agilent Technologies, Lexington, MA) high-throughput mass spectrometry to screening of ~100,000 compounds against JmjD2c. 15 However, despite the throughput advances of the RapidFire system relative to liquid chromatography-mass spectrometry (sample times of ~8 s/well), 16,17 the system is still not adequate for largescale HTS in a reasonable time frame. One could enable HTS by RapidFire with access to multiple systems in parallel; however, it is unlikely that capital investment in a single technology at this scale would occur within the pharmaceutical industry today.…”

Section: Introductionmentioning

confidence: 99%

Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

et al. 2014

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Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true highthroughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

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High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

Cited by 39 publications

References 19 publications

Development of an Inhibitor Screening Platform via Mass Spectrometry

Development of an Inhibitor Screening Platform via Mass Spectrometry

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

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