Mass spectrometry-based screening can be applied to a wide range of
targets, including targets that use substrates such as lipids, fatty
acids, phospholipids, steroids, prostaglandins and other compounds not
generally amenable to conventional screening techniques.
Introduction A successful
high-throughput screening (HTS) campaign requires development of an assay
in which a desirable biological outcome can be measured. Ideally, an HTS
assay facilitates use of the native biological substrate of a target, does
not require radioisotopes and provides accurate and precise quantification
without the need for compromises in assay design.
Mass spectrometry (MS) allows for the detection of a very wide range of
compounds based on mass-to-charge ratio. We demonstrate that MS can be
applied to many challenging HTS targets, including the lipoxygenase family
of enzymes.
Figure 1. Twenty-four screening
samples analyzed by RapidFire™ Lead Discovery in 2 min. Data
contains two IC50 curves, followed by high and low
controls.
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Experimental Conditions
The RapidFire™ (BioTrove, Woburn, MA, USA) ultra high-throughput MS
system has been described previously (1). Briefly, it is an integrated
sample purification and injection system that can operate at throughputs
of up to 3 s per sample. Over 10,000 samples per day can be routinely
analyzed per instrument. Example data are shown in Figure 1.
A MS-based assay that directly
quantifies the conversion of arachidonic acid to
5,8,11,13-Eicosatetraenoic acid (15(S)-HETE) by 15-lipoxygenase was
developed. Test compounds that attenuate the formation of 15(S)-HETE can
be identified through the decreased percent conversion.
Five known inhibitors of
15-lipoxygenase (quercetin, fisetin, nordihydroguaiaretic acid [NDGA],
tannic acid and ebselen) were tested to determine relative potencies.
Triplicate, 7-point log dilutions starting at a test compound
concentration of 1 mM were prepared for each of the five inhibitors.
Samples and controls consisted of 192 samples (2 × 96-well microtiter
plates). RapidFire™ Lead Discovery analysis of the two plates was
completed in 16.5 min., or an average throughput of 5.2 s per sample.
Figure 2. IC50 curves for
five known inhibitors of 15-lipoxygenase.
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Results
IC50 curves are shown in Figure 2. The four-plant based
polyphenolic compounds – fisetin, quercetin, NDGA and tannic acid –
had IC50 values of 18.9 µM, 10.7 µM, 1.7 µM and 0.2 µM,
respectively. Ebselen, a selenium-containing irreversible inhibitor, had
an IC50 value of 2.5 µM. These values are within ranges
reported in the literature (2, 3).
Conclusions
MS used in the RapidFire™ Lead Discovery system enables direct, accurate
quantitation of the percent conversion in a solution-phase reaction.
Because MS can quantify analytes based on molecular weight, label-free
detection can be applied to native biological substrates. The use of
native substrates provides biologically relevant data and streamlines the
assay development process. Development of surrogate or
radioactively-labeled substrates or indirect detection schemes is
eliminated.
References
1. C.C. Özbal et al., Assay and Drug Dev. Technol. 2,
373–381 (2004).
2. C.D. Sadik, H. Sies and T. Schewe,
Biochem. Pharmacol. 65, 773–781 (2003).
