Lead Discovery for Microsomal Prostaglandin E Synthase Using a Combination of High-Throughput Fluorescent-Based Assays and RapidFire Mass Spectrometry

Abstract

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

“…The speed and efficiency of the HT-MS/MS can allow for experiments that would otherwise be deemed "untenable" under normal circumstances. The HT system has been validated as suitable for many drug discoveries [58][59][60][61][62][63][64][65] and ADME (Absorption, Distribution, Metabolism and Excretion) based applications [66]. The main drawback of this method is that disaccharides with identical molecular weights cannot be distinguished.…”

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

“…15 Although this method has previously been used for routine assay of KMO activity, 10,13 the use of the RF-MS system provided the increased throughput desirable for a lead discovery campaign. Mass spectrometer protocols were established for the simultaneous detection of Kyn and 3-HK, giving no signal cross-talk between the channels, thus allowing the percentage conversion to be calculated.…”

“…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.…”

The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

“…As this could bias the percent conversion values generated, a correction factor of 0.16 was applied to the ACh data to bring it in line with the Ch data, as has been shown previously for another MS-based assay where product and substrate gave very different intensities. 22 Metal 1536-format AnchorChip MALDI targets are available, but for the work described here, a plastic prespotted 384-well AnchorChip (PAC) target, wiped clean with ethanol and water to create a blank surface, was used. This was because our overall aim was to understand the greatest density at which targets could be spotted with samples, to minimize plate movements into and out of the mass spectrometer, and we did not want to restrict ourselves to the…”