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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.

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 high-throughput 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.

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.

Standard methods of monitoring the growth kinetics of anaerobic microorganisms are generally impractical when there is a protracted or indeterminate period of active growth, and when high numbers of samples or replications are required. As part of our studies of the adaptive evolution of a simple anaerobic syntrophic mutualism, requiring the characterization of many isolates and alternative syntrophic pairings, we developed a multiplexed growth monitoring system using a combination of commercially available electronics and custom designed circuitry and materials. This system automatically monitors up to 64 sealed, and as needed pressurized, culture tubes and reports the growth data in real-time through integration with a customized relational database. The utility of this system was demonstrated by resolving minor differences in growth kinetics associated with the adaptive evolution of a simple microbial community comprised of a sulfate reducing bacterium, Desulfovibrio vulgaris, grown in syntrophic association with Methanococcus maripaludis, a hydrogenotrophic methanogen.