Scintillation proximity assays (SPAs) have become a powerful tool for high-throughput screening (HTS) because they can measure the activity and binding of very diverse classes of drug targets. By applying the basic principles of ligand-receptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, high-throughput assays and screen millions of compounds. SPAs are enabled by the diversity of radiolabeled molecules and affinity tags that are commercially available. These synthetic radiotracers allow for minimal disturbance of the natural binding interactions. This article will present a comprehensive review of the technique and provide detailed information on its applications related to HTS, highlighting the major uses and giving some suggestions for future research.

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential.  Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.

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A mix-and-read FlashPlate (PerkinElmer, Waltham, MA) assay for the enzyme farnesyl pyrophosphate (FPP) synthase (FPPS) was developed to rapidly measure both steps in the synthesis of FPP from dimethylallyl pyrophosphate (DMAPP). The assay used either DMAPP or geranyl pyrophosphate (GPP) and [(3)H]isopentenyl pyrophosphate ([(3)H]IPP) as substrates, and measured the FPPS-catalyzed conversion of these into [(3)H]FPP or [(3)H]GPP by capturing the products onto a phospholipid-coated scintillating microtiter plate and monitoring the product formation in a charge coupled device imager. The Michaelis-Menten parameters-k(cat) GPP (38/min), K(m) IPP (0.6 microM), and K(m) GPP (0.7 microM)-were consistent with previous studies using difficult phase separation techniques. The 50% inhibitory concentrations of various nitrogen-containing bisphosphonates (N-BPs) were determined and were also consistent with prior literature. Without precedent, weaker inhibition (5 microM) of the non-N-BPs was also detected. In preincubation studies, the potency of the N-BPs, and specifically zoledronate, increased slowly over time by 100-fold. This potency shift was reversed significantly by the inclusion of GPP with zoledronate. Zoledronate was uncompetitive with respect to IPP. Thus, these studies were consistent with prior structural and thermodynamic studies, and suggest a rapid formation of a lower-affinity complex between zoledronate and the GPP binding site, followed by the formation of a very tight complex of zoledronate and enzyme, which excludes further binding of GPP. Furthermore, one of the substrates from the first step in the catalytic cycle, DMAPP, was identified as a 1 microM inhibitor of the second step of the catalysis, suggesting that the FPP two-step synthesis is regulated by DMAPP.

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