Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors.

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.

Screening for mevalonate biosynthetic pathway inhibitors using sensitized bacterial strains.

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.

Scintillation proximity assays in high-throughput screening.

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.

Farnesyl pyrophosphate synthase: real-time kinetics and inhibition by nitrogen-containing bisphosphonates in a scintillation assay.

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.

Statistical evaluation of a self-deconvoluting matrix strategy for high-throughput screening of the CXCR3 receptor.

In high-throughput screening (HTS), compounds can be tested in self-deconvoluting matrices (SDMs) of 10 compounds per well. The SDM setup is based upon a systematic mixing of compound samples such that each compound appears twice in the screening assay, in two independent mixtures. In order to test the quality of the SDM approach, we compared it with a standard single-compound screening approach. In a CXCR3 scintillation proximity assay, we performed five multiple screening trials of 26,400 compounds at a 10 microM screening concentration to estimate false positive and false negative rates in the compound population. No potent hits (<6.2 microM IC50) were missed in any screening method. Forty-eight percent of all actives were found in every screening trial independent of compound handling method. The SDM strategy had an average of 25 false positives and 15 false negatives as compared with an average of 34 false positives and 15 false negatives with a more conventional single-compound screening approach. Most of the variability resulted from day-to-day variation around the hit cutoff criterion, rather than from any particular screening technique. In the two most extreme examples, a compound with a 7.5 microM IC50 was missed in one out of two mixture trials, and a compound with a 6.2 microM IC50 was missed in one out of three single-compound trials. In the CXCR3 assay presented herein, the SDM screening method had better predictive value than the single-compound screening approach.