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Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed.

Background l -3,4-dihydroxyphenylalanine ( l -DOPA)-induced dyskinesia (LID) are a complication of chronic dopamine replacement therapy in Parkinson's disease (PD). Recent studies have suggested that the mechanisms underlying development and expression of LID in PD may involve epigenetic changes that include deacetylation of striatal histone proteins. We hypothesised that inhibition of histone deacetylase, the enzyme responsible of histone deacetylation, would alleviate LID. Methods Four female common marmoset (Callithrix jacchus) were rendered parkinsonian by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Following stabilisation of the parkinsonian phenotype, marmosets were primed to exhibit dyskinesia with chronic administration of l -DOPA. We then investigated the effects of the brain-penetrant histone deacetylase inhibitor, RGFP109 (30 mg/kg p.o. once daily for 6 days), on LID and l -DOPA anti-parkinsonian efficacy. Results RGFP109 had no acute effects on dyskinesia after single or 6 days once-daily treatment (both P > 0.05). However, one week following cessation of RGFP109, dyskinesia and duration of ON-time with disabling dyskinesia were reduced by 37% and 50%, respectively (both P l -DOPA alone. There was no change in anti-parkinsonian actions of, or ON-time duration afforded by, l -DOPA (P > 0.05). Conclusions Histone deacetylation inhibition may represent a novel approach to reverse established LID in PD and improve quality of the anti-parkinsonian benefit provided by l -DOPA.

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.

The voltage-gated sodium channel Nav1.7 is one of the most validated pain targets, as gain-of-function mutations cause excessive pain, whereas loss-of-function mutations produce insensitivity to pain. Targeting Nav1.7 holds great promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 selective chemical scaffolds exist. So far millions of compounds have been screened by using fluorescence membrane potential (MP) assays, but very few selective hits have been identified. Here we show that the conventional MP approach, which uses Nav1.7 WT channel and the activator veratridine, is intrinsically flawed: it is biased toward non-selective pore blockers which compete with veratridine, and fails to detect highly potent, selective compounds such as the aryl sulfonamide PF-771. By using a mutant channel and an alternative activator, we developed a novel method that not only robustly detects known Nav1.7 blockers, but also decreases the number of non-selective pore blocker hits. We conducted a high-throughput screen using this methodology and identified novel Nav1.7-selective blockers.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B 2 receptor. In this study, we have examined the relationship between B 2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg 9 removal from BK and its analogs on E 2 receptor binding. The ratios of binding constants ( K i +Arg K i −Arg ) for the Arg 9 containing compounds and the corresponding des-Arg 9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg 9 -BK and Lys°-BK/des-Arg 9 -Lys°-BK.

A new basic antigen with a molecular weight of 36.5 kDa and a pI of 8.65 was purified from short ragweed pollen using ammonium sulfate precipitation, Q-sepharose chromatography and S-sepharose chromatography. The purified protein, herein referred to as Ambrosia artemisiifolia basic antigen (AaBA), migrated as a single band in SDS-polyacrylamide gel electrophoresis and isoelectric focusing, and as a single peak on reverse phase HPLC. AaBA reacted with sera from humans allergic to ragweed pollen including pooled NIH serum. AaBA and two major acidic allergens, Amb a I (antigen E) and Amb a II (antigen K), displayed immunological cross-reactivity inasmuch as they competed significantly with each other in a competition ELISA. This suggests that at least some major epitopes in all three antigens are similar; however, other possibilities like adjacent location of different epitopes cannot be ruled out at this time. Molecular weight, amino acid composition and sequence data suggest that AaBA is very similar to Amb a II whereas it is significantly different from other basic antigens purified from ragweed pollen.

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.