An 11-year-old with mallet finger.

An 11-year-old girl presented with pain and deformity in her right little finger distal interphalangeal joint (DIPJ). She was active in several sports including hurling and had a history of dyspraxia with frequent minor soft tissue injuries which had not required hospital assessment. Her mother was concerned about the possibility of a recent injury.Examination showed flexion deformity of the right fifth finger with complete loss of extension at the DIPJ. There was mild swelling and tenderness of the DIPJ with no bruising, erythema or warmth. An X-ray was performed (figure 1). emermed;35/11/679/F1F1F1Figure 1An teroposterior (AP) and lateral radiographs of the right little finger. QUESTION: What is the diagnosis?Salter-Harris type 1 fracture of distal phalanxDystelephalangyExtensor digiti minimi tendon injuryClinodactyly.

2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors.

A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-gamma (INF-gamma) production.

Utilizing Low-Volume Aqueous Acoustic Transfer with the Echo 525 to Enable Miniaturization of qRT-PCR Assay.

Quantitative reverse transcription PCR (qRT-PCR) is a valuable tool for characterizing the effects of inhibitors on viral replication. The amplification of target viral genes through the use of specifically designed fluorescent probes and primers provides a reliable method for quantifying RNA. Due to reagent costs, use of these assays for compound evaluation is limited. Until recently, the inability to accurately dispense low volumes of qRT-PCR assay reagents precluded the routine use of this PCR assay for compound evaluation in drug discovery. Acoustic dispensing has become an integral part of drug discovery during the past decade; however, acoustic transfer of microliter volumes of aqueous reagents was time consuming. The Labcyte Echo 525 liquid handler was designed to enable rapid aqueous transfers. We compared the accuracy and precision of a qPCR assay using the Labcyte Echo 525 to those of the BioMek FX, a traditional liquid handler, with the goal of reducing the volume and cost of the assay. The data show that the Echo 525 provides higher accuracy and precision compared to the current process using a traditional liquid handler. Comparable data for assay volumes from 500 nL to 12 µL allowed the miniaturization of the assay, resulting in significant cost savings of drug discovery and process streamlining.

Probing the primary screening efficiency by multiple replicate testing: a quantitative analysis of hit confirmation and false screening results of a biochemical assay.

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability.

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.

A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-microl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening.

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.