Ryan P. Bingham

Real Experiences of uHTS: A Prototypic 1536-Well Fluorescence Anisotropy-Based uHTS Screen and Application of Well-Level Quality Control Procedures

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

Developments in fluorescence lifetime-based analysis for ultra-HTS

Abstract Homogeneous fluorescence detection methods are proving key in the establishment of miniaturized ultra-HTS (uHTS). Fluorescence lifetime-based methods seem set to provide an additional dimension to these approaches and are likely to represent a significant further advance both in terms of increasing measurement robustness and in opening up completely new types of uHTS assay format.

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. (Journal of Biomolecular Screening 2003:19-33)

A Modular, Fully Integrated Ultra-High-Throughput Screening System Based on Confocal Fluorescence Analysis Techniques

The rapid increase in size of compound libraries, as well as new targets emerging from the Human Genome Project, require progress in ultra-high-throughput screening (uHTS) systems. In a joint effort with scientists and engineers from the biotech and the pharmaceutical industry, a modular, fully integrated system for miniaturized uHTS was developed. The goal was to achieve high data quality in small assay volumes (1-4 μL) combined with reliable and unattended operation. Two new confocal fluorescence readers have been designed. One of the instruments is a 4-channel confocal fluorescence reader, measuring with 4 objectives in parallel. The fluorescence readout is based on single-molecule detection methods, allowing high sensitivity at low tracer concentrationsand delivering an information-rich output. The other instrument isa confocal fluorescence im aging reader, where the imagesare analyzed in terms of generic patternsand quantified in units of intensity per pixel. Both readers are spanning the application range from assays with isolated targets in homogenous solution or membrane vesiclebased assays (4-channel reader) to cell-based assays (imaging reader). Results from a comprehensive test on these assay types demonstrate the high quality and robustness of this screening system.

The discovery of in vivo active mitochondrial branched-chain aminotransferase (BCATm) inhibitors by hybridizing fragment and HTS hits

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

Discovery and Optimization of Potent, Selective, and in Vivo Efficacious 2-Aryl Benzimidazole BCATm Inhibitors

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

Structurally diverse mitochondrial branched chain aminotransferase (BCATm) leads with varying binding modes identified by fragment screening

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.

Pharmacological disruption of the MID1/α4 interaction reduces mutant Huntingtin levels in primary neuronal cultures.

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntingtons Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.

Confocal detection in ultra-high throughput screening

Today, ultra-high throughput screening (uHTS) methods enable screening of 100,000 samples/day or more to meet the demands for speed, with assay volumes miniaturised to 10 /spl mu/l or below to reduce reagent consumption. Various methods have been employed and are now implemented on a common instrumental platform (EVOscreen/spl trade/ by Evotec Technologies, Hamburg), which allows efficient selection of the best read-out mode for particular assay types, or even multiplexing of methods. Fluorescence correlation spectroscopy (PCS) is sensitive to changes in diffusion as seen when a fluorescent ligand binds to a receptor protein, for example. Fluorescence intensity distribution analysis (FIDA) allows detection of changes in specific brightness which could be caused for example by quenching, fluorescence resonance energy transfer (FRET) or accumulation of fluorophores on particles with multiple binding sites. By extending this method to 2-channel detection (2D-FIDA) using a polarising beamsplitter, anisotropy changes can be monitored, which is often the method of choice for small-ligand binding assays. Finally, using pulsed laser sources and single-photon counting electronics, fluorescence lifetimes can be measured providing a robust read-out parameter in various assay formats. We will present examples of assays and screens based on these detection modes which include the most important target classes and cover a variety of assay types. Several examples for these assay types and their validation for uHTS will be discussed.