High-throughput mechanistic screening of non-equilibrium inhibitors by a fully automated data analysis pipeline in early drug-discovery.

Recent efforts for increasing the success in drug discovery focus on an early, massive, and routine mechanistic and/or kinetic characterization of drug-target engagement as part of a design-make-test-analyze strategy. From an experimental perspective, many mechanistic assays can be translated into a scalable format on automation platforms and thereby enable routine characterization of hundreds or thousands of compounds. However, now the limiting factor to achieve such in-depth characterization at high-throughput becomes the quality-driven data analysis, the sheer scale of which outweighs the time available to the scientific staff of most labs.

Acoustic Mist Ionization Mass Spectrometry for Ultrahigh-Throughput Metabolomics Screening.

Incorporating safety data early in the drug discovery pipeline is key to reducing costly lead candidate failures. For a single drug development project, we estimate that several thousand samples per day require screening (<10 s per acquisition). While chromatography-based metabolomics has proven value at predicting toxicity from metabolic biomarker profiles, it lacks sufficiently high sample throughput.

A Novel High-Throughput FLIPR Tetra-Based Method for Capturing Highly Confluent Kinetic Data for Structure-Kinetic Relationship Guided Early Drug Discovery.

Target engagement by small molecules is necessary for producing a physiological outcome. In the past, a lot of emphasis was placed on understanding the thermodynamics of such interactions to guide structure-activity relationships. It is becoming clearer, however, that understanding the kinetics of the interaction between a small-molecule inhibitor and the biological target [structure-kinetic relationship (SKR)] is critical for selection of the optimum candidate drug molecule for clinical trial.

Information-rich high-throughput cellular assays using acoustic mist ionisation mass spectrometry.

Cellular metabolites and phospholipids contain a vast amount of information about the current state of a cell, and are a useful resource for understanding the effects of drug candidates in vitro. Typical human cell-based assays in early drug discovery rely on simple readouts such as cell viability, or focus on single end-points revealed by an antibody or other label-based technologies. We introduce a generic 384-well plate-based workflow for data-rich cellular assays using facile sample preparation and direct analysis by acoustic mist ionization mass spectrometry (AMI-MS).

Acoustic Mist Ionization Platform for Direct and Contactless Ultrahigh-Throughput Mass Spectrometry Analysis of Liquid Samples.

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector.

Nanoparticles and intracellular applications of surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectrocopy (SERS) offers ultrasensitive vibrational fingerprinting at the nanoscale. Its non-destructive nature affords an ideal tool for interrogation of the intracellular environment, detecting the localisation of biomolecules, delivery and monitoring of therapeutics and for characterisation of complex cellular processes at the molecular level. Innovations in nanotechnology have produced a wide selection of novel, purpose-built plasmonic nanostructures capable of high SERS enhancement for intracellular probing while microfluidic technologies are being utilised to reproducibly synthesise nanoparticle (NP) probes at large scale and in high throughput.

High-Throughput Screening Using Mass Spectrometry within Drug Discovery.

In order to detect a biochemical analyte with a mass spectrometer (MS) it is necessary to ionize the analyte of interest. The analyte can be ionized by a number of different mechanisms, however, one common method is electrospray ionization (ESI). Droplets of analyte are sprayed through a highly charged field, the droplets pick up charge, and this is transferred to the analyte.

Novel Acoustic Loading of a Mass Spectrometer: Toward Next-Generation High-Throughput MS Screening.

High-throughput, direct measurement of substrate-to-product conversion by label-free detection, without the need for engineered substrates or secondary assays, could be considered the "holy grail" of drug discovery screening. Mass spectrometry (MS) has the potential to be part of this ultimate screening solution, but is constrained by the limitations of existing MS sample introduction modes that cannot meet the throughput requirements of high-throughput screening (HTS). Here we report data from a prototype system (Echo-MS) that uses acoustic droplet ejection (ADE) to transfer femtoliter-scale droplets in a rapid, precise, and accurate fashion directly into the MS.

Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing.

Design and synthesis of novel lactate dehydrogenase A inhibitors by fragment-based lead generation.

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors.