Advances in high-throughput mass spectrometry in drug discovery

Abstract

High-throughput (HT) screening drug discovery, during which thousands or millions of compounds are screened, remains the key methodology for identifying active chemical matter in early drug discovery pipelines. Recent technological developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabelled biomolecules in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label-free MS assays are often cheaper, faster, and more physiologically relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label-free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compounds in cell lines, and even primary cells. Finally, we will give an outlook on how technological advances will increase the future use and the capabilities of mass spectrometry in drug discovery.

Keywords: MALDI-TOF; affinity selection; drug discovery; high-throughput screening; mass spectrometry.

Figure 1. Schematic of main ionization techniques employed for HTS‐MS

(A) Surface‐based: MALDI. Samples are co‐crystallized with a matrix on a conductive target plate. Laser shots are used to activate matrix molecules and evaporate analyte and matrix. In the reactive cloud, protons are transferred from the matrix to ionize the analyte molecules (Karas et al, 1985). SAMDI. Components of an enzymatic reaction (either enzymes or substrates) are immobilized onto self‐assembled monolayers (SAMs) in an array format, and upon irradiation with a laser, the monolayers are desorbed from the surface through cleavage of the thiolate‐gold bond and ionized (Gurard‐Levin et al, 2011). (B) Electrospray‐based: ESI. The analytes are dissolved in a liquid carrier phase, and a high voltage is applied to the tip of the metal capillary relative to the mass spectrometer's sampling cone. The electric field causes the dispersion of the sample solution resulting in nebulization. Charged droplets containing the analytes are generated at the exit of the electrospray tip. The solvent of the droplets is vaporized by a drying gas or heat and the charged analytes are guided by a potential gradient toward the analyzer region of the MS (Fenn et al, ; El‐Aneed et al, 2009). AMI. An acoustic transducer and charging cone are used to generate nanolitre‐sized charged droplets that are guided through an ion transfer line into a MS (Sinclair et al, 2015). ADE‐OPI. A pulse of acoustic energy ejects sample droplets upward into the inverted OPI, where a fluid pump delivers carrier solvent to a sample capture region. The sample is captured, diluted, and guided to MS by conventional ESI (Zhang et al, 2021).

Figure 2. Types of high‐throughput mass spectrometry drug discovery assays

(A) Enzyme activity screening by mass spectrometry. In vitro reactions of enzymes with substrates are stopped at appropriate time points and the resulting mixture analysed by mass spectrometry to identify substrate to product conversion. Addition of chemical compounds that affect the reaction are identified by reduced product conversion. (B) Affinity Selection Mass Spectrometry. Compounds bind to a protein of interest and non‐binding compounds are removed by size‐exclusion chromatography. Binding compounds are identified by mass spectrometry. (C) Cellular and phenotypic screening by mass spectrometry. Cellular phenotypes of “healthy” and “diseased” controls are defined by a read‐out of a cellular “fingerprint” of specific biomolecules. Chemical compounds that shift the “diseased” phenotype to “healthy” are considered hits.

Figure 3. Fragment‐based drug discovery assays

(A) Summary of amino acid targeting moieties with the reactive warheads highlighted in red. (B) Overview of a reactive fragment covalently binding to a protein target of interest and how LC–MS and LC–MS/MS contribute to the measurement of binding affinity, localization of covalently bound residue as well as kinetic and competition studies.

Figure 4. Cellular phenotypic assays by MALDI‐TOF mass spectrometry

Cells or extracts of cells are spotted by liquid handling robots onto a MALDI target. MALDI‐TOF MS analysis of these samples defines “fingerprints” of “healthy” and “diseased” controls. Characterization of these complex fingerprints, potentially through machine learning or dimensionality reduction analysis (such as principal component analysis), allows the identification of biomarkers specific for the phenotypes. Changes of these biomarkers can be used as a read‐out for a drug discovery screen against many chemical moieties. Since the readout produces information‐rich multi‐dimensional data, the use of known inhibitors and cytotoxic compounds can be used to multiplex and identify novel compounds in these pathways.