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Since its conception in 1985, matrix-assisted laser desorption/ionization (MALDI), coupled with time-of-flight (TOF) mass spectrometry (MS), has benefitted a wide range of applications, including protein sequencing, mapping biomolecules in tissues, identifying microorganisms, and analyzing thousands of compounds in biochemical assays. The technology’s high sensitivity and mass resolving power has had a major impact in the field of drug discovery and development, particularly with regard to visualizing drugs and their metabolites in tissues, and identifying disease biomarkers.
High-throughput screening (HTS) has made significant contributions to early-stage drug discovery over the years by rapidly testing large numbers of compounds for identification of hits that can be progressed into leads, with the potential to modulate a specific biological pathway. Automated HTS and ultrahigh-throughput screening (uHTS) provides researchers with the ability to screen large compound libraries against a continuously increasing number of targets. HTS can be carried out using an array of technologies, including fluorescence, nuclear magnetic resonance (NMR), affinity chromatography, surface plasmon resonance (SPR), and DNA microarrays.
More recently, however, MS has been used to rapidly screen through compound libraries of up to a million substances. In particular, the highly automated workflows, high-volume microplate reading and handling systems, and rapid assay screening times have made MALDI-TOF MS a popular technology in the drug discovery pipeline.1-3
A time-saving technique
HTS is often the first step in finding new drugs. A single protein target that is believed to play an important role in disease is established for subsequent identification of small molecules that are binding to or inhibiting that protein. For the past 10–20 years, systematic screening methods for identifying small-molecule chemicals that block these single proteins, also known as biochemical assays, have largely been carried out in the pharmaceutical industry using fluorescent methods. A key challenge with this technique is its susceptibility to interference from compounds that are colored or fluorescent themselves or that quench fluorescence. Counter-screening in the form of extensive confirmation assays is therefore required to validate hits from fluorescent HTS, adding increased costs and time delays to the drug discovery process. Studies have shown the capabilities of MALDI-TOF MS for HTS and uHTS applications.4
MALDI-MS-based assays in drug discovery
Researchers at the Center for Mass Spectrometry and Optical Spectroscopy (CeMOS) at Mannheim Technical University in Germany use MALDI MS for cellular drug discovery assays. When the group first began using this technology, mass spectrometers were much slower than what modern instruments can currently achieve. Despite this, the laboratory set out to develop cell-based assays, which are a mainstay in pharmaceutical R&D due to their closer resemblance to drug interactions within tissues, but have not been possible with MS readouts.
The team at CeMOS applied industrial assay development rules to MALDI-based cellular assays and pioneered this technique in the scientific and industrial community. The industry is interested in this technique because a cell more closely resembles the context in which the drug has to act in the body—it has to be able to enter cells. This cannot be accessed with biochemical assays, which would also require secondary screens to test compounds in cells.
For this reason, researchers are focusing on whole cell (WC)-MALDI-TOF MS for cell-based drug profiling to monitor the response of candidate drugs in the body. Scientists at CeMOS have been using WC-MALDI-TOF MS to monitor the concentration-responses of small-molecule drugs, and to determine cellular drug potencies. In a recent study, the group introduced a workflow for the discovery of pharmacodynamics markers, such as small-molecule lipid/metabolite markers, and demonstrated its use for cellular drug-response monitoring of BCR-ABL tyrosine kinase inhibitors—the first-line treatment for chronic myelogenous leukemia (CML)—such as imatinib in K562 leukemia cells.5
Using MALDI-TOF MS and MALDI magnetic resonance MS (MRMS) in a novel workflow, the group could automatically acquire and evaluate drug-concentration responses of lipids/metabolites in whole cells, enabling several functional markers of K562 cell differentiation to be monitored simultaneously. K562 cells were treated with different concentrations of imatinib and were measured according to the workflow to study the effect of this BCR-ABL tyrosine kinase inhibitor on the MS lipid/metabolite fingerprints in these cells. MALDI MRMS enables the direct identification of low-molecular-mass compounds and was used following MALDI-TOF MS to remeasure candidate marker molecules at ultrahigh resolution (see Figure 1; image reproduced with permission from Ref. 5 and the Creative Commons Attribution 4.0 International License).
Figure 1 – Identification of candidate response markers using ultrahigh-resolution MALDI MRMS. Spots that show highest feature of interest intensity were remeasured and fragmented using a solariX 7 T XR FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA) with R = 500,000. For this reason, a spot containing cells treated with 1 μM
imatinib was used for remeasurement and fragmentation of the feature m/z
616.1766 (a), and a spot containing DMSO-treated cells was used for fragmentation of the feature m/z
826.5722 (b).The future of drug discovery
The ongoing development of MALDI MS technology is driving newer drug discovery techniques, such as MALDI-based cellular assays. Institutes such as the Mannheim Technical University are furthering the field of drug discovery through collaborative partnerships with industry, as well as investing in powerful instrumentation. Many companies are now realizing the capabilities of MALDI MS, and are interested in collaborating with institutes with strong pharmaceutical backgrounds, such as CeMOS. Ongoing discussions in the field between peers in different companies and regular industry events organized by CeMOS help to foster the development of future drug discovery innovations.
For more information about Mannheim Technical University, please visit https://www.english.hs-mannheim.de/the-university.html.
For more information about Bruker’s MALDI mass spectrometry solutions, please visit https://www.bruker.com/products/mass-spectrometry-and-separations/maldi-toftof.html.
References
- De Cesare, V.; Johnson, C. et al. The MALDI-TOF E2/E3 ligase assay as universal tool for drug discovery in the ubiquitin pathway. Cell Chem. Biol. Sept 2018, 25(9), 1117–27.e4; doi: 10.1016/j.chembiol.2018.06.004.
- Winter, M.; Ries, R. et al. Automated MALDI target preparation concept: providing ultra-high-throughput mass spectrometry-based screening for drug discovery. SLAS Technol. Apr 2019, 24(2), 209–21; doi: 10.1177/2472630318791981.
- Beeman, K.; Baumgärtner, J. et al. Integration of an in situ MALDI-based high-throughput screening process: a case study with receptor tyrosine kinase c-MET. SLAS Discov. Dec 2017, 22(10), 1203–10; doi: 10.1177/2472555217727701.
- Haslam, C.; Hellicar, J. et al. The evolution of MALDI-TOF mass spectrometry toward ultra-high-throughput screening: 1536-well format and beyond. J. Biomolec. Screen. 2016, 21(2), 176–86.
- Weigt, D.; Sammour, D.A. et al. Automated analysis of lipid-drug response markers by combined fast and high-resolution whole cell MALDI mass spectrometry biotyping. Sci. Reports 2018, 8, 11260; doi:10.1038/s41598-018-29677-z.
Rohan Thakur, Ph.D., is executive vice president at Bruker Daltonics, 40 Manning Rd., Billerica, MA 01821, U.S.A.; e-mail: [email protected]; www.bruker.com. Dr. Carsten Hopf is professor of Drug Discovery in the Department of Biotechnology at the Mannheim Technical University, Germany.