Advances in high‐throughput mass spectrometry in drug discovery
2022; Springer Nature; Volume: 15; Issue: 1 Linguagem: Inglês
10.15252/emmm.202114850
ISSN1757-4684
AutoresMaría Emilia Dueñas, Rachel E. Heap, Melanie Leveridge, Roland S. Annan, Frank Büttner, Matthias Trost,
Tópico(s)Analytical Chemistry and Chromatography
ResumoReview14 December 2022Open Access Advances in high-throughput mass spectrometry in drug discovery Maria Emilia Dueñas Corresponding Author Maria Emilia Dueñas [email protected] orcid.org/0000-0003-3411-4068 Laboratory for Biomedical Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Rachel E Peltier-Heap Rachel E Peltier-Heap orcid.org/0000-0002-1665-8216 Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Melanie Leveridge Melanie Leveridge Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Writing - original draft, Writing - review & editing Search for more papers by this author Roland S Annan Roland S Annan Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Frank H Büttner Frank H Büttner Drug Discovery Sciences, High Throughput Biology, Boehringer Ingelheim Pharma GmbH&CoKG, Biberach, Germany Contribution: Conceptualization, Writing - original draft, Writing - review & editing Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X Laboratory for Biomedical Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Maria Emilia Dueñas Corresponding Author Maria Emilia Dueñas [email protected] orcid.org/0000-0003-3411-4068 Laboratory for Biomedical Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Rachel E Peltier-Heap Rachel E Peltier-Heap orcid.org/0000-0002-1665-8216 Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Melanie Leveridge Melanie Leveridge Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Writing - original draft, Writing - review & editing Search for more papers by this author Roland S Annan Roland S Annan Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Frank H Büttner Frank H Büttner Drug Discovery Sciences, High Throughput Biology, Boehringer Ingelheim Pharma GmbH&CoKG, Biberach, Germany Contribution: Conceptualization, Writing - original draft, Writing - review & editing Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X Laboratory for Biomedical Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Maria Emilia Dueñas *,1,†, Rachel E Peltier-Heap2,†, Melanie Leveridge2, Roland S Annan2, Frank H Büttner3 and Matthias Trost *,1 1Laboratory for Biomedical Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK 2Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, UK 3Drug Discovery Sciences, High Throughput Biology, Boehringer Ingelheim Pharma GmbH&CoKG, Biberach, Germany † These authors contributed equally to this work *Corresponding author. Tel: +44 191 2088983; E-mail: [email protected] *Corresponding author. Tel: +44 191 2087009; E-mail: [email protected] EMBO Mol Med (2023)15:e14850https://doi.org/10.15252/emmm.202114850 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Glossary BLAZE mode The name of the RapidFire hardware modification that improves the speed of the system by enabling cycling times of 2.5 s per sample Chemoproteomics A broad set of techniques used to identify and characterize the mode of action of a drug. This can include quantitative MS-based proteomics Data-independent acquisition MS A recently developed global MS-based proteomics strategy that first isolates precursor ions into pre-defined isolation windows, which are then fragmented and analysed Fragment-based drug discovery Method used to develop potent small-molecule compounds starting from fragments binding weakly to targets Limited proteolysis MS Used to measure protein structural transitions directly in biological matrices and on a proteome-wide scale Mechanism of action Refers to the specific biochemical interaction through which a drug substance produces its pharmacological effect PhAbit PhotoAffinity bits. A reversible ligand with a photoreactive warhead incorporated to facilitate covalent binding Phosphoproteomics Proteomics analysis that seeks to determine the overall level of protein phosphorylation and the identity of proteins, which are phosphorylated, and amino acid residues, which hold the phosphate group RapidFire Is a proprietary automated microfluidic sample collection and purification system that interfaces directly to standard ESI-MS instruments. This system uses high-speed robotics to directly aspirate fluidic samples from 96- or 384-well screening plates, rapidly removes non-volatile assay components such as salts, buffers and detergents in an online fractionation step, and delivers purified analytes to the mass spectrometer Size exclusion chromatography A chromatographic separation technique that separates analytes by size, and, therefore, relative to molecular weight Thermal proteome profiling A quantitative MS-based proteomics tool used to monitor the melting profile of thousands of proteins simultaneously Warhead A reactive group that is strategically incorporated onto a reversible ligand to facilitate the formation of a covalent bond to a target biomolecule Introduction The drug discovery and development pipeline is an interdisciplinary process that engages multiple phases of research to facilitate the generation of effective therapies (Mohs & Greig, 2017). The historical aspects of the traditional drug discovery pipeline have been extensively reviewed and demonstrate the advantages and challenges of drug discovery throughout R&D including productivity, attrition, and evolution of new technologies (Moffat et al, 2017; Vincent et al, 2022). The drug discovery phase contains the target identification and validation phase, as well as hit finding, typically through high-throughput screening (HTS) campaigns employing large compound libraries of several hundred thousands of compounds. At the end of this phase, chemistry is performed to optimize the activity and physicochemical properties of the molecule, both of which influence its in vivo behavior as it relates to potency, clearance, and safety. Early adoption of new technologies can be critical to improving R&D as there are often lengthy cycle times and high failure rates of drug discovery projects prior to pre-clinical development. There is, therefore, a focus across industry and academia on the development of more biologically relevant and diverse approaches to the discovery of chemical starting points, to address both the success rates and pace of research. Mass spectrometry (MS) is a powerful, versatile technique with applications spanning the full spectrum of the drug discovery and development pipeline. For example, MS techniques such as proteomics, metabolomics and analysis of clinical tissue samples are an important part of target validation, as well as later in discovery where these techniques can be used to gain insight into a compound's cellular mechanism of action (MoA). During lead optimization, MS has for decades played the central role in determining both the structure and pharmacokinetic properties of compounds. MS is also increasingly important in the target identification step of the drug discovery pipeline. For example, limited proteolysis-coupled MS (Schopper et al, 2017) is routinely used to determine proteome-wide specificity and uncover small molecule binding sites, thermal proteome profiling (Franken et al, 2015) for small molecule target finding, and data-independent acquisition MS for HT analysis of cell systems for global proteomics and phosphoproteomics (Kitata et al, 2021). Despite MS being a powerful tool within the overall drug discovery process, its application to HT screening has lagged, often due to a lack of throughput and lack of associated automation. Current HTS assays are often performed using fluorescence and chemiluminescence-based detection modalities that although HT, are susceptible to compound-dependent screening artefacts leading to false positives or negatives (Winter et al, 2018). Here, MS presents itself as an attractive alternative technology as it is already an established, sensitive, and versatile technique in research for the analysis of small and large biomolecules. A key advantage of MS has been the potential to build label-free assays that improve hit confirmation rates and ultimately accelerate the drug discovery process. HTS-MS has been demonstrated to be an effective tool for removing potential detection-based false positives and thus mitigating sources of assay interference (Adam et al, 2015). From orthogonal to traditional hit-finding approaches, MS presents the opportunity to explore alternative hit-identification strategies that focus on detecting protein-target binders, or compounds that directly modulate cellular function to reverse or treat a disease phenotype. The aim of this review is to provide an overview of the recent developments in HT-MS for drug discovery. We outline how these advancements in MS have enabled the development of HTS-MS platforms and their applications. Finally, we provide an outlook of how technological advances could further drive alternative capabilities of MS in drug discovery. Basic principles of mass spectrometry instrumentation MS is an analytical technique that measures both the mass-to-charge ratio (m/z) and abundance of ions to generate a mass spectrum that can in turn yield chemically relevant information such as empirical mass or structure about a particular analyte. In its simplest form, a mass spectrometer consists of an ionization source coupled to a mass analyzer and detector. The ion source transfers sample molecules into the gas phase as charged ions which then are transferred into a mass analyzer. Here, ions are separated based on their m/z and detected, thus generating a mass spectrum. As not only the m/z but also the number of detected ions is recorded, MS can be a highly quantitative technique with a linear range of up to ~105 (Collings et al, 2014). HT-MS-based readouts in drug discovery have been largely dominated by instruments comprising of solid-phase extraction (SPE) coupled to electrospray ionization (ESI), or surface-based techniques such as matrix-assisted laser/desorption ionization (MALDI). Self-assembled monolayers (SAMs) coupled with desorption/ionization (SAMDI), as well as some more recent approaches such as acoustic mist ionization (AMI), and acoustic droplet ejection (ADE) open port interface (OPI) MS have been added to the toolbox. These principles are described in Fig 1 (surface-based, Fig 1A and electrospray-based Fig 1B). Each of these ionization techniques can be combined with different mass analyzers to access different levels of mass resolution, dynamic ranges, analysis time, and sample throughput. For a detailed review, please see Challen and Cramer (2022). 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, 1989; 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). Download figure Download PowerPoint Mass spectrometry screening assays for drug discovery Biochemical and functional assays to identify inhibitors of enzymes Once target proteins have been identified as a potential drug target in a specific disease, biochemical in vitro assays are often performed to identify molecules that modulate protein function. For protein targets that are enzymes, target inhibition or activation can be measured via the generation of a product, or the decrease of a substrate, in a biochemical reaction (Fig 2A). Unlike most traditional biochemical assays, MS allows the direct, label-free quantitative measurement of both substrate and product in these in vitro assays, as long as a mass shift occurs; therefore, most enzyme targets are principally amenable for mass spectrometric analysis. In recent years, ion mobility separation has been integrated within new HTS capable mass spectrometers, thus enabling the separation of complex and isobaric compounds such as lipid classes (Djambazova et al, 2020). This will likely broaden the development of HTS-compatible MS assays for challenging enzymes, such as isomerases, in future years. 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. Download figure Download PowerPoint For ESI, different technologies such as the RapidFire system in BLAZE-mode (Bretschneider et al, 2019) or ADE-OPI MS approach have been described for enzymatic-type assays (Häbe et al, 2020; Simon et al, 2021a). The versatility of the instrument setup allows the analysis of many different biomolecules including lipids (Highkin et al, 2011; Dittakavi et al, 2020), peptides (Hutchinson et al, 2011; Liddle et al, 2020), and metabolites (Soulard et al, 2008; Maxine et al, 2009), from a wide range of matrix systems including blood, plasma (Highkin et al, 2011; Bretschneider et al, 2017) and cell lysates (Gordon et al, 2016; Dittakavi et al, 2020). Ambient ionization, such as desorption electrospray ionization (DESI), which commonly does not require sample preparation, is a new and attractive alternative for HT analysis. DESI-MS displays remarkably high salt tolerance, making this technique ideal for the analysis of complex samples without any sample preparation. Using DESI, samples are ionized outside of a mass spectrometer under native conditions. Due to its ability to rapidly scan a surface, DESI-MS has been amenable to HT applications (Wleklinski et al, 2018), at rates approaching 10,000 reactions per hour, and for the analysis of enzymatic reactions directly from the bioassay matrix (Morato et al, 2020). The development of instrumentation and improvements in sample preparation have enabled MALDI- time-of-flight (TOF) MS to rival the more conventional HTS assays with throughputs of 10–20 samples per second for conventional MALDI (Haslam et al, 2015) and liquid atmospheric pressure MALDI (Krenkel et al, 2022) reported. The first drug discovery studies using the high speed (1,536 spots in less than 8 min) of these new-generation MALDI-TOF mass spectrometers for drug discovery was the development of a HTS compatible assay to study the specificity and drugability of deubiquitylases (DUBs; Ritorto et al, 2014). In this work, individual DUBs were incubated with ubiquitin dimers of different linkage type and the quantitation of mono-ubiquitin using an isotopically labelled internal standard enabled the determination of DUB specificity, and this was further applied to drug screening. This assay was unique to the field as it used native substrates, rather than the previously used rhodamine fluorescently labelled reagents (Hassiepen et al, 2007), and also had the potential to be expanded to a HT drug screening platform. HT MALDI-TOF MS assays targeting post-translational modifications have grown rapidly in the past decade as the technique can be applied to potentially any reaction that involves a mass change. This importantly allows label-free quantitation, a gold standard for assays in the drug discovery field with respect to simplicity and cost. Successful MALDI-TOF MS assays now include the study of kinases (Beeman et al, 2017; Heap et al, 2017), methyltransferases (Guitot et al, 2017), and phosphatases (Winter et al, 2018). Most of the MALDI-TOF-based HTS-compatible approaches conducted so far have focused on in vitro assays with simple readouts (with often just a single substrate and product) and have been limited to peptide/protein-centric activity assays (Ritorto et al, 2014; Guitot et al, 2017; Heap et al, 2017; De Cesare et al, 2018; Winter et al, 2018; Simon et al, 2020). Applying this technology for cellular assays and metabolomics-based drug discovery remains a challenge mostly due to (i) interference from matrix peaks in the low-mass range, (ii) matrix-dependent analyte selectivity, and (iii) limited metabolite coverage due to low sensitivity of certain classes of metabolites. Although, recently, individual metabolites such as trimethylamine (Winter et al, 2019), acetylcholine (Chandler et al, 2016), 3-methoxytyramine (Winter et al, 2022), and cyclic GMP-AMP (at a throughput of ~60,000 samples per day; Simon et al, 2020) have been used in MALDI-TOF HTS campaigns, new tools and methods need to be developed to meet the opportunities and challenges toward HT metabolic profiling for drug discovery. The SAMDI technology is a promising strategy for HTS that uses the same MALDI-TOF MS instrumentation but in a more targeted approach where immobilized proteins are used to capture substrates or products (Gurard-Levin et al, 2011). Although generally not label-free, as the protein needs to have a tag to be immobilized, this technology enables the specific capture of analytes and is well suited for measuring a broad range of enzyme activities as SAMs can be customized to use a variety of immobilization chemistries (Mrksich, 2008). An exemption to this statement is traceless-SAMDI (Helal et al, 2018). This work introduced a truly label-free approach for analysing HT reactions by using a photogenerated carbene to non-selectively attach molecules to the SAMs, from which can then be analyzed by MS. SAMDI has also been used for in vitro recombinant enzyme/substrate screen on diverse enzyme classes, such as methyltransferases (Swalm Brooke et al, 2013), glycosyltransferases (Ban et al, 2012), and deacetylases (Gurard-Levin et al, 2010). Selected publications describing HTS compatible MS assays can be found in Table 1. Table 1. Selected publications describing HTS MS-compatible assays in drug discovery. Enzyme Substrate Product Platform Citation Phosphatidylserine decarboxylase Phosphatidylserine Phosphatidylethanolamine RapidFire Forbes et al (2007) ERAP1 Peptide Peptide RapidFire Liddle et al (2020) Acetyl-coenzyme A carboxylase Sphingosine in whole blood Sphingosine-1-phosphate RapidFire Maxine et al (2009) Autotaxin Lysophosphatidyl choline Lysophosphatidic acid RapidFire Soulard et al (2008) Histone lysine demethylase Trimethylated peptide Demethylated peptide RapidFire Hutchinson et al (2011) Histone deacetylase Acetylated peptide Peptide AMI-MS Sinclair et al (2019) Histone acetyltransferase Peptide and acetyl-CoA cofactor Acetylated peptide AMI-MS Belov et al (2020) Diacylglycerol acyltransferase 2 Diolein and oleoyl-CoA triolein ADE-OPI MS Wen et al (2021) Cyclic GMP-AMP synthase GTP + ATP Cyclic GMP-AMP ADE-OPI MS Simon et al (2020) Deubiquitylases Diubiquitin Ubiquitin MALDI-TOF Ritorto et al (2014) E3-ligases Diubiquitin Ubiquitin MALDI-TOF De Cesare et al (2018) and De Cesare et al (2020) Kinases Peptide Phosphopeptide MALDI-TOF Beeman et al (2017) and Heap et al (2017) Methyltransferases Peptide Methylated peptide MALDI-TOF Guitot et al (2017), Guitot et al (2014) and Haslam et al (2015) Phosphatases Phosphopeptide Peptide MALDI-TOF Winter et al (2018) Acetylcholinesterase Acetylcholine Choline MALDI-TOF Haslam et al (2015) Cyclic GMP-AMP synthase GTP + ATP Cyclic GMP-AMP MALDI-TOF Simon et al (2020) Anthrax lethal factor Peptide Peptide SAMDI Min et al (2004) Sirtuin 3 Acetylated peptide Peptide SAMDI Patel et al (2015) Methyltransferases Peptide Methylated peptide SAMDI Swalm Brooke et al (2013) Glycosyltransferases Saccharides Oligosaccharides SAMDI Ban et al (2012) Deacetylases Acetylated peptide Peptide SAMDI Gurard-Levin et al (2010) Isocitrate dehydrogenase 1 Isocitrate α-ketoglutarate MALDI + ESI Radosevich et al (2022) Catechol-O-methyltransferase Dopamine 3-methoxytyramine MALDI-TOF Winter et al (2022) Affinity and binding assays Affinity selection mass spectrometry (ASMS) is a HT and cost-effective binding assay that enables rapid screening of a large number of compounds against a specific target biomolecule of interest (Prudent et al, 2021). In a traditional HT ASMS approach (Fig 2B), the biomolecular target is typically present in molar excess relative to the potential ligands that are then captured by the protein. Non-bound ligands are separated from the protein using usually either an affinity enrichment or size exclusion chromatography (SEC). Bound ligands are then dissociated from the target protein and identified by their accurate mass with a suitable MS technique. Alternatively, ASMS can also be employed as an assay to further characterize ligand-binding properties, such as to demonstrate proof of binding as well as performing competition experiments (Simon et al, 2021b). ASMS has emerged over the past two decades as a strategy complimentary to functional HTS assays (Annis et al, 2007). This approach leverages the label-free and direct detection capability of MS and is most often coupled to SEC. In particular, it has been widely adopted in industry due to its scalability and led to the development of fully automated systems, such as the Automated Ligand Identification System (Annis et al, 2004), as well as the SpeedScreen system (Muckenschnabel et al, 2004; Zehender et al, 2004; Zehender & Mayr, 2007). Typically, a 1 million compound screen with a pooling strategy can take 5–7 days with follow-up experiments ranging 1–3 weeks to re-confirm and characterize compound binding depending on the strategy employed. The rationale behind the affinity selection approach is that binding must precede activity, therefore, the identification of small molecule binders can be a surrogate to reading out activity in a traditional HT biochemical assay during the first stages of a hit ID campaign. Advantageously, this can identify ligands that exhibit multiple MoA, potentially identifying agonists and antagonists in a single screen. An ASMS HTS can often be less complex to develop than a traditional biochemical HTS and can accommodate targets where very little knowledge of protein function or structure exists. By designing ASMS specific collections or mass encoded libraries, a broader screening of chemical space could be possible to reduce complex downstream deconvolution and redundancies. (Prudent et al, 2021). MS has been instrumental in the development of ASMS strategies and HT screens of more than one million compounds have been achieved across in-solution ASMS platforms. These include a diverse range of targets like beta-secretase (Coburn et al, 2004), G-protein coupled receptors (Whitehurst & Annis, 2008), RNA polymerase (Walker et al, 2017), CHK1 (Comess et al, 2006) and to probe druggable target space within the NF-kβ pathway (Kutilek et al, 2016). These screens have historically been performed using pools of 100–2,000 compounds and analysis on high-resolution MS instruments. This approach, although HT, does suffer a few analytical challenges. Typically, protein concentrations in the micromolar range are needed and good protein solubility over 12–24 h is critical, which can be problematic for some targets like membrane proteins. Furthermore, the use of large pools of compounds can increase overall DMSO concentration, reduce assay sensitivity, and could also denature the target protein structure. More recently, HTS capable MALDI-TOF MS platforms that use faster instrument scanning speeds have been used to screen smaller pools of compounds by ASMS. This includes the SEC MALDI-TOF MS platform proposed by Simon et al (2021b), as well as a SAMDI-TOF MS approach, both of which use pools of tens of compounds rather than hundreds, yet can still reach the same sample throughput. Covalent fragment assays in drug discovery Fragment-based drug discovery (FBDD) is an established, versatile strategy in drug discovery that aims to develop novel drugs from small, low molecular weight starting points. Sensitive technologies, including surface plasmon resonance, nuclear magnetic resonance, and MS, have been used to detect the binding or activity of these fragments. An excellent example of this approach is the discovery of vemurafenib, a selective inhibitor of the oncogenic target B-RAF (Tsai et al, 2008). Advantages of FBDD often include reduced experimental costs, as well as novel strategies to developing new drugs that harness advances in HT chemistry. One aspect of FBDD where MS technology has been instrumental is the development of reactive or covalent fragment screening strategies. This approach exploits the advances made in synthesis of small molecule libraries that can then be coupled to covalent warheads to accelerate screening efforts (Lu et al, 2021). Using small covalent fragments to probe biological systems and poorly cha
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