Advances in high‐throughput mass spectrometry in drug discovery

EMBO Molecular Medicine, Journal Year: 2022, Volume and Issue: 15(1)

Published: Dec. 14, 2022

Review14 December 2022Open Access Advances in high-throughput mass spectrometry drug discovery Maria Emilia Dueñas Corresponding Author [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, review & editing Search more papers by this author Rachel E Peltier-Heap orcid.org/0000-0002-1665-8216 Discovery Analytical, Screening Profiling and Mechanistic Biology, GSK R&D, Stevenage, Melanie Leveridge Roland S Annan Supervision, Project administration, Frank H Büttner Drug Sciences, High Throughput Boehringer Ingelheim Pharma GmbH&CoKG, Biberach, Germany Matthias Trost orcid.org/0000-0002-5732-700X Information *,1,†, Peltier-Heap2,†, Leveridge2, Annan2, Büttner3 *,1 1Laboratory 2Discovery 3Drug † These authors contributed equally to work *Corresponding author. Tel: +44 191 2088983; E-mail: 2087009; EMBO Mol Med (2023)15:e14850https://doi.org/10.15252/emmm.202114850 See the Glossary abbreviations used article. PDFDownload PDF of article text main figures. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract High-throughput (HT) screening discovery, during which thousands or millions compounds are screened, remains key methodology identifying active chemical matter early pipelines. Recent technological developments (MS) automation have revolutionized application MS use HT screens. methods allow targeting unlabelled biomolecules assays, thereby expanding breadth targets assays can be developed compared traditional approaches. Moreover, these label-free often cheaper, faster, physiologically relevant than competing assay technologies. In review, we will describe current techniques explain their advantages disadvantages. We highlight power vitro its setting up multiplexed cellular phenotypic providing an exciting new tool cell lines, even primary cells. Finally, give outlook on how advances increase future capabilities discovery. BLAZE mode The name RapidFire hardware modification that improves speed system enabling cycling times 2.5 s per sample Chemoproteomics A broad set identify characterize action a drug. This include quantitative MS-based proteomics Data-independent acquisition recently global strategy first isolates precursor ions into pre-defined isolation windows, then fragmented analysed Fragment-based Method develop potent small-molecule starting from fragments binding weakly Limited proteolysis Used measure protein structural transitions directly biological matrices proteome-wide scale Mechanism Refers specific biochemical interaction through substance produces pharmacological effect PhAbit PhotoAffinity bits. reversible ligand with photoreactive warhead incorporated facilitate covalent Phosphoproteomics Proteomics analysis seeks determine overall level phosphorylation identity proteins, phosphorylated, amino acid residues, hold phosphate group Is proprietary automated microfluidic collection purification interfaces standard ESI-MS instruments. uses high-speed robotics aspirate fluidic samples 96- 384-well plates, rapidly removes non-volatile components such as salts, buffers detergents online fractionation step, delivers purified analytes spectrometer Size exclusion chromatography chromatographic separation technique separates size, and, therefore, relative molecular weight Thermal proteome profiling monitor melting profile proteins simultaneously Warhead reactive is strategically onto formation bond target biomolecule Introduction development pipeline interdisciplinary process engages multiple phases research generation effective therapies (Mohs Greig, 2017). historical aspects been extensively reviewed demonstrate challenges throughout R&D including productivity, attrition, evolution technologies (Moffat et al, 2017; Vincent 2022). phase contains identification validation phase, well hit finding, typically (HTS) campaigns employing large compound libraries several hundred compounds. At end chemistry performed optimize activity physicochemical properties molecule, both influence vivo behavior it relates potency, clearance, safety. Early adoption critical improving there lengthy cycle high failure rates projects prior pre-clinical development. There is, focus across industry academia biologically diverse approaches points, address success pace research. powerful, versatile applications spanning full spectrum pipeline. For example, proteomics, metabolomics clinical tissue important part validation, later where gain insight compound's mechanism (MoA). During lead optimization, has decades played central role determining structure pharmacokinetic also increasingly step limited proteolysis-coupled (Schopper 2017) routinely specificity uncover small molecule sites, thermal (Franken 2015) data-independent systems phosphoproteomics (Kitata 2021). Despite being powerful within process, lagged, due lack throughput associated automation. Current HTS using fluorescence chemiluminescence-based detection modalities although HT, susceptible compound-dependent artefacts leading false positives negatives (Winter 2018). Here, presents itself attractive alternative technology already established, sensitive, biomolecules. advantage potential build improve confirmation ultimately accelerate process. HTS-MS demonstrated removing detection-based thus mitigating sources interference (Adam 2015). From orthogonal hit-finding approaches, opportunity explore hit-identification strategies detecting protein-target binders, modulate function reverse treat disease phenotype. aim provide overview recent HT-MS outline advancements enabled platforms applications. could further drive Basic principles instrumentation analytical measures mass-to-charge ratio (m/z) abundance generate turn yield chemically information empirical about particular analyte. simplest form, consists ionization source coupled analyzer detector. ion transfers molecules gas charged transferred analyzer. separated based m/z detected, generating spectrum. As not only but number detected recorded, highly linear range ~105 (Collings 2014). HT-MS-based readouts largely dominated instruments comprising solid-phase extraction (SPE) electrospray (ESI), surface-based matrix-assisted laser/desorption (MALDI). Self-assembled monolayers (SAMs) desorption/ionization (SAMDI), some acoustic mist (AMI), droplet ejection (ADE) open port interface (OPI) added toolbox. described Fig 1 (surface-based, 1A electrospray-based 1B). Each combined different analyzers access levels resolution, dynamic ranges, time, throughput. detailed please see Challen Cramer (2022). Figure 1. Schematic employed HTS-MS(A) Surface-based: MALDI. Samples co-crystallized matrix conductive plate. Laser shots activate evaporate analyte matrix. cloud, protons ionize (Karas 1985). SAMDI. Components enzymatic reaction (either enzymes substrates) immobilized self-assembled array format, upon irradiation laser, desorbed surface cleavage thiolate-gold ionized (Gurard-Levin 2011). (B) Electrospray-based: ESI. dissolved liquid carrier voltage applied tip metal capillary spectrometer's sampling cone. electric field causes dispersion solution resulting nebulization. Charged droplets containing generated at exit tip. solvent vaporized drying heat guided gradient toward region (Fenn 1989; El-Aneed 2009). AMI. An transducer charging cone nanolitre-sized transfer line (Sinclair ADE-OPI. pulse energy ejects upward inverted OPI, fluid pump capture region. captured, diluted, conventional ESI (Zhang Download figure PowerPoint Biochemical functional inhibitors Once identified disease, function. enzymes, inhibition activation measured via product, decrease substrate, (Fig 2A). Unlike most allows direct, measurement substrate product long shift occurs; enzyme principally amenable spectrometric analysis. years, mobility integrated capable spectrometers, complex isobaric lipid classes (Djambazova 2020). likely broaden HTS-compatible challenging isomerases, years. 2. Types assays(A) Enzyme spectrometry. reactions substrates stopped appropriate time points mixture conversion. Addition affect reduced Affinity Selection Spectrometry. Compounds bind interest non-binding removed size-exclusion chromatography. Binding (C) Cellular phenotypes "healthy" "diseased" controls defined read-out "fingerprint" Chemical phenotype considered hits. ESI, BLAZE-mode (Bretschneider 2019) ADE-OPI approach enzymatic-type (Häbe 2020; Simon 2021a). versatility instrument setup many lipids (Highkin 2011; Dittakavi 2020), peptides (Hutchinson Liddle metabolites (Soulard 2008; Maxine 2009), wide blood, plasma Bretschneider lysates (Gordon 2016; Ambient ionization, desorption (DESI), commonly does require preparation, DESI-MS displays remarkably salt tolerance, making ideal without any preparation. Using DESI, outside under native conditions. Due ability scan surface, (Wleklinski 2018), approaching 10,000 hour, bioassay (Morato improvements preparation MALDI- time-of-flight (TOF) rival throughputs 10–20 second MALDI (Haslam atmospheric pressure (Krenkel 2022) reported. studies (1,536 spots less 8 min) new-generation MALDI-TOF spectrometers was compatible study drugability deubiquitylases (DUBs; Ritorto work, individual DUBs were incubated ubiquitin dimers linkage type quantitation mono-ubiquitin isotopically labelled internal determination DUB specificity, screening. unique substrates, rather previously rhodamine fluorescently reagents (Hassiepen 2007), had expanded platform. post-translational modifications grown past decade potentially involves change. importantly quantitation, gold respect simplicity cost. Successful now kinases (Beeman Heap 2017), methyltransferases (Guitot phosphatases Most MALDI-TOF-based conducted so far focused simple (with just single product) peptide/protein-centric (Ritorto 2014; Guitot De Cesare 2018; Winter Applying metabolomics-based challenge mostly (i) peaks low-mass range, (ii) matrix-dependent selectivity, (iii) metabolite coverage low sensitivity certain metabolites. Although, recently, trimethylamine 2019), acetylcholine (Chandler 2016), 3-methoxytyramine 2022), cyclic GMP-AMP (at ~60,000 day; 2020) campaigns, tools need meet opportunities metabolic SAMDI promising same targeted products Although generally label-free, needs tag immobilized, enables suited measuring activities SAMs customized variety immobilization chemistries (Mrksich, 2008). exemption statement traceless-SAMDI (Helal introduced truly analysing photogenerated carbene non-selectively attach SAMs, analyzed MS. recombinant enzyme/substrate screen classes, (Swalm Brooke 2013), glycosyltransferases (Ban 2012), deacetylases 2010). Selected publications describing found Table MS-compatible Substrate Product Platform Citation Phosphatidylserine decarboxylase Phosphatidylethanolamine Forbes al (2007) ERAP1 Peptide (2020) Acetyl-coenzyme carboxylase Sphingosine whole blood Sphingosine-1-phosphate (2009) Autotaxin Lysophosphatidyl choline Lysophosphatidic Soulard (2008) Histone lysine demethylase Trimethylated peptide Demethylated Hutchinson (2011) deacetylase Acetylated AMI-MS Sinclair (2019) acetyltransferase acetyl-CoA cofactor Belov Diacylglycerol acyltransferase 2 Diolein oleoyl-CoA triolein Wen (2021) Cyclic synthase GTP + ATP Deubiquitylases Diubiquitin Ubiquitin (2014) E3-ligases (2018) Kinases Phosphopeptide Beeman (2017) Methyltransferases Methylated (2017), Haslam (2015) Phosphatases Acetylcholinesterase Acetylcholine Choline Anthrax lethal factor Min (2004) Sirtuin 3 Patel Swalm (2013) Glycosyltransferases Saccharides Oligosaccharides Ban (2012) Deacetylases Gurard-Levin (2010) Isocitrate dehydrogenase α-ketoglutarate Radosevich (2022) Catechol-O-methyltransferase Dopamine selection (ASMS) cost-effective rapid against (Prudent ASMS 2B), biomolecular present molar excess ligands captured protein. Non-bound usually either affinity enrichment size (SEC). Bound dissociated accurate suitable technique. Alternatively, ligand-binding properties, proof performing competition experiments (Simon 2021b). emerged over two complimentary (Annis 2007). leverages direct capability SEC. particular, widely adopted scalability led fully systems, Automated Ligand Identification System 2004), SpeedScreen (Muckenschnabel 2004; Zehender Mayr, Typically, million pooling take 5–7 days follow-up ranging 1–3 weeks re-confirm depending employed. rationale behind must precede activity, binders surrogate reading out stages ID campaign. Advantageously, exhibit MoA, agonists antagonists screen. accommodate very little knowledge exists. By designing collections encoded libraries, broader space possible reduce downstream deconvolution redundancies. instrumental screens one achieved in-solution platforms. like beta-secretase (Coburn G-protein receptors (Whitehurst Annis, 2008), RNA polymerase (Walker CHK1 (Comess 2006) probe druggable NF-kβ pathway (Kutilek 2016). historically pools 100–2,000 high-resolution approach, suffer few challenges. concentrations micromolar needed good solubility 12–24 h critical, problematic membrane proteins. Furthermore, DMSO concentration, sensitivity, denature structure. More faster scanning speeds smaller ASMS. includes SEC platform proposed (2021b), SAMDI-TOF tens hundreds, yet still reach Covalent fragment (FBDD) aims novel drugs small, points. Sensitive technologies, plasmon resonance, nuclear magnetic MS, detect fragments. excellent example vemurafenib, selective inhibitor oncogenic B-RAF (Tsai Advantages FBDD experimental costs, developing harness chemistry. One aspect strategies. exploits made synthesis warheads efforts (Lu poorly cha

Language: Английский

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Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

Journal of Proteome Research, Journal Year: 2023, Volume and Issue: 22(8), P. 2629 - 2640

Published: July 13, 2023

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due engagement requires high quantitative accuracy consistent detection. Isobaric tandem mass tags (TMTs) are used multiplex samples increase quantification precision TPP analysis by data-dependent acquisition (DDA). However, advances data-independent (DIA) can provide higher sensitivity protein coverage with reduced costs sample preparation steps. Herein, we explored the performance different DIA-based label-free approaches compared TMT-DDA for shift quantitation. Acute myeloid leukemia cells were treated losmapimod, known inhibitor MAPK14 (p38α). Label-free DIA approaches, particularly library-free mode DIA-NN, comparable ability detect losmapimod one its downstream targets, MAPKAPK3. Using quantitation is cost-effective alternative labeled pipeline.

Language: Английский

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Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(5), P. 1898 - 1905

Published: Jan. 27, 2024

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry SARS-CoV-2. Both and ACE2 are highly glycosylated, which can regulate binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling glycosylation effects on binding between RBD ACE2. Overall, it was found that affects more strongly than does glycosylation. affinity improved after desialylation or partial deglycosylation (N690) ACE2, while decreased degalactosylation. form dimers in solution, bind tightly monomers. dimerization dimeric also be by Partial increased a factor 2, suggesting its high potential neutralizing method described work provided simple way analyze protein–protein interaction without sample purification. It widely used rapid glycosylation-related diseases study multiple interactions aggregates single system.

Language: Английский

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Proteomic Analysis Reveals Trilaciclib-Induced Senescence

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: 23(6), P. 100778 - 100778

Published: April 26, 2024

Trilaciclib, a CDK4/6 inhibitor, was approved as myeloprotective agent for protecting bone marrow from chemotherapy-induced damage in extensive-stage small cell lung cancer (ES-SCLC). This is achieved through the induction of temporary halt cycle cells. While it has been studied various types, its potential haematological cancers remains unexplored. research aimed to investigate efficacy trilaciclib cancers. Utilizing mass spectrometry-based proteomics, we examined alterations induced by chronic myeloid leukaemia (CML) line, K562. Interestingly, promoted senescence these cells rather than death, observed acute (AML), lymphoblastic (ALL), and myeloma In K562 cells, hindered progression proliferation stabilising downregulating cycle-related proteins, along with concomitant activation autophagy pathways. Additionally, trilaciclib-induced also non-small carcinoma line (NSCLC), A549. These findings highlight trilaciclib's therapeutic option underscore need carefully balance modulation CML treatment, well NSCLC.

Language: Английский

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Proteomic Analysis Reveals Trilaciclib-Induced Senescence

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 14, 2024

ABSTRACT Trilaciclib, a CDK4/6 inhibitor, was approved as myeloprotective agent for protecting bone marrow from chemotherapy-induced damage in extensive-stage small cell lung cancer (ES-SCLC). This is achieved through the induction of temporary halt cycle cells. While it has been studied various types, its potential haematological cancers remains unexplored. research aimed to investigate efficacy trilaciclib cancers. Utilizing mass spectrometry-based proteomics, we examined alterations induced by chronic myeloid leukaemia (CML) line, K562. Interestingly, promoted senescence these cells rather than death, observed acute (AML), lymphoblastic (ALL), and myeloma In K562 cells, hindered progression proliferation stabilising downregulating cycle-related proteins, along with concomitant activation autophagy pathways. Additionally, trilaciclib-induced also non-small carcinoma line (NSCLC), A549. These findings highlight trilaciclib’s therapeutic option underscore need carefully balance modulation CML treatment, well NSCLC. GRAPHIC

Language: Английский

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NUDCD3 deficiency disrupts V(D)J recombination to cause SCID and Omenn syndrome

Science Immunology, Journal Year: 2024, Volume and Issue: 9(95)

Published: May 24, 2024

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for single deleterious missense variant the gene NudC domain–containing 3 ( NUDCD3 ) . Two infants had severe combined immunodeficiency with complete absence and B cells (T - SCID), whereas nine showed classical features Omenn syndrome (OS). Restricted antigen receptor usage residual lymphocytes suggested impaired V(D)J recombination. Patient reduced expression protein diminished ability to support RAG-mediated recombination vitro, was associated pathologic sequestration RAG1 nucleoli. Although mouse model bearing homologous led milder immunologic abnormalities, absolutely required healthy humans.

Language: Английский

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A Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Feb. 15, 2023

Abstract Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due engagement requires high quantitative accuracy consistent detection. Isobaric tandem mass tags (TMT) are used multiplex samples increase quantification precision TPP analysis by data-dependent acquisition (DDA). However, advances data-independent (DIA) can provide higher sensitivity protein coverage with reduced costs sample preparation steps. Herein, we explored the performance different DIA-based label-free (LFQ) approaches compared TMT-DDA for shift quantitation. Acute myeloid leukaemia (AML) cells were treated losmapimod, known inhibitor MAPK14 (p38α). Label-free DIA approaches, particularly library-free mode DIA-NN, comparable or better than ability reproducibly detect losmapimod one its downstream targets, MAPKAPK3. Using quantitation is cost-effective alternative labelled pipeline.

Language: Английский

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A Concise Review on Analytical Methods for Determination of Nilotinib

Current Analytical Chemistry, Journal Year: 2023, Volume and Issue: 19(7), P. 513 - 530

Published: Aug. 1, 2023

Abstract: Nilotinib hydrochloride is a tyrosine kinase inhibitor licensed to treat chronic myelogenous leukemia in patients with the Philadelphia Chromosome (Ph+). Researchers at Novartis Pharmaceuticals discovered novel inhibitors that are effective against imatinib-resistant BCR-ABL mutations. As consequence, was discovered. Several analytical approaches were employed address quantitative as well qualitative assessment of from diverse biological and pharmaceutical matrices during development Nilotinib. The literature search conducted by evaluating publications reporting on nilotinib methodologies 2006 2022. This review briefly summarizes drug profile, viz. stereochemistry, mechanism action, resistance, pharmacokinetics, pharmacodynamics, side effects, several techniques used assess dosage form, bulk, fluids. determination using methods important for therapeutic monitoring, optimizing dosage, ensuring safety efficacy, conducting comparative studies. A variety gathered examined, including spectroscopy, electrophoresis, voltammetry, Raman differential scanning calorimetry, X-ray diffraction, chromatography, hybrid techniques. They also useful studying pharmacokinetics drug. These play crucial role personalized treatment myeloid other conditions where used.

Language: Английский

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