Target discovery and Validation - Role of proteomics Shivanshu Bajaj 2.7k views 30 slides Tools for target identification and validation Dr. sreeremya S 1.6k views 13 slides Role of genomics proteomics, and bioinformatics. High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: interaction with IQ motif-containing factors. In the pharmaceutical industry, proteomics has long been utilized as a drug-discovery tool to help understand changes in protein profiles for disease states or protein expression in relation to genomic studies for target discovery or identification [ 1 ]. In embarking on assembling this review, we dissected the literature and interviewed colleagues for where they see this field evolving and having an influence in biotechnology and pharmaceutical research. The emerging role of RNA as a therapeutic target for small molecules. This paper reports how chemoproteomics enabled the discovery that thalidomide binds to an E3 ligase complex. Ligand and target discovery by fragment-based screening in human cells. This is exemplified by a recently introduced method called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ) which combines isobaric labels and synthetic peptides to enable sample and peptide multiplexing within a sensitive targeted assay [Citation19]. Data from Phase 3 clinical trials is also critical for reverse translation, understanding not only what pathways and disease pathologies are impacted by the successful drug, but also which pathways and pathologies remain unchanged thus providing potential targets for future drug discovery. In parallel to label-free detection methods for low level proteomic analysis, a method called Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) has gained significant momentum. At present, proteomics is used pre-clinically for target identification and characterization, drug candidate selection and characterization, and clinically for biomarker discovery and development. Methods 18, 8491 (2021). Biol. Mol. Annu. 251, 100112 (2020). A class of biocompatible reactions commonly used to join small, modular molecule units. Capturing the spatial proteome that is, the localizations of proteins and their dynamics at the subcellular level is therefore essential for a complete understanding of cell biology. Mol. Specht, H. & Slavov, N. Transformative opportunities for single-cell proteomics. An alternative strategy is to analyze candidate transcript expression within databases specialized in normal tissue expression, such as the Genotype-Tissue Expression (GTEX) project. A mammalian protein targeted by G1-arresting rapamycinreceptor complex. Rikova, K. et al. 120, 1432 (2011). These proteins can be further selectively conjugated to affinity reagents, nanoparticles or fluorophores, for a variety of biochemical or proteomic applications [Citation201]. The power of proteomics technology will lead to new clinical markers of disease, new protein therapeutics, and new drug targets. On the other side, increasing throughput will enable screening applications to proactively generate protein interaction profiles for compounds in screening libraries. Mol. Nat. Biol. Biophysics in drug discovery: impact, challenges and opportunities. The promise and peril of chemical probes. Future Med. In addition, these approaches offer an experimental framework to demonstrate target engagement in cells, model organisms and ultimately the patient in this case often using a more targeted detection and quantitation of the protein of interest to increase sensitivity and throughput. Covering an even larger target spectrum are probes that target solvent exposed reactive amino acids in general, e.g. Therefore, TPD drug discovery projects rely heavily on proteomics for target identification and compound characterization and optimization. Proteomics has evolved to address increasingly complex biological questions, unravel new intracellular signaling pathways leading to new therapeutic targets and has helped decipher key pathway modulators and biomarkers [Citation1]. 10, eaau5516 (2018). Wildsmith et al. Drug Discov. Quantitative label label-free analysis employed MaxQuant. Google Scholar. These therapeutically relevant dark matter antigens are of interest for both cancer vaccine and T cell therapy approaches, where common, tumor specific antigens represent ideal targets. 10, 305312 (2014). For more information, or to register for this event, visit The Role of Protein Analytics in the Advancement of Biotherapeutic Drug Discovery & Development.. ABOUT XTALKS. Nat. Nat. Challenges remain for nanopore sequencing, particularly the discrimination of post-translationally modified amino acid residues. Am. In addition, an estimate of the level of validation needed to support the biomarker is indicated, ranging from low to high where low refers to biomarkers used for internal decision making, medium refers to biomarkers that are submitted to regulatory agencies to support the filing, and high refers to biomarkers that impact diagnostics and companion diagnostics. Proc. Specificity of protein covalent modification by the electrophilic proteasome inhibitor carfilzomib in human cells. Accurate quantitation tools have come a long way in the past decade, moving from binary SILAC experiments to 16-plex TMT and beyond. At the forefront of this movement is MassIVE.quant, a community resource of quantitative mass spectrometry-based proteomics datasets [Citation64]. Nat. Parker, C. G. et al. This shift has occurred not only in target discovery but also in many other areas of the process, including patient treatment and care [6]. 27, 34513453 (2019). eLife 7, e38430 (2018). DIA-MS quantified a total of 734 plasma proteins, 379 of which were observed in more than 25% of the samples while Olink detected 728 proteins in at least 25% of the samples. Chemoproteomics. Biochemistry 46, 350358 (2007). Nature 473, 337342 (2011). LC-MS/MS was performed in the DIA and DDA modes using a Q Exactive HF instrument (Thermo) and affinity proteomics used the Olink PEA platform to measure the relative abundance of 736 protein analytes. Nat. Metabolites and short chain fatty acids for example, represent the downstream products of multiple interactions between various genes, transcripts, and proteins. 404, 1521 (2007). Describes the interdependency between compound structures and protein binding affinities. The discovery of biomarker candidates, analytical validation, and biomarker validation depends on the ultimate intended use of the biomarker and required a more nuanced approach than the triangular paradigm associated with diagnostic discovery. This association makes functional sense because GNS is a Ca2+-activated actin-severing protein and citrate is a calcium chelator. 11, 1124.e4 (2020). This is followed by 3) separation of compound-interacting proteins from the rest of the proteome by a variety of means including affinity enrichment or detection of changes in protein stability upon compound binding. Validation of MRM assays are well established and guidance documents are available [Citation174176]. Opin. Nature 567, 257261 (2019). The schematic on the right outlines the steps involved in the drug discovery process. Zhao, Q. et al. Meissner, F., Geddes-McAlister, J., Mann, M. et al. 9, 36883700 (2010). A biomarker used to identify individuals who are more likely than similar individuals without the biomarker to experience a favorable or unfavorable effect from exposure to a medical product or an environmental agent. Mellacheruvu, D. et al. Ghaemmaghami, S., Fitzgerald, M. C. & Oas, T. G. A quantitative, high-throughput screen for protein stability. Chem. The mechanism of photoaffinity labeling. Google Scholar. All of the methods tested performed well, identifying between 34003800 plasma proteins. Nat. This simplified MS workflow was successfully used to validate protein biomarkers for diagnosis of colorectal cancer [Citation180] and has the potential to significantly improve the discovery to validation gap. The arginine carrier ensured the peptide spent a sufficient amount of time in the pore and enabled sequencing of 13 of 20 proteinogenic amino acids. PubMed Contact Dermat. Soc. Murale, D. P., Hong, S. C., Haque, M. M. & Lee, J.-S. Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs). Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. & Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has been collecting proteomics data on tumor and normal adjacent tissue (NAT) for many years [Citation60,Citation61] and recently an application programming interface (API) was released to facilitate programmatic access to the data [Citation62]. Biol. AI-driven Deep Visual Proteomics defines cell identity and heterogeneity. Mund, A. et al. Like many scientific fields, proteomics is currently undergoing a machine learning revolution. Science 358, eaan4368 (2017). And imagine if one could rapidly analyze all of the proteoforms from just tens of cells from a xenograft model, or a few microliters of biofluid in a high throughput manner? Therefore, approaches that enable live cell applications are increasingly gaining popularity. The promise of multi-omics workflows to decipher intricate cellular signaling mechanisms at a cellular level has held great promise, however it is only now that we see the true union of genomic sequencing technologies with proteomics, metabolomics and other cellular readouts as analytical tools become more sensitive, and software analysis enables integration of these data sets in a meaningful way. Biol. Phosphoproteomics reveals that Parkinsons disease kinase LRRK2 regulates a subset of Rab GTPases. Nat. Youn, J. Y. et al. Krastel, P. et al. 47, 513539 (2007). Roscovitine targets, protein kinases and pyridoxal kinase. 18, 40274037 (2019). Nature 545, 505509 (2017). 46, D645D648 (2018). Huttlin, E. L. et al. Bioorg. Urine [Citation159] and stool [Citation160] in particular have the additional advantage of being noninvasive, simple to collect, store and transport. Go, C. D. et al. Nat. A machine learning-based chemoproteomic approach to identify drug targets and binding sites in complex proteomes. Lundby, A. et al. This article redefines the chemogenomics target space by covalent ligands. The resulting throughput challenges have led to the introduction of compressed workflows where individual treatment conditions, e.g. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. The proteogenomic landscape of curable prostate cancer. Photoaffinity-labeling (PAL) allows the interrogation of compound-protein interactions in living cells since a typical PAL probe consists of three elements: the pharmacophore responsible for target binding, a functional group for installing an affinity handle and a photoreactive moiety (e.g., diazirine, benzophenone) that allows proximity-based covalent labeling of the interacting protein(s) upon cell irradiation. Choudhary, C. et al. This article highlights the current status of the proteomics field, and how it supports drug discovery and development. Nature Reviews Drug Discovery Sinha, A. et al. Cell Syst. Becher, I. et al. 17, 25652571 (2018). Nat. 12, 759770 (2021). 10, 507519 (2011). Mol. Finally, global proteomic profiling has seen renewed interest in the context of compound target identification and mechanism of action studies. Finally, 4) the interacting proteins are detected and quantified vs. an untreated control using typically quantitative mass spectrometry. These include issues related to the discovery sample set; including insufficient size, lack of appropriate controls, and changes in the patient population between discovery and validation experiments. Flanagan, M. E. et al. Chemical proteomic profiling of bromodomains enables the wide-spectrum evaluation of bromodomain inhibitors in living cells. Nature 369, 756758 (1994). Nature 468, 790795 (2010). Janes, M. R. et al. PLoS ONE 8, e61551 (2013). The classical chemoproteomics workflow for target deconvolution is based on a compound pulldown step using an immobilized variant of the compound of interest. This is particularly important as new therapeutic modalities, such as cellular therapies, aim to target proteins upregulated in tumor tissue (tumor associated antigens, TAA) or the mutated cancer proteins. J. G. Kaur et al. Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Chem. recently demonstrated that combining proteomic, metabolomic, and lipidomic measurements in plasma with transcriptomic analysis of leukocytes revealed 219 biomolecules strongly associated with COVID-19 status and severity [Citation47]. SLAS Discov. 9, 15401553 (2010). Nat. Zhang, J. et al. And while multi-omic integration is still evolving, examples of disease-relevant studies are starting to emerge. Jafari, R. et al. suggests that including a carrier proteome decreases the dynamic range of quantification [Citation18]. Proteomics technologies present a means of providing evidence early in a drug discovery program that a target is not worthwhile, yielding significant cost savings to pharma companies whilst also ultimately helping patients and the healthcare systems . However, this does mean that it is standard to only identify less than half of the spectra in a typical bottom up workflow. Chem. The cellular thermal shift assay for evaluating drug target interactions in cells. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. These matrices are well suited for biomarker discovery and can readily be incorporated into large clinical trials. Nat. 12, 20402050 (2017). This is a pivotal process, and a lot rides on its success, thus, efficiency is absolutely critical, but mainly for two key points: Rexer, B. N. et al. Reducing sample preparation time and the number of adherent surfaces that come into contact with it, can all contribute to more sensitive analyses. recently been used to identify RBBP9 as a valacyclovir-activating enzyme [Citation89], highlighting the fact that chemoproteomics can identify functionally relevant binding events other than the efficacy target. Chem. Hornbeck, P. V. et al. Automated sample preparation with SP3 for low-input clinical proteomics. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Proteomics in the pharmaceutical and bio . 2. Transl. Biol. 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