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Expanding the Drug Target Universe: How Next-Generation Proteomics Is Transforming Oncology Research

Protein structure.
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Effective target identification and validation lie at the heart of modern drug discovery. Yet the current repertoire of cancer therapeutics is still focused on a relatively small subset of targets compared with the overall “druggable” biology within cancer cells.


Genetic technologies such as genomics and transcriptomics have opened up a wider range of novel targets in cancer over recent years. However, they cannot capture what is happening at the phenotypic level as cancer grows and spreads. The missing piece of this picture is the proteome – the ultimate output of the genome and the target of most therapeutics.


A number of studies presented at the 2023 American Association for Cancer Research (AACR) annual meeting by mass spectrometry specialists Biognosys and collaborators showcase how next-generation proteomics tools are expanding the universe of targets in cancer, unlocking new biology and novel therapeutics, and validating drug binding and mechanism of action.


Exploring patient-derived tumor models in search of new targets


Patient-derived tumor models, including patient-derived xenograft (PDX) and in vitro 3D organoid counterparts (PDXO), are widely recognized as predictive preclinical cancer models closely recapitulating both tumor complexity, patient response and drug resistance. These models are, therefore, ripe territory in the hunt for new targets for next-generation cancer therapies.


Mass spectrometry proteomics provides opportunities to explore known and novel pathways in these models. This is especially true of pathways modulated by post-translational modifications such as phosphorylation, which cannot be detected using conventional genomic or transcriptomic analysis.


In a recent study, Biognosys’ high-throughput unbiased discovery mass spectrometry proteomics platform, TrueDiscovery™, was used to explore the impact of various targeted therapies on genetically specified PDX and PDXO models. This included testing the effect of a novel KRAS inhibitor (AMG510) in a non-small cell lung cancer (NSCLC) model carrying a KRAS G12C mutation, the BCR-ABL TKI ponatinib in a colorectal model with a RET fusion gene, and the EGFR tyrosine kinase inhibitor afatinib in a lung NSCLC model with EGFR exon 19 deletions.


Overall, 16,797 proteins and 35,403 phospho-sites were robustly identified and quantified in the PDX samples, and 12,578 proteins and 40,754 phospho-sites across the PDXO models. These data enabled the validation of expected pathways of drug response and resistance in these cancers, such as RAF/MEK/ERK and PI3K/AKT/mTOR, and also revealed new ones, including downregulation of CDK activity and DNA double-strand break repair.


Probing challenging drug targets


Advances in structural mass spectrometry proteomics techniques are bringing powerful new approaches for drug target validation and deconvolution. Chief among these is limited proteolysis mass spectrometry (LiP-MS), which allows probing of drug-target interactions with peptide-level resolution.


In LiP-MS, free and drug-treated cell extracts are digested with proteases to produce distinctive peptide “fingerprints” that are then analyzed with mass spectrometry, revealing detailed information about the drug binding site and potential mode of action. It can be applied even to targets such as large complexes, multimers and membrane proteins, which can be difficult to analyze with other methods.


Powered by LiP-MS, Biognosys’ TrueTarget™ platform is a unique proprietary tool for probing structural changes across the complete proteome without the need for compound modification or labeling. For example, in collaboration with InterAx Biotech, Biognosys applied TrueTarget to identify the target of a novel compound aimed at multi-pass transmembrane G-protein-coupled receptors, with atypical chemokine receptor 3 (ACKR3) identified as the most likely hit. Additionally, 82 other proteins were found to be altered in drug-treated samples, including ACKR3 downstream pathways, helping to confirm the target and providing valuable insights into the underlying biological effect of the drug.


In another study presented at AACR 2023 in collaboration with Cedilla Therapeutics, a high-resolution form of LiP-MS (HR-LiP) was used to map the binding site of the inhibitor JQ1 within the 200 kDa multi-domain transcriptional regulator bromodomain-containing protein 4 (BRD4) – an important target in cancer. The site showed a close correlation with the previously characterized binding location of the drug, proving the validity of the method in a large protein complex.


HR-LiP was also used to examine the binding of a novel small molecule activator, SMER28, to the endoplasmic reticulum associated ATPase Valosin-containing protein (VCP, also known as p97). VCP is a potent inducer of autophagy that forms oligomeric complexes within the cell and is of great interest as a potential therapeutic target in cancer and other diseases. Peptide analysis showed that SMER28 is highly likely to bind between the substrate binding domain and ATPase domain, a previously demonstrated activation site for VCP (figure 1), confirming the binding location and shedding light on the likely mechanism of action.


Figure 1: HR-LiP peptides (red, orange) predict the likely binding site of SMER28 (yellow) to be between the substrate binding domain (green) and the ATPase domain (purple, blue) of VCP. Credit: Lidia Wrobel, Department of Medical Genetics, Cambridge Institute for Medical Research & UK Dementia Research Institute, University of Cambridge, Cambridge Institute for Medical Research.


Mass spectrometry proteomics: A vital tool for modern drug discovery


It is an exciting time for drug discovery in oncology, with the opportunity to explore previously uncharted territory across the proteome in the search for new targets. Underpinning all this progress is the need to have a solid understanding of the underlying biology and drug-target interactions, from accurate mapping of compound binding sites to understanding the mechanism of action and identifying likely off-target effects.


Mass spectrometry proteomics offers many advantages at all stages of the drug discovery process – from pathway exploration and target identification through unbiased proteome-wide analysis with TrueDiscovery to deconvolution and validation with TrueTarget – accelerating and de-risking novel therapies on the journey from the lab to the clinic.


Biognosys is the leading provider of mass spectrometry proteomics solutions for all stages of the drug development pipeline, from initial discovery to clinical trials. Find out more at biognosys.com.