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Journal articleLueg G, Zhang J, Faronato M, et al., 2025,
MYC deregulation sensitizes cancer cells to N-myristoyltransferase inhibition
, Cell Reports, Vol: 44, ISSN: 2211-1247Human N-myristoyltransferases (NMTs) catalyze N-terminal protein N-myristoylation and are promising targets in cancer, with an emerging mechanistic rationale for targeted therapy. Here, we screened 245 cancer cell lines against IMP-1320, a potent NMT inhibitor (NMTi), and conducted pathway-level analyses to identify that deregulated MYC increases cancer cell sensitivity to NMTis. Proteomics on detergent-enriched membrane fractions in MYC or MYCN-deregulated cancer cell models revealed that cell death is associated at least in part with loss of membrane association of mitochondrial respiratory complex I. This is concurrent with loss of myristoylation and degradation of the complex I assembly factor NDUFAF4, and induction of mitochondrial dysfunction, driven by MYC or MYCN-deregulation. NMTis eliminated or suppressed MYC- and MYCN-driven tumors in vivo without overt toxicity, suggesting that this constitutive co-translational protein modification can be targeted in MYC-driven cancers.
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Journal articleGan J, Dabhade P, Wijne C, et al., 2025,
Identification and characterization of nanobodies specific for the human ubiquitin-like ISG15 protein
, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 301 -
Journal articleSoday L, Seripracharat C, Gray JL, et al., 2025,
Discovery and validation of a novel class of necroptosis inhibitors targeting RIPK1
, ACS Chemical Biology, Vol: 20, Pages: 1527-1543, ISSN: 1554-8929Necroptosis is a form of programmed cell death that, when dysregulated, is associated with cancer and inflammatory and neurodegenerative diseases. Here, starting from hits identified from a phenotypic high-throughput screen for inhibitors of necroptosis, we synthesized a library of compounds containing a 7-phenylquinoline motif and validated their anti-necroptotic activity in a novel live-cell assay. Based on these data, we designed an optimized photoaffinity probe for target engagement studies and through biochemical and cell-based assays established receptor-interacting kinase 1 (RIPK1) as the cellular target, with inhibition of necroptosis arising from the prevention of RIPK1 autophosphorylation and activation. X-ray crystallography and mass spectrometry revealed that these compounds bind at the hinge region of the active conformation of RIPK1, establishing them as type I kinase inhibitors. In addition, we demonstrated in vitro synergy with type III kinase inhibitors, such as necrostatin-1 and found that lead compounds protected mice against acute inflammation in necroptosis models in vivo. Overall, we present a novel pharmacophore for inhibition of human RIPK1, a key protein involved in necroptosis, and provide a photoaffinity probe to explore RIPK1 target engagement in cells.
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Journal articleShchepinova MM, Richardson R, Houghton JW, et al., 2025,
Spatiotemporally resolved GPCR interactome uncovers unique mediators of receptor agonism
, Cell Chemical Biology, Vol: 32, Pages: 722-735.e7, ISSN: 2451-9448Cellular signaling by membrane G protein-coupled receptors (GPCRs) is governed by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome, as it evolves over time and space in response to an agonist, provide a unique perspective on pleiotropic signaling decoding and functional selectivity at the cellular level. In this study, we utilized proximity-based APEX2 proteomics to investigate the interaction network of the luteinizing hormone receptor (LHR) on a minute-to-minute timescale. We developed an analytical approach that integrates quantitative multiplexed proteomics with temporal reference profiles, creating a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is finely regulated spatially, leading to the identification of putative interactors, including the Ras-related GTPase RAP2B, which modulate both receptor signaling and post-endocytic trafficking. This work provides a valuable resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments.
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Journal articleTate EW, Bickel JK, Ahmed AIS, et al., 2025,
Macrocyclic peptide probes for immunomodulatory protein CD59: potent modulators of bacterial toxin activity and antibody-dependent cytotoxicity
, Angewandte Chemie, ISSN: 0044-8249CD59 is an immunomodulatory cell surface receptor associated with human disease. Despite its importance in complement regulation and bacterial pathogenesis, CD59 remains a challenging therapeutic target. Research to date has focused on antibody or protein-based strategies. Here we present a new approach to target CD59 using macrocyclic peptides with low nanomolar affinity for CD59. Through X-ray crystallographic studies and structure-activity relationship (SAR) studies, we identify key interactions that are essential for binding and activity. We find that the macrocyclic peptide CP-06 adopts a beta-hairpin structure and binds CD59 through an intermolecular beta-sheet, mimicking protein–protein interactions of biologically relevant CD59 interaction partners. We create dimeric and lipidated macrocyclic peptide conjugates as enhanced cell-active CD59 inhibitors and show that these probes can be used to modulate both complement-mediated killing of human cells and lytic activity of bacterial virulence factors. Together, our data provide a starting point for future development of macrocyclic peptides to target CD59 activity in diverse cellular contexts.
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Journal articleDate A, Wall A, Zhang P, et al., 2025,
Affinity-based protein profiling of MDM2 inhibitor Navtemadlin
, Chemical Science, Vol: 16, Pages: 6886-6894, ISSN: 2041-6520Navtemadlin is a potent inhibitor of the p53-MDM2 protein–protein interaction, which plays a critical role in the proliferation of p53-wildtype tumours. Whilst Navtemadlin has progressed to multiple Phase III clinical trials in oncology, little has been disclosed regarding its selectivity for MDM2 in cells. Here, we report the synthesis and validation of photoactivatable clickable probes of Navtemadlin, and their application to de novo target discovery for Navtemadlin through affinity-based protein profiling. MDM2 was robustly identified as the main target, across two cell lines, using two distinct probe designs. While off-targets were identified, these were not consistent across cell lines and probe designs, consistent with a high degree of selectivity for the target protein. Whole proteome profiling experiments across different time points confirmed p53-mediated phenotypic activity and revealed novel expression patterns for key proteins in the p53 pathway.
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Journal articleMendez A, Bolling C, Taylor S, et al., 2025,
Structure of Plasmodium vivax N-myristoyltransferase with inhibitor IMP-1088: exploring an NMT inhibitor for antimalarial therapy
, Acta Crystallographica Section F: Structural Biology and Crystallization Communications Online, Vol: 81, ISSN: 1744-3091Plasmodium vivax is responsible for the most widespread form of malaria and approximately 2.5 billion people, or over one-third of the world's population, are at risk of P. vivax infection (Battle et al., 2019[Battle, K. E., Lucas, T. C. D., Nguyen, M., Howes, R. E., Nandi, A. K., Twohig, K. A., Pfeffer, D. A., Cameron, E., Rao, P. C., Casey, D., Gibson, H. S., Rozier, J. A., Dalrymple, U., Keddie, S. H., Collins, E. L., Harris, J. R., Guerra, C. A., Thorn, M. P., Bisanzio, D., Fullman, N., Huynh, C. K., Kulikoff, X., Kutz, M. J., Lopez, A. D., Mokdad, A. H., Naghavi, M., Nguyen, G., Shackelford, K. A., Vos, T., Wang, H., Lim, S. S., Murray, C. J. L., Price, R. N., Baird, J. K., Smith, D. L., Bhatt, S., Weiss, D. J., Hay, S. I. & Gething, P. W. (2019). Lancet, 394, 332-343.]). In humans, P. vivax can enter a dormant liver phase, which allows it to survive in various climates, including tropical and temperate regions, and contributes to its extensive geographical prevalence (Battle et al., 2019[Battle, K. E., Lucas, T. C. D., Nguyen, M., Howes, R. E., Nandi, A. K., Twohig, K. A., Pfeffer, D. A., Cameron, E., Rao, P. C., Casey, D., Gibson, H. S., Rozier, J. A., Dalrymple, U., Keddie, S. H., Collins, E. L., Harris, J. R., Guerra, C. A., Thorn, M. P., Bisanzio, D., Fullman, N., Huynh, C. K., Kulikoff, X., Kutz, M. J., Lopez, A. D., Mokdad, A. H., Naghavi, M., Nguyen, G., Shackelford, K. A., Vos, T., Wang, H., Lim, S. S., Murray, C. J. L., Price, R. N., Baird, J. K., Smith, D. L., Bhatt, S., Weiss, D. J., Hay, S. I. & Gething, P. W. (2019). Lancet, 394, 332-343.]). P. vivax infection significantly impacts the quality of life of infected individuals, causing cyclical episodes of fever and weakness, representing a substantial burden in endemic countries due to treatment costs and productivity loss. P. vivax can persist in human hosts as hypnozoites in the liver that can cause relapses that can extend over several months or years (Flannery et al., 2022[Flannery, E.
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Journal articleZhang J, Kallemeijn WW, Hassan S, et al., 2025,
Chemical proteomic approaches to investigate N-myristoylation.
, Methods Enzymol, Vol: 718, Pages: 215-240The protein lipidation event N-myristoylation is catalyzed by the N-myristoyltransferase (NMT) enzymes and occurs on over 200 proteins with N-terminal glycines. The modification controls the localization, stability, function and interactions of its substrate proteins and has been implicated in the regulation of multiple biological processes and disease pathologies. Understanding how the N-myristoylated proteome is altered in these pathologies and in response to pharmacological NMT inhibition is therefore critical to understand how N-myristoylation regulates basic biology and how its pharmacological inhibition may be optimally leveraged in clinical settings. Chemical proteomic approaches have emerged as powerful methods to profile protein post-translational modifications, including lipidation, on a proteome-wide scale. This chapter describes two complementary chemical proteomic methods to assess N-myristoylation. The metabolic labelling approach is based on the uptake and incorporation of bio-orthogonal myristic acid analogues into NMT substrates, allowing the selective labelling, enrichment and detection of these proteins. In contrast, the second method is based on the ability of Sortase A to modify N-terminal glycines with a substrate peptide, allowing the incorporation of an enrichment handle in NMT substrates lacking N-myristoylation, for example due to pharmacological NMT inhibition. Detailed, up-to-date protocols for both chemical proteomic approaches are described in this chapter and encompass all steps of the workflows, including cell treatment, sample preparation and data analysis.
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Journal articleHassan S, Zhang J, Kallemeijn W, et al., 2025,
Chemical proteomic approaches to investigate S-prenylation.
, Methods Enzymol, Vol: 719, Pages: 299-316Protein S-prenylation is a post-translational lipid modification that occurs at C-terminal cysteines. While S-prenylation has been widely studied in the context of disease biology and drug discovery, only in the last decade have robust proteomic techniques emerged to study the complex dynamics between different classes of protein prenyltransferases, significantly aiding the development and application of tools and inhibitors. Herein, we describe the use of S-prenylation probes as a chemical proteomic technique to interrogate S-geranylgeranylation and S-farnesylation, the two classes of protein prenylation, from sample preparation to mass spectrometry analysis.
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Journal articleOcasio CA, Baggelaar MP, Sipthorp J, et al., 2024,
A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation
, Nature Biotechnology, Vol: 42, Pages: 1548-1558, ISSN: 1087-0156The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.
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Contact
Prof. Ed Tate
GSK Chair in Chemical Biology
Department of Chemistry
Molecular Sciences Research Hub, White City Campus,
82 Wood Lane, London, W12 0BZ
e.tate@imperial.ac.uk
Tel: +44 (0)20 759 + ext 43752 or 45821