Proteins are the so-called workhorses of the cell. Although their quantity can be accurately analysed by advanced mass spectrometry techniques, some features of proteins are still hidden, such as their activity state, ability to bind drugs, or certain post-translational modifications. The goal of the project »Chemical Probes« is to gain insight into the importance of these processes in biomedical questions using a chemical proteomics approach.
Researchers use synthetic chemistry to design and synthesise chemical probes for the covalent modification of proteins. The aim is to enable or enhance their analysis. Specifically, chemical probes can aid in the identification of novel drug targets and their binding sites. They can also help in visualising the location of diseased tissue. With novel chemical probes aimed against proteases, a class of enzymes capable of breaking up proteins, but also the proteins themselves, the project intends to facilitate novel strategies for biomarkers and drug development, particularly the identification of targets for drugs.
Project goals
Overall, the aims of the project are:
The development of novel synthetic strategies and novel chemical probes
The application of these probes for the detection of proteases and other enzymes
The project has a specific focus on target identification by tandem mass spectrometry making use of cleavable linker strategies. Cleavable linkers attach the protein of interest to the chemical probe – but in a way that the two can easily be separated again. Thus the cleavable linkers help separate the cargo (protein of interest) from the vehicle (chemical probe).The project also looks at the application of chemical probes in imaging.
The chemical probes that are being developed in this project contain different types of detection tags: fluorophores for (high-resolution) fluorescence microscopy and alkyne”mini-tags” for bioorthogonal chemistry - high-yielding chemical reactions that can occur in living cells without perturbing their normal chemistry. These alkyne detection tags are only two atoms in size and minimise cell permeability issues. They are amenable to the click chemistry-mediated introduction of any desired tag. Additionally, the alkyne group enables coherent anti-stokes raman scattering (CARS) microscopy imaging of the targets without functionalisation by a large fluorophore, which can influence the chemical properties and bioactivity of the molecule.
Chemical probes for aspartic proteases
Through synthetic chemistry, researchers at ISAS have developed reagents that can modify complex natural products into chemical probes. They also managed to devise novel chemical probes for aspartic proteases, a small, but biomedically important class of proteases. Despite representing a small number of proteases, aspartic proteases play an important role in human physiology and pathological processes. For example, presenilin is the catalytic component of the gamma-secretase complex, which is associated with Alzheimer’s disease. It is also an emerging target in cancer therapy, because of its involvement in the signalling pathway involved in malignant cell growth and cell death (Notch signalling).
In addition, aspartic proteases of infectious agents are attractive drug targets, such as human immunodeficiency virus (HIV) protease as well as plasmepsins from the malaria-causing parasite Plasmodium falciparum. The novel chemical probes will help further characterise the roles of these enzymes. Through the design and full solid-support synthesis of probes that bind to their target in response to activation by light (photoaffinity labelling probes), aspartic proteases can be covalently labelled and detected on gel or by mass spectrometry.
Biomarker for breast cancer
In breast cancer cells, the scientists detected another aspartic protease, cathepsin D. It has been linked to poor prognosis in breast cancer and is a potential biomarker. The researchers also found potential interaction partners. In addition, they discovered that sequestosome-1, a protein involved in the degradation of cells (autophagy), is a cathepsin D substrate. The chemical probes could help to shed light on this degradation pathway in the future.
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ChemBioChem, Vol. 24, No. 21, 2023, P. e202300444
Verhelst S, Prothiwa M.
Chemical Probes for Profiling of MALT1 Protease Activity
https://doi.org/10.1002/cbic.202300444
ChemBioChem, Vol. 24, No. 1, 2023, P. e202300418
Yang J, Carvalho LA, Ji S, Chen S, Moreira R, Verhelst S.
4-Oxo-β-Lactams as Novel Inhibitors for Rhomboid Proteases
https://doi.org/10.1002/cbic.202300418
Organic & Biomolecular Chemistry, Vol. 21, No. 32, 2023, P. 6498-6502
Ji S, Verhelst S.
Furin-targeting activity-based probes with phosphonate and phosphinate esters as warheads
https://doi.org/10.1039/d3ob00948c
ACS Omega, Vol. 8, No. 28, 2023, P. 25487-25495
Korovesis D, Gaspar VP, Beard HA, Chen S, Zahédi RP, Verhelst SHL.
Mapping Peptide-Protein Interactions by Amine-Reactive Cleavable Photoaffinity Reagents
https://doi.org/10.1021/acsomega.3c03064
Angewandte Chemie (International ed. in English), Vol. 62, No. 29, 2023, P. e202305093
Li B, Su K, Van Meervelt L, Verhelst SHL, Ismalaj E, De Borggraeve WM, Demaerel J.
Ex situ Generation of Thiazyl Trifluoride (NSF3 ) as a Gaseous SuFEx Hub
https://doi.org/10.1002/anie.202305093
ACS Chemical Biology, Vol. 18, No. 4, 2023, P. 686-692
Chen S, Liang C, Li H, Yu W, Prothiwa M, Kopczynski D, Loroch S, Fransen M, Verhelst SHL.
Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification
https://doi.org/10.1021/acschembio.2c00946
ACS Chemical Biology, Vol. 18, No. 3, 2023, P. 456-464
Kahler JP, Aloi VD, Aliaga JM, Kerselaers S, Voets T, Vriens J, Verhelst SHL, Barniol-Xicota M.
Clotrimazole-Based Modulators of the TRPM3 Ion Channel Reveal Narrow Structure-Activity Relationship
https://doi.org/10.1021/acschembio.2c00672
Israel Journal of Chemistry, Vol. 63, No. 3-4, 2023
Yang J, Korovesis D, Ji S, Kahler JP, Vanhoutte R, Verhelst S.
Efficient Synthesis of an Alkyne Fluorophosphonate Activity-Based Probe and Applications in Dual Colour Serine Hydrolase Labelling
https://doi.org/10.1002/ijch.202200094
Chemical Science, Vol. 14, No. 7, 2023, P. 1666-1672
Vanhoutte R, Barniol-Xicota M, Chiu W, Vangeel L, Jochmans D, De Jonghe S, Zidane H, Barr HM, London N, Neyts J, Verhelst SHL.
Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells
https://doi.org/10.1039/d2sc04147b
Biomolecules, Vol. 13, No. 2, 2023
Houdou M, Jacobs N, Coene J, Azfar M, Vanhoutte R, van den Haute C, Eggermont J, Daniels V, Vangheluwe P, Verhelst SHL.
Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System
https://doi.org/10.3390/biom13020337
International Journal of Molecular Sciences , Vol. 24, No. 3, 2023
Sun J, Ru J, Ramos-Mucci L, Qi F, Chen Z, Chen S, Cribbs AP, Deng L, Wang X.
DeepsmirUD: Prediction of Regulatory Effects on microRNA Expression Mediated by Small Molecules Using Deep Learning
https://doi.org/10.3390/ijms24031878
Neuropathology and Applied Neurobiology, Vol. 49, No. 1, 2023, P. e12877
Phan V, Hathazi D, Preusse C, Czech A, Freier E, Shema G, Zahedi RP, Roos A.
Molecular mechanisms in chloroquine-exposed muscle cells elucidated by combined proteomic and microscopic studies
https://doi.org/10.1111/nan.12877
Journal of Medicinal Chemistry, Vol. 65, No. 20, 2022, P. 13660-13680
Codony S, Entrena JM, Calvó-Tusell C, Jora B, González-Cano R, Osuna S, Corpas R, Morisseau C, Pérez B, Barniol-Xicota M, Griñán-Ferré C, Pérez C,…
Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain
https://doi.org/10.1021/acs.jmedchem.2c00515
ACS Medicinal Chemistry Letters, Vol. 13, No. 7, 2022, P. 1144-1150
Vanhoutte R, Verhelst SHL.
Combinatorial Optimization of Activity-Based Probes for Acyl Protein Thioesterases 1 and 2
https://doi.org/10.1021/acsmedchemlett.2c00174
Chemical Science, Vol. 13, No. 8, 2022, P. 2270-2279
Li B, Voets L, Van Lommel R, Hoppenbrouwers F, Alonso M, Verhelst SHL, De Borggraeve WM, Demaerel J.
SuFEx-enabled, chemoselective synthesis of triflates, triflamides and triflimidates
https://doi.org/10.1039/d1sc06267k
Journal of Materials Chemistry B, Vol. 40, 2021, P. 8512-8517
Yang L, Chen S, Yi D, Chen Q, Zhang J, Xie Y, Sun H.
Synthesis and fluorescence properties of red to near-infrared emitting push-pull dyes based on benzodioxazole scaffolds
https://doi.org/10.1039/D1TB01189H
Cell Death and Differentiation, 2021, P. 230-245
Rufo N, Korovesis D, Van Eygen S, Derua R, Garg AD, Finotello F, Vara-Perez M, Rozanc J, Dewaele M, de Witte PA, Alexopoulos LG, Janssens S,…
Stress-induced inflammation evoked by immunogenic cell death is blunted by the IRE1α kinase inhibitor KIRA6 through HSP60 targeting
https://doi.org/10.1038/s41418-021-00853-5
RSC Chemical Biology, Vol. 2, No. 4, 2021, P. 1285-1290
Kahler JP, Verhelst SHL.
Phosphinate esters as novel warheads for activity-based probes targeting serine proteases
https://doi.org/10.1039/d1cb00117e
ChemBioChem, Vol. 22, No. 13, 2021, P. 2206-2218
Korovesis D, Beard HA, Mérillat C, Verhelst SHL.
Probes for Photoaffinity Labelling of Kinases
https://doi.org/10.1002/cbic.202000874
ChemBioChem, Vol. 22, No. 9, 2021, P. 1578-1581
Yang J, Mendowicz RJ, Verhelst S.
Tagged benzoxazin-4-ones as novel activity-based probes for serine proteases
https://doi.org/10.1002/cbic.202000848
Molecular Omics, Vol. 17, No. 2, 2021, P. 197-209
Beard HA, Korovesis D, Chen S, Verhelst SHL.
Cleavable linkers and their application in MS-based target identification
https://doi.org/10.1039/d0mo00181c
RSC Advances, Vol. 11, No. 7, 2021, P. 4196-4199
Van Kersavond T, Konopatzki R, van der Plassche MAT, Yang J, Verhelst SHL.
Rapid synthesis of internal peptidyl α-ketoamides by on resin oxidation for the construction of rhomboid protease inhibitors
https://doi.org/10.1039/d0ra10614c