Thèse Inhibition Sélective de Sous-Types de Protéasomes Associés à Différents Régulateurs H/F - Doctorat.Gouv.Fr

Établissement : Université de Toulouse École doctorale : BSB - Biologie, Santé, Biotechnologies Laboratoire de recherche : IPBS - Institut de Pharmacologie et Biologie Structurale Direction de la thèse : Julien MARCOUX ORCID 0000000173217436 Début de la thèse : 2027-01-04 Date limite de candidature : 2026-07-15T23:59:59 Ce projet de doctorat vise à développer une troisième génération d'inhibiteurs du protéasome hautement sélectifs afin de réduire la toxicité des traitements actuels contre les maladies inflammatoires et les cancers. Les travaux seront réalisés au sein de l'équipe ProteoToul Research de l'Institut de Pharmacologie et de Biologie Structurale (IPBS, CNRS Toulouse).

Le protéasome est un complexe cellulaire essentiel chargé de dégrader les protéines endommagées ou inutiles et de maintenir l'homéostasie cellulaire. Comme la dégradation protéique régule l'immunité, l'inflammation et la prolifération cellulaire, les dysfonctionnements du protéasome sont impliqués dans de nombreuses pathologies, notamment les cancers et les maladies auto-immunes. Des inhibiteurs du protéasome sont déjà utilisés pour traiter certaines cancers, mais les inhibiteurs de première génération agissent de manière non spécifique sur tous les protéasomes, entraînant d'importants effets secondaires.

Le projet se concentre sur deux complexes protéasomiques spécifiques : l'immunoprotéasome associé à PA28 (i20S-PA28), impliqué dans les maladies inflammatoires et auto-immunes, et le protéasome constitutif associé à PA28 (c20S-PA28), impliqué dans la progression tumorale et l'échappement immunitaire. Plutôt que de bloquer directement les sites catalytiques, le projet propose d'inhiber les interactions entre le protéasome et ses régulateurs afin d'obtenir une meilleure sélectivité thérapeutique.

Le travail est structuré en trois axes principaux. Le premier consiste à identifier des inhibiteurs sélectifs parmi une centaine de composés dérivés de trois molécules prometteuses déjà détectées lors de criblages préliminaires. Des essais basés sur la fluorescence permettront d'évaluer leur efficacité in vitro puis dans des modèles cellulaires.

Le deuxième axe étudie les effets cellulaires globaux des inhibiteurs sélectionnés grâce à la protéomique quantitative. Des analyses par spectrométrie de masse compareront les cellules traitées et non traitées afin d'identifier d'éventuels effets hors cible et de vérifier la spécificité des inhibiteurs.

Le troisième axe vise à comprendre le mode d'action des inhibiteurs et à localiser leurs sites de liaison. Plusieurs approches biophysiques avancées seront utilisées, notamment la photométrie de masse, la protéolyse limitée couplée à la spectrométrie de masse (LiP-MS) et la spectrométrie de masse native. Des études préliminaires de cryo-microscopie électronique permettront également d'évaluer la faisabilité d'une caractérisation structurale des complexes inhibiteur-protéasome.

Ce projet devrait permettre d'identifier des inhibiteurs hautement sélectifs présentant moins d'effets secondaires que les thérapies actuelles. Ces molécules pourraient ouvrir la voie à de nouvelles stratégies thérapeutiques contre les maladies auto-immunes, l'inflammation chronique, le rejet de greffe et certains cancers, tout en améliorant la compréhension des mécanismes de régulation du protéasome. The human proteasome is a multi-protein complex essential for protein degradation and cellular homeostasis. Its deregulation is linked to neurodegenerative diseases, inflammation, and cancer. Since 2003, three FDA-approved inhibitors have targeted its catalytic sites for treating multiple myeloma and mantle cell lymphoma, but they cause severe side effects by fully blocking proteasomal activity. Developing selective inhibitors for specific proteasome subtypes offers a promising alternative.
Proteasome heterogeneity stems from alternative subunits, regulatory interactions, and post-translational modifications. Its catalytic core (std20S) consists of 14 subunits, while the immunoproteasome (i20S), induced by interferon-, enhances antigen presentation. Human cells contain mixtures of std20S, i20S, and intermediate complexes. Due to first-generation inhibitors' low specificity and i20S involvement in diseases, selective inhibitors have emerged, showing promise in graft rejection, cancer, and autoimmune disorders.
Proteasome subtypes interact with activators like 19S, PA28, PA28, and PA200, which enhance substrate access. Though 20S alone has low activity, these activators can boost its function significantly. PA28 and PA28 increase 20S activity up to 50 times, forming hybrid complexes involved in antigen processing, oxidative stress response, cancer, and autoimmune diseases. This PhD project funded by the ANR (Proteasom-inhib) aims to develop a 3rd generation of inhibitors specific to PA28-associated 20S proteasome subtypes in an auto-inflammatory context. Targeting these subtypes via their activators could significantly reduce the toxicity seen with 1st and 2nd generation drugs. Work package 1: Identifying effective and selective proteasome inhibitors
The team previously identified three inhibitors in a preliminary screen that showed specificity for i20S-PA28 or c20S-PA28. These did not inhibit the proteasome on its own, so were hypothesised to target the complexes via their regulator. Based on the three initial hits, ~100 analogue molecules have been designed, and these will first be tested in vitro for their efficacy in inhibiting proteasome activity. This will be done using fluorogenic peptides which, when cleaved by the proteasome, fluoresce [10][7]. Fluorescence can therefore be used as a measure of proteasome enzymatic activity upon treatment with the inhibitor compounds. For this assay, recombinant proteasomes and regulators will be expressed from HEK293T and E. coli, respectively. i20S and c20S will be immunopurified using MCP21 antibody [7], whereas PA28 and PA28 will be purified through sequential chromatography. From this activity assay, 10-20 of the most effective inhibitors will be selected in cellulo activity experiments.
A fluorescence assay involving GFP-tagged substrates of the proteasome-regulator complex would be used to measure the efficacy of the inhibitors in cellulo, as upon cleavage by the 20S, the GFP-tagged substrates fluoresce. Selectivity of the candidates to i20S-PA28 or c20S-PA28 would be validated using knockouts of PA28 and PA28. Keratinocytes and THP1 reporter cell lines will be used for this activity assay, as they contain substrates of the proteasome that aggregate together when activated.
Work package 2: Assessing the proteome-wide effects of i20S-PA28 and c20S-PA28 inhibition
Once the two most effective and selective inhibitors for each complex have been identified, proteome-wide studies can be conducted, with the focus on identifying off-target effects. Bottom-up proteomics will be conducted on the chosen cell lines after treatment with the candidate inhibitors that are selective for the proteasome complex present in that cell line. For changes in the proteome after c20S-PA28 inhibition, HEK293 cells will be used, as they naturally contain no immunoproteasome. For inhibition of i20S-PA28 however, HEK293 cells transfected with immunoproteasome subunits (HEK293-EBNA) will be used, as overexpression of these subunits represses constitutive subunits leading to complete replacement of c20S by i20S [4]. Cells will be treated with a candidate inhibitor, lysed, and the analysed by mass spectrometry (MS). The relative intensity of proteins will then be compared to untreated cells. Data-independent acquisition (DIA) on an Orbitrap Exploris (Thermo Fisher Scientific) will be used to provide comprehensive coverage of the proteome.
Work package 3: Uncovering inhibitor mode of action and localisation of binding
The final work package consists of integrating biophysical and proteomic methods. First. mass photometry will be performed on purified i20S-PA28 and c20S-PA28 complexes treated with the candidate inhibitors, to accurately determine the mass of regulator-bound, or unbound, proteasomes in solution [11].
In an approach to identify inhibitors binding sites and detect off-target binding, limited proteolysis MS (LiP-MS) will be conducted. This technique involves partially digesting cell lysate with proteinase K (PK) in the presence or absence of an inhibitor, followed by full tryptic digestion. If inhibitor binding prevents cleavage, it will generate conformotypic peptides, i.e. peptides specific for bound and unbound states, which have different MS/MS spectra [12]. LiP-MS experiments will be conducted on the HEK cell lines and mass spectrometer described in work package 2.
To provide insight into which subunit the drug is binding to and the stoichiometry of inhibitor binding, native top-down MS coupled to charge detection (MS-CD) will be used, analysing both the complex as a whole and dissociating it into monomers. MS-CD can directly measure the charge (and therefore mass) of large protein complexes, which results in the detection of small mass shifts corresponding to one or more drug molecules binding [13]. This would be done using the purified i20S-PA28 and c20S-PA28 complexes on a UHMR mass spectrometer (Thermo Fisher Scientific).
Finally, in order to locate and visualise the precise binding site of the drug, a structure of the drug-bound complex would have to be solved, either by cryo-EM or X-ray crystallography. Cyro-EM grids will be prepared for preliminary screening using the purified proteasome-regulator complexes treated with the candidate inhibitors. However, the structure will not be solved during this project, as it is not feasible within the timeline.

Nous recherchons un(e) candidat(e) titulaire d'un master en biochimie et/ou chimie analytique.

Le/la candidat(e) idéal(e) possède une expérience confirmée en culture cellulaire (lignées cellulaires bactériennes et/ou humaines), en expression et purification de protéines et en spectrométrie de masse. Une expertise en biologie structurale serait un atout. Un bon niveau d'anglais (écrit et oral) est également requis.