Information on endowment from the state budget or a state special purpose fund
The project is financed by the state budget.

Name of the programme or fund
IMPLEMENTATION DOCTORATE PROGRAMME IVTH EDITION

Name of the project
Implementation Doctorate Programme at the Małopolska Center of Biotechnology of the Jagiellonian University - IVth edition - path within the Doctoral School of Exact and Natural Sciences

Project manager
dr Sebastian Glatt

Value of the endowment
PLN 648 154.70

Total investment cost
PLN 648 154.70

Brief description of the project
Project goals:
Targeted protein degradation (TPD) means highly specific pharmacological removal of proteins responsible for advancing pathological state in humans. The technology has great therapeutic potential as it targets proteins that lack any enzymatic function or active spots, and that are typically not available to conventional small molecule inhibitors. Moreover, unlike traditional drugs, molecules using TPD cause the phenomenon of drug resistance less frequently and require much smaller therapeutic doses. As of today, several strategies for targeted protein degradation have been developed (the common denominator of which is the use of low molecular weight compounds ("degraders") to induce the formation of pathological protein complexes with E3 ubiquitin ligases. Due to the chemically induced approximation, pathological proteins can then be tagged with poly-ubiquitin chains and directed for degradation in the proteasome.

The technological problem that will be solved within the project is to determine the geometry and structures of the complexes consisting of E3 ligases, degraders and pathological proteins. This is essential because complex formation alone does not guarantee that degradation will be achieved in the model cell lines and it is often not known what causes the inactivity. To search for new degraders, the identification of compounds that recruit E3 ligase into pathological proteins in in vitro systems is typically made at the start of a project. These compounds very often do not show any degradation activity in cellular tests and the goal of further research is to achieve degradation through chemical modification of the compounds. Such process is laborious and risky; poor absorption of compounds into the cell interior or the unproductive architecture of the degradation complex is assumed to be a likely cause of inactivity. Determining the structure of the complex with E3 ligase will have a significant impact on the choice of further optimization strategy; in the case of compounds inducing a productive complex with E3 ligase, it will be possible to focus on optimizing the properties of ADMET, while for non-optimal geometry of the complex (e.g. when the catalytic subunit of E3 ligase is placed far away from the pathological protein), it will be reasonable to introduce changes in the chemical topology of the degrader.

The main result will be the determination of the cryo-EM structure that will provide information on the domain ordering and overall architecture of the studied protein complexes.

Main goal:

• development of a sample preparation strategy and high resolution cryoEM data collection for the ubiquitin ligases of the culin family and their complexes, which will have a complex structure with intrinsic flexibility and limited thermodynamic stability. This is a problem that can be solved, as presented in recent publications.
•  

Dissertation objectives:

• Optimization of sample preparation procedures for cryoEM, including freezing conditions, stabilization of weak and dynamic complexes by genetic fusion or chemical cross-linking
• Attempts to measure, analyze and define the cryoEM structure for at least 3 members of the cullin family

Schedule for the implementation of scientific work

The research task of the doctoral student will be to determine the optimal conditions of the chemical environment in order to promote co-crystallization of complexes between the target protein degraders and their protein partners. Despite recent technical advances and successes, this remains a tedious and difficult task, especially in the case of low solubility effector compounds or in the presence of a dynamic complex which may adopt many different conformations. However, the problem is possible to solve, as evidenced by the results referenced in the literature (eg Farnaby, W., Koegl, M., Roy, M.J. et al. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat Chem Biol 15, 672–680 (2019). https://doi.org/10.1038/s41589-019-0294-6, Gadd, M., Testa, A., Lucas, X. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514–521 (2017). https://doi.org/10.1038/nchembio.2329) and the inner experience of Captor Therapeutics.

Main research objective:

• Purification of at least 3 different recombinant cullin family E3 ligases and their target proteins for degradation Crystallization trials for minimal E3-Protac-target ternary complexes
• Establishing optimal methods for co-purification and enrichment of complexes
• Design and production of cullin fusion proteins (e.g. adapter-receptor complexes) to improve protein homogeneity and increase the stability of the cullin complexes
• Collection of diffraction data, analysis and determination of the crystal structure

The results obtained during the project may be possible for publication, because widely described model systems, e.g. the VHL-MZ1-BRD4 complex will be used for preliminary experiments. (Nat Chem Biol 2017 May;13(5):514-521. doi: 10.1038/nchembio.2329). In addition, the timeframe of the doctoral studies will coincide with the schedule of projects implemented at Captor Therapeutics and at least 3 of the currently realized projects will end before 2024, which will open the way for further publications.

Schedule for the implementation of scientific work