Structure-based design of Anaphase Promoting Complex/Cyclosome (APC/C) inhibitors as leads for drug development: an experimental and computational approach
Biochemistry
Bioinformatics and Theoretical Biology
Structural Biology
Final Report Abstract
Most cancer cells proliferate rapidly and in an uncontrolled manner compared to healthy cells. Therefore, specific targeting of fast-proliferating cells is a well-established and successful principle of current cancer therapeutics. During proliferation cells grow, duplicate their genome and finally divide into two equal daughter cells. The process of cell division is termed mitosis and is tightly regulated by a key ubiquitin E3 enzyme, the anaphase promoting complex or cyclosome (APC/C). Without the APC/C cells cannot divide and are eventually eliminated by programmed cell death. Thus, it is not surprising that APC/C has been identified as a promising target for cancer therapy. Up to date no direct APC/C inhibitors are in the clinic. In part, this reflects that the APC/C belongs to the so-called hard-to-drug proteome as its enzymatic activity is not mediated by a small well-defined enzymatic pocket that could easily be targeted my small molecule inhibitors but, instead, it is driven by larger and more complex protein-protein interactions. In the work funded by this DFG research project we combined our biochemical and structural knowledge on APC/C enzymology and APC/C protein-protein interactions to pursue a computer-aided structurebased rational drug design approach to obtain inhibitory lead molecules to target APC/C biological function. In the first part of our design strategy, and making use of available structural information and molecular modeling and dynamics simulation techniques, we investigated at atomic detail the proteinprotein interactions in which APC/C is involved and exploited the differences and similitudes in binding exhibited by ligands and related ubiquitin ligases versus known naturally occurring inhibitors in order to identify plausible attack points and determine key interacting features. Next, by applying a templatebased rescaffolding strategy, we reduced the identified binding hot spots to a minimum set of functionalities that could ensure the required disposition in the three-dimensional space of the most prominent interactions to recognize the APC11 RING domain. Initially, three different suitable naturallike molecular scaffolds extensively used in ligand design were considered for our designs (i.e. β-hairpin, helix-turn and helix-turn-helix), and they were gradually rationally modified through iterative experimental and computational optimization cycles in which stability, specificity, toxicity and cell permeability properties were considered. For this purpose, we applied state-of-the-art medicinal chemistry strategies and structure-based de novo design methods. In iterative computational and experimental cycles, and from first to third generation, the best candidates were optimized and selected for synthesis and experimental evaluation for APC/C inhibition. We fully reconstituted APC/C activity in a test tube and showed that 3 generations of APC/C inhibitors (iAPC11) block its activity with increasing potency and specificity. We determined pharmacokinetic parameters of iAPC11 (e.g. IC50) and identified a “lead molecule” for further development and assessment in living cells. Indeed, the lead iAPC11 molecule induced a mitotic arrest cancer cell lines representative of small lung, cervical, fibrosarcoma and colon cancers. Notably, fibrosarcoma cells appear susceptible to iAPC11 induced cell death after the mitotic arrest – a highly desirable feature in cancer therapy. Several challenges arising through the development of our inhibitors and their application in living cells resulted in the establishment of new technologies in a collaborative fashion, and they advanced our knowledge about APC/C enzymology beyond the original proposed work: To enable screening of iAPC11 molecules independently of their cell membrane permeability features in living cells we developed a new, now patented method, which by mechanical perforation of the cellular membrane enables molecule delivery into the cell. Our finding that iAPC11 molecules also prevented APC/C activation by the ubiquitin chain elongating E2 enzyme UBE2S initiated research into the interplay of APC/C and UBE2S. Together, our work shows that, through such an interdisciplinary approach, novel molecules targeting a hard-to-drug ubiquitin enzyme can be developed as leads for further advancement in living cells and eventually model organisms and the clinic. Through our consecutive optimization of APC/C inhibitors we also identified general principles of E3 ubiquitin enzyme inhibition that can exploited in the future to target a larger class of E3 enzymes in humans. The de novo designed iAPC11 inhibitors have been patented to drive their future commercialization and translation into biotechnological and clinical applications. Altogether, our work resulted thus far in three publications and 2 patents.
Publications
- (2019). Autoinhibition Mechanism of the Ubiquitin-Conjugating Enzyme UBE2S by Autoubiquitination. Structure 27, 1195– 1210.e1197
Liess, A.K.L., Kucerova, A., Schweimer, K., Yu, L., Roumeliotis, T.I., Diebold, M., Dybkov, O., Sotriffer, C., Urlaub, H., Choudhary, J.S., et al.
(See online at https://doi.org/10.1016/j.str.2019.05.008) - (2020). Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S. Sci Signal 13
Liess, A.K.L., Kucerova, A., Schweimer, K., Schlesinger, D., Dybkov, O., Urlaub, H., Mansfeld, J., and Lorenz, S.
(See online at https://doi.org/10.1126/scisignal.aba8208) - (2021). Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation. Lab Chip 21, 2437–2452
Uvizl, A., Goswami, R., Gandhi, S.D., Augsburg, M., Buchholz, F., Guck, J., Mansfeld, J., and Girardo, S.
(See online at https://doi.org/10.1039/D0LC01224F) - “Bereich Mathematik und Naturwissenschaften der Technische Universität Dresden: Activity Regulation and Pharmacological Inhibition of the Anaphase Promoting Complex/Cyclosome. 2021
Alena Uvizl
