Due to its dual role as an apoptosis inhibitor and mitotic regulator, Survivin is accepted as a nodal protein hub, with high relevance in basic cell and translational cancer biology. Particularly, our work on the impact of Survivin's nuclear export signal not only resulted in several publications but also provided the basis for innovative chemico-genetic translational interference strategies. Successfully embedded in collaborative DFG research projects, such as the RTG/GRK1739 Radiation Biology and the CRC/SFB1093 Supramolecular Chemistry on Proteins, we are currently pursuing the concept of supramolecular inhibition and innovative nanocarrier based delivery tools. In addition, recent publications revealed a so far unknown role in the DNA damage response and as such now set the stage for in-depth investigations of Survivin's role in cancer biology and therapy response. Here, we will additionally focus on Survivin’s apoptotic/mitotic switch, whether it really exists as proposed and how it can be induced. This has so far eluded experimental investigation, although it would be of utmost importance to finally understand how Survivin decides between its two (or more) biological roles.
Recently, we succeeded in the development of supramolecular inhibitors targeting the Survivin/Histone H3 binding site and the Survivin's dimer interface, partially overlapping with its nuclear export signal (NES), thus blocking the pivotal interaction with the export receptor Crm1 ( see below).
Although less is known about the molecular (patho)mechanisms of the threonine protease Taspase1, the ZMB’s outstanding expertise in protease research represents an ideal platform to study its unresolved impact on ‘Cell fate decisions in development and disease’. Employing cutting-edge live cell microscopy (in cooperation with our high-end microscopes implemented at the Imaging Center Campus Essen) and biosensor systems, we not only uncovered novel mechanisms regulating Taspase1’s structure activity relationships (SAR), but also identified previously unknown targets with cellular functions in e.g., transcriptional regulation and cell differentiation and migration. Hence, Taspase1 also represents an important molecular switch determining cell fate and in addition serves as a valuable model in protease research to unravel novel fine-tuning mechanisms with translational relevance.
In the collaborative framework of the DFG CRC/SFB1093 Supramolecular Chemistry on Proteins, we are currently pursuing the concept of supramolecular inhibition of pivotal Taspase1 protein interactions. Here, we particularly focus on its Importin-mediated nuclear transport, but also on the interplay of prerequisites for autocatalytic activation of the protease and proteolytic activity in general. Is it possible to interfere with Importin-related nucleolar association and at which steps of the protease activation process? And finally, how is substrate specificity for only one or a certain subset of Taspase1 target(s) achieved and at which developmental stages?
Recently, we succeeded in the development of supramolecular binders blocking the pivotal interaction of Taspase1's nuclear import signal (NLS) with the import receptor Importin α ( see below).
Cell delivery systems & Nanoparticles
Together with the Faculty of Chemistry and as a member of CENIDE, we develop small ligands as transporters for nucleic acids and proteins and optimize their cellular uptake mechanism. A long-term goal of this project is to achieve not only efficient cell transfection, but also cell-specific delivery by e.g., the addition of distinct targeting sequences. In addition, there has been recent success in the use of nanoparticles as carriers for insoluble or poorly soluble drugs.
As nanoparticles bind proteins in biological fluids, such as the blood system, knowledge of the biomolecule corona covering the surface of the nanoparticles is critical for the success of nano-biomedical applications, including tumor targeting and in vivo imaging. For one, we could provide the first time-resolved, standardized analysis of the protein corona formed on different types of nanomaterials. Moreover, particularly humans are increasingly exposed to nanomaterials via the inhalative and oro-gastrointestinal route, representing also major infection paths for fungal spores and enteric bacterial pathogens. Just recently we substantiated the (patho)biological consequences of microbe-nanomaterial interactions and thus propose potential nanomaterial applications as anti-biotics or anti-mycotics.
Complementing the strategies pursued together with the Faculty of Chemistry we venture on the field of cell-penetrable VHH single domain antibodies (so-called nanobodies) as nanoscale biological binders. Combining our expertise in cell delivery systems, protein modification and cellular localization signals we explore possibilities to kidnap proteins from the site of action in order to reduce their functional availability.
- A bivalent supramolecular GCP ligand enables blocking of the Taspase1/Importin α-interaction
- Inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers
- Inhibition of the Survivin-Histone H3 interaction
- Inhibition of the Taspase1/Importin α interaction by PEGylated oligomers
- Inhibition of the protease Taspase1 by nanoparticles
- TFIIA is controlled by a “cleave-and-run” Exportin-1/Taspase 1-switch
- Cancer-cell-specific drug delivery by a tumor-homing conjugate
A bivalent supramolecular GCP ligand enables blocking of the Taspase1/Importin α-interaction.
Threonine aspartase 1 (Taspase1) is a protease overexpressed in numerous tumor cell lines and has a furthering effect on tumor development. Its knockdown is well tolerated in adult human tissue but not in tumor cells. Thus, Taspase1 is classified as a non-oncogene-addiction protease and represents a promising target in cancer therapy. However, there is only a limited number of inhibitors available to target the proteolytic activity of this unique threonine aspartase. Therefore, we recently started to explore a novel interference strategy targeting its interaction with Importin α as pivotal part of the intracellular Taspase1 activation process. In line with that, we now rationally developed a bivalent supramolecular disruptor of this interaction utilizing two copies of a guanidiniocarbonylpyrrole(GCP)-motif called 2GC. The compound was effective in the low micromolar range as reflected by the respective IC50 values in pull-down experiments and comparable EC50 values revealing its toxicity against tumor cells. To validate our rational design, we varied important structural charateristics of the inhibitor: bivalent vs. monovalent design, different derivations (cbz vs. alloc) and exchange of the basic GCP units by arginine. Comprehensive studies including all those compound variations indeed only marked our GCP-motif containing bivalent ligand as an effective disruptor of the Taspase1/Importin α-interaction, thereby validating the importance of each modular component as well as our rational design strategy.
Inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers.
Survivin’s dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin’s nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed us to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.
Meiners A, Bäcker S, Hadrovic I, Heid C, Beuck C, Ruiz-Blanco Y.B, Mieres-Perez J, Pörschke M, Grad J.-N, Vallet C, Hoffmann D, Bayer P, Sanchez-Garcia E,* Schrader T,* Knauer SK* (2020). Specific inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers. *corresponding authors, Nat Commun 12:1505. doi: 10.1038/s41467-021-21753-9
Inhibition of the Survivin-Histone H3 interaction.
The protein Survivin is highly upregulated in most cancers and considered to be a key player in carcinogenesis. Here, we explored a supramolecular approach based on the guanidiniocarbonyl pyrrole cation that serves as a highly specific anion binder. Based on a focused library we were able to identify L1 as a potent ligand to target the cancer-relevant protein Survivin by disrupting the protein-protein interaction with Histone H3. NMR spectroscopy enabled us to verify binding of the ligand to Survivin’s Histone H3 binding site, which mediates the interaction with Histone H3 in the early stages of mitosis and is crucial for cell proliferation. In addition, microscopic and cellular assays revealed that the interaction between the two proteins was successfully decreased in a cellular environment. This resulted in an increasing number of mitotic defects and consequently in a reduction of cancer cell proliferation that was confirmed to be caused by a specific inhibition of Survivin inside the cells. Further studies now focus on the development of additional ligands in order to target other functionally relevant protein–protein interactions of Survivin. In addition, multivalency is explored as an approach to further improve and optimize L1 regarding specificity and affinity.
Inhibition of the Taspase1/Importin α interaction by PEGylated oligomers.
Here, a novel strategy to inhibit the oncologically relevant protease Taspase1 is explored by developing PEGylated macromolecular ligands presenting the supramolecular binding motif guanidiniocarbonylpyrrole (GCP). Notably, Taspase1 requires interaction of its nuclear localization signal (NLS) with import receptor Importin α. We could demonstrate that our PEGylated ligands effectively disrupted the functionally relevant interaction in a concentration-dependent manner, thereby exploiting steric shielding as a so far unexplored inhibition mechanism for this protease. Future studies now include investigations concerning the potential selectivity of the ligands as well as their potential for cellular studies. As a first prerequisite, ligands were tested in a cell viability assay and showed no general toxicity.
Inhibition of the protease Taspase1 by nanoparticles.
When nanoparticles enter a physiological environment, they rapidly adsorb biomolecules, in particular cellular proteins. This biological coating, the so-called nanoparticle protein corona, undoubtedly affects the biological identity and potential cytotoxicity of the nanomaterial. To elucidate a possible impact on the adsorbed biomolecules, we focused on an important group of players in cellular homeostasis, namely proteolytic enzymes. We could demonstrate that amorphous silica nanoparticles are not only able to bind to the oncologically relevant threonine protease Taspase1 as revealed by microscale thermophoresis and fluorescence anisotropy measurements, but moreover inhibit its proteolytic activity in a non-competitive manner. As revealed by temperature-dependent unfolding and CD spectroscopy, binding did not alter the stability of Taspase1 or its secondary structure. Noteworthy, inhibition of protein function seems not to be a general feature of nanoparticles, as several control enzymes were not affected in their proteolytic activity. Our data suggests that nanoparticles bind Taspase1 as an αβ-dimer in a single layer without conformational change, resulting in noncompetitive inhibition that is either allostery-like or occludes the active site. Nanoparticle-based inhibition of Taspase1 could be also achieved in cell lysates and in live cells as shown by the use of a protease-specific cellular cleavage biosensor. Collectively, we could demonstrate that nanoparticles could not only bind but also selectively inhibit cellular enzymes, which might explain observed cytotoxicity but might serve as a starting point for the development of nanoparticle-based inhibitors as therapeutics.
TFIIA is controlled by a “cleave-and-run” Exportin-1/Taspase 1-switch.
Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA’s nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA’s nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.
Schrenk C, Fetz V, Vallet C, Heiselmayer C, Schröder E, Hensel A, Hahlbrock A, Wünsch D, Goesswein D, Bier C, Habtemichael N, Schneider G, Stauber RH, Knauer SK* (2018). TFIIA transcriptional activity is controlled by a “cleave-and-run” Exportin-1/Taspase 1-switch. JMCB, 10(1): 33–47.*corresponding author
Cancer-Cell-Specific Drug Delivery by a Tumor-Homing Conjugate.
Tumor-targeted drug delivery is highly important for improving chemotherapy, as it reduces the dose of cytotoxic agents and minimizes the death of healthy tissues. Here, we have developed a cancer cell-specific delivery system by using simple Schiff’s base ligation chemistry to generate a semi-labile conjugate of Gossypol and a cancer type-specific cell penetrating peptide (CPP) as a vector. Utilizing the aldehyde moiety of gossypol, a tumor homing CPP (RLYMRYYSPTTRRYG) was attached through a semi-labile imine linker, which was cleaved in a traceless fashion under aqueous conditions. This potent conjugate enabled selective killing of MCF-7 breast-cancer cells in contrast to e.g., HeLa cells or healthy fibroblasts. Furthermore, the solubility of gossypol was improved, which made handling of the conjugates for cellular studies very convenient, as potentially harmful solubilizing agents like DMSO were not necessary. Importantly, the presented drug delivery strategy does not rely on any external stimulus to initiate drug release and activation. A particular advantage of this imine linkage is the convenient half-life of 10 hours in aqueous media. Hopefully these results accelerate the applicability of gossypol particularly in tumor targeted chemotherapy. In future research cell line-derived xenograft (CDX) mouse models should be established to evaluate the in vivo efficacy of the CPP-gossypol conjugate. In a broader sense, the reported approach demonstrates that CPPs with tumor homing properties can be easily ligated to gossypol without the need for an additional linker. Therefore, it should be easy to expand the scope of this approach to other cancer types, for which appropriate homing peptides can be identified.