4 research outputs found

    Cytosolic delivery and characterization of monobodies interfering with SH2 domain-phosphotyrosine interactions

    Get PDF
    Targeted cancer therapy is a very promising concept; however, small molecule inhibitors only exist for a limited number of oncoproteins and resistance development limits their clinical use. Different engineered non-antibody scaffolds are being explored to develop potent protein-based inhibitors of oncoproteins, which can overcome these hurdles. The first goal of this thesis was to characterize monobodies, engineered binders based on the fibronectin type 3 domain, which were selected to bind the SH2 domains of 6 members of the Src kinase family that play a role in different types of cancer. I have shown that the monobodies bind specifically to their target in cells by performing tandem affinity purifications with subsequent mass spectrometry analysis. When expressed in T cells, the Lck-targeting monobodies ML1 and ML3 inhibit the phosphorylation of Zap70, a kinase which is activated downstream of T cell activation. Moreover, the fusion of ML1 or ML3 to VHL, the substrate receptor of an E3 ubiquitin ligase, resulted in the targeted degradation of Lck. The selected monobodies therefore have great potential to interfere with cancer cell signaling and to be used in therapeutic approaches. The development of protein-based inhibitors is currently limited to extracellular targets due to the inability of proteins to cross the cell membrane. Many approaches to achieve cellular delivery are hampered by non-selective cytotoxicity or endosomal entrapment of the protein cargo. The second aim of my thesis was to explore different methods to deliver functional monobodies into cancer cells. Firstly, I tested cell penetrating poly-disulfides (CPDs). CPD-monobody adducts showed toxic effects on the tested cell line, and mainly followed an endocytic uptake route leading to lysosomal degradation, when used at subtoxic concentrations. Secondly, bacterial toxins have naturally evolved to deliver their payload into the cytoplasm of host cells. Both Shiga-like toxins and Exotoxin A from Pseudomonas aeruginosa are taken up into the cytosol via a retrograde trafficking route, avoiding endosomal entrapment. To deliver monobodies to the cytosol, we have tested a combination of their non-toxic subunits: The B subunit of Shiga-like toxin (Stx2B), binding to the sphingolipid Gb3 in the cell membrane, and the translocation domain of Exotoxin A (TDP). We could show that recombinant Stx2B-TDP-Monobody fusion proteins are taken up in HeLa cells, which naturally express Gb3. Colocalization analyses with markers for endocytic compartments demonstrated that the major part of the monobodies escape early endosomes and are not degraded in lysosomes. A recombinant fusion protein of the Stx2B-TDP construct with the monobody AS25, which allosterically inhibits Bcr-Abl kinase, leads to apoptosis in Bcr-Abl-dependent chronic myeloid leukemia cells. Furthermore, the delivery of the VHL-ML3 fusion protein into Jurkat T cells results in decreased Lck levels. The delivery of both constructs is dependent on Gb3 on the cell surface as well as the Stx2B-TDP subunits. These results demonstrate that functional monobodies can be delivered to the cytosol and bind to their target protein in cells. This method could facilitate the development of protein-based inhibitors of intracellular proteins for cancer treatment

    Targeted protein degradation through cytosolic delivery of monobody binders using bacterial toxins

    Get PDF
    Monobodies are small engineered binding proteins that, upon expression in cells, can inhibit signaling of cytosolic oncoproteins with outstanding selectivity. Efficacy may be further increased by inducing degradation of monobody targets through fusion to the von Hippel-Lindau (VHL) substrate receptor of the Cullin2-E3 ubiquitin ligase complex. However, potential therapeutic use is currently limited, because of the inability of monobody proteins to cross cellular membranes. Here, we use a chimeric bacterial toxin, composed of the Shiga-like toxin B (Stx2B) subunit and the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA-II) for delivery of VHL-monobody protein fusions to target endogenous tyrosine kinases in cancer cells. Depending on the expression of the Stx2B receptor Gb3 on the cell surface, we show that monobodies are taken up by an endocytic route, but are not degraded in lysosomes. Delivery of monobodies fused to a nuclear localization signal resulted in accumulation in the nucleus, thereby indirectly, but unequivocally, demonstrating cytosolic delivery. Delivery of VHL fused to monobodies targeting the Lck tyrosine kinase in T-cells resulted in reduced Lck protein levels, which was dependent on the expression of Gb3. This led to the inhibition of proximal signaling events downstream of the T-cell receptor complex. This work provides a prime example of the delivery of a stoichiometric protein inhibitor of an endogenous target protein to cells and inducing its degradation without the need of genetic manipulation of target cells. It lays the foundation for further in vivo exploitation of this delivery system

    Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

    No full text
    The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. (C) 2017 The Authors. Published by Elsevier Ltd
    corecore