Probing the surface of eukaryotic cells using combinatorial toxin libraries

AbstractThe success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells

An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1A^IYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells.</p> <p>Results</p> <p>SLT-1A^IYSNKLMwas able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1A^IYSNKLMadministered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1A^IYSNKLMreadily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1A^IYSNKLMwith DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1A^IYSNKLMtreatment alone (115 day median survival versus 46 and 47 days respectively; <it>P </it>values < 0.001). SLT-1A^IYSNKLMis stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice.</p> <p>Conclusions</p> <p>These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1A^IYSNKLMcan specifically kill human melanoma cells <it>in vitro </it>and <it>in vivo</it>.</p

Characterization and localization of glomerular factor X activator in murine lupus nephritis

grantor: University of TorontoGlomerular fibrin deposition and induction of cellular procoagulant activity (PCA) are associated with both human and murine glomerulonephritis (GN). In order to examine the relationship between murine GN and glomerular PCA, we compared glomerular PCA in normal mice (Balb/cJ) and in three strains of lupus mice (MRL/lpr female, NZBxWF\sb1 female, and BXSB male) that develop immune complex mediated GN spontaneously. In young mice (age 6-8 weeks), at a time when there was no histologic evidence of renal disease, there was no difference in the spontaneous glomerular PCA between the normal and the lupus prone mice. However, older (5-8 months) autoimmune mice, but not Balb/cJ mice showed a significant augmentation in glomerular PCA, which coincided well with the histologic appearance of severe GN. Glomerular PCA was characterized as a direct activator of factor X because (a) expression of its activity required the presence of factors X, V and II (prothrombin), and (b) incubation of glomerular lysates with \sp{125}I labeled factor X revealed factor Xa generation. Factor X activator was characterized as a serine protease and the molecular weight was found to be 66 kD. Immunohistochemical studies using H4 localized factor X activator to the glomerular mesangium and capillary wall of 4 to 6 month old diseased MRL/lpr mice. There was a correlation between the expression of glomerular factor X activator, severity of nephritis and the presence of fibrin in the glomeruli. Furthermore, immunogold labeled H4 bound to the dense deposits in the sub-endothelial and mesangial regions as well as to the macrophages and altered endothelial-like cells of diseased glomeruli.M.Sc

Cell Targeted Ribosome Inactivating Proteins Derived from Protein Combinatorial Libraries

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.Ph

An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

Abstract Background Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells. Results SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1AIYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. Conclusions These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo