Abstract
Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli—a concept that could lead to efficient production of highly multifunctional drugs in the future.
Highlights
Developing new cytostatic drugs with enhanced anticancer effectiveness but reduced side effects in non-cancerous tissues is still the central goal of cancer research
The specific cleavage site (PLGLYL) we chose for MMP-2 and MMP-9 was a sequence described by Albright et al 2005 [22], which has low cleavage/activation by non-tumor proteases, especially by neprilysin, a membrane metalloendopeptidase highly expressed throughout the body
Two steps were crucial in designing this fusion protein: incorporation of the XTEN polypeptide as expressible variant of polyethylene glycol (PEG) and deactivation of Killin, which is otherwise difficult to express in E. coli
Summary
Developing new cytostatic drugs with enhanced anticancer effectiveness but reduced side effects in non-cancerous tissues is still the central goal of cancer research. One strategy to achieve this goal is to modulate the biodistribution of active drugs by reducing their accumulation in critical organ tissues sensitive to side effects while enhancing their uptake into malignant tumors. This can be accomplished by enhancing the hydrophilic properties through coupling of polymers like polyethylene glycol (PEG) to the active agent in order to reduce the unspecific uptake by normal tissues, which can increase the circulation time in PLOS ONE | DOI:10.1371/journal.pone.0157193. This can be accomplished by enhancing the hydrophilic properties through coupling of polymers like polyethylene glycol (PEG) to the active agent in order to reduce the unspecific uptake by normal tissues, which can increase the circulation time in PLOS ONE | DOI:10.1371/journal.pone.0157193 June 13, 2016
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