In silico and in vitro evaluation of a PE38 and Nb-based recombinant immunotoxin targeting the GRP78 receptor in
Mona Khoshbakht1, Mohammad Mahdi Forghanifard1, Hossein Aghamollaei2
1Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.
Abstract:
Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post-treatment issues. Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps. Two constructs were designed from a single-variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU-145 and breast cancer MDA-MB-468 cell lines were used as GRP78-positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme-linked immunosorbent assay, and western blot. The results showed that in the DU-145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time-dependent. In MDA-MB-468 and HEK-293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78-positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78-positive tumor cells.
Insights
Researchers designed novel recombinant immunotoxins targeting GRP78 receptors on cancer cells. These targeted therapies show potential for effective cancer treatment by inducing cancer cell death while sparing healthy cells.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Cancer remains a significant global health challenge despite advanced therapies.
- Targeted drug delivery enhances treatment efficacy and minimizes side effects.
- The GRP78 receptor is a potential target on tumor cells for selective therapy.
Purpose of the Study:
- To design and evaluate novel recombinant immunotoxins targeting the GRP78 receptor on cancer cells.
- To create a fusion protein combining nanobody domains with PE exotoxin for targeted cancer therapy.
- To assess the therapeutic potential of these constructs using bioinformatics and in vitro methods.
Main Methods:
- Bioinformatics tools were used for designing and evaluating two recombinant protein constructs.
- Constructs comprised nanobody domains and PE exotoxin domains (II, Ib, III).
- In vitro assays (MTT, ELISA, Western blot) evaluated cytotoxicity, binding affinity, internalization, and apoptosis in GRP78-positive (DU-145) and negative (MDA-MB-468) cell lines.
Main Results:
- The designed recombinant immunotoxins demonstrated dose- and time-dependent cytotoxicity specifically in GRP78-positive DU-145 prostate cancer cells.
- No significant cytotoxicity was observed in GRP78-negative MDA-MB-468 breast cancer cells or HEK-293 cells.
- In vitro findings correlated with in silico bioinformatics predictions, validating the design approach.
Conclusions:
- The developed recombinant immunotoxins show therapeutic promise for targeting GRP78-positive tumors.
- These agents can potentially eradicate cancer cells through targeted internalization and apoptosis induction.
- The study highlights the efficacy of combining bioinformatics with experimental validation for novel cancer therapeutic development.
