Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation
| Autor: | Daria Nawrocka, Aleksandra Sokolowska-Wedzina, Karolina Jendryczko, Mateusz Adam Krzyscik, Natalia Porębska, Marta Pozniak, Malgorzata Zakrzewska, Lukasz Opalinski, Jacek Otlewski, Anna Szlachcic |
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| Rok vydání: | 2021 |
| Předmět: |
General Chemical Engineering
Antineoplastic Agents Protein Engineering Endocytosis General Biochemistry Genetics and Molecular Biology Maleimides chemistry.chemical_compound Bacterial Proteins Protein Domains Sortase Neoplasms Humans Cytotoxic T cell Sulfhydryl Compounds Cell Death General Immunology and Microbiology Chemistry General Neuroscience Protein engineering Aminoacyltransferases Immunoglobulin Fc Fragments Vesicular transport protein Cysteine Endopeptidases Monomethyl auristatin E Biochemistry Sortase A Cancer cell Fibroblast Growth Factor 2 Oligopeptides |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61918 |
| Popis: | Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers. |
| Databáze: | OpenAIRE |
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