Abstract
A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.
Highlights
Published: 28 December 2021Methotrexate (MTX) is an antiproliferative and immunosuppressive agent widely and effectively used against a broad spectrum of diseases, primarily cancerous tumors and certain autoimmune diseases such as psoriasis and rheumatoid arthritis [1]
We found that bovine serum albumin (BSA)–S–S–MTX is slightly less potent than BSA–(CH2)6–MTX, both in the sensitive and the insensitive glioma cell lines (Figure 4)
We have engineered, synthesized, and studied conjugates of bovineserum albumin linked to methotrexate (BSA–MTX conjugates)
Summary
Methotrexate (MTX) is an antiproliferative and immunosuppressive agent widely and effectively used against a broad spectrum of diseases, primarily cancerous tumors and certain autoimmune diseases such as psoriasis and rheumatoid arthritis [1]. A variety of tumor cells develop resistance to MTX upon treatment with agents, affecting several mechanistic pathways, including impaired transport of MTX into cells [4,5] Solving these deficiencies and increasing MTX selectivity toward inflamed, diseased, or malignant tissues is of primary clinical significance. Binding MTX to other macromolecules, such as anti-TNFα antibodies, might be clinically beneficial in autoimmune and inflammatory diseases such as arthritis, for example, where MTX is administrated at low dosages separately from anti-TNFα antibodies Generating such an antibody–MTX conjugate may reduce side effects and improve efficacy as well as provide better treatment tolerance. Two glioma cell lines are investigated here, for the first time, in the context of albumin conjugates to align with other research in our lab, in which new compounds and methods that enable blood–brain barrier (BBB) opening, and the entrance of large therapeutic molecules such as albumin are being developed [14,15,16,17,18]
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