Conjugation of Folate via Gelonin Carbohydrate Residues Retains Ribosomal-inactivating Properties of the Toxin and Permits Targeting to Folate Receptor Positive Cells

Abstract

Conjugation of folate to proteins permits receptormediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SHfolate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10 ؊10 M). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.

Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF–saporin

“…The binding forms rapidly and is convenient for proof-of-concept studies as those performed in paper I and II. One of the obstacles of conjugating a targeting ligand to a protein-toxin is loss of effect of the toxic moiety (Atkinson et al 2001). Linking of EGF to saporin through the biotin-streptavidin binding did not influence the RIP activity of saporin (paper I), and this binding seems therefore promising for screening and evaluation of targeting ligands for PCI-mediated delivery of protein toxins.…”

Molecular tumor targeting of gelonin by fusion with F3 peptide

“…It is a 30 kDa glycoprotein that was first derived from the seeds of Gelonium multiflorum. As a ribosome-inactivating protein (RIP) toxin, gelonin inhibits protein synthesis via the cleavage of a single adenine residue (A 4324 ) in the 28S ribosomal RNA [23] . The potency of gelonin in inhibiting protein translation is so high that even a single gelonin molecule can kill one tumor cell after cellular uptake [23] .…”

“…As a ribosome-inactivating protein (RIP) toxin, gelonin inhibits protein synthesis via the cleavage of a single adenine residue (A 4324 ) in the 28S ribosomal RNA [23] . The potency of gelonin in inhibiting protein translation is so high that even a single gelonin molecule can kill one tumor cell after cellular uptake [23] . However, despite the high potency of gelonin, its clinical translation as an anticancer drug remains a challenge because of its poor cellular uptake.…”

Preparation, characterization and targeting of micronized 10-hydroxycamptothecin-loaded folate-conjugated human serum albumin nanoparticles to cancer cells

“…Importantly, HSA offers the benefits of being able to carry functional groups (ie, amino and carboxylic groups) that could be used for surface modifications. 25 Therefore, folate as a drug targeting ligand has often been used in the recent years for cancer-targeted drug delivery because of its high affinity (10 −10 M) 26 through association with albumin.…”