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Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction
1Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.
Insights
Researchers enhanced human ribonucleases (hRNAse) and eosinophil-derived neurotoxins (EDN) to resist inhibitors. Transferrin conjugates showed significantly increased cancer cell toxicity, offering a novel targeted cancer therapy approach.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Nonmammalian RNase A superfamily members show anticancer activity linked to ribonuclease inhibitor (RI) resistance.
- Human ribonucleases (hRNAse) and eosinophil-derived neurotoxins (EDN) are sensitive to RI, limiting their therapeutic potential.
Purpose of the Study:
- To enhance the anticancer activity of hRNAse and EDN by increasing their resistance to RI.
- To develop targeted cancer therapies using modified human ribonucleases.
Main Methods:
- Mutated hRNAse and EDN to introduce cysteine residues at sites interacting with RI, distant from catalytic domains.
- Created thioether-linked conjugates by coupling mutated ribonucleases to transferrin for cellular targeting and RI blocking.
- Assessed the enzymatic activity, RI resistance, and cytotoxicity of the engineered conjugates against cancer cells.
Main Results:
- Engineered hRNAse and EDN conjugates demonstrated resistance to RI while retaining enzymatic activity.
- Transferrin-conjugated rhRNase(Gly89-->Cys) exhibited a 5000-fold increase in toxicity against U251 cells compared to wild-type hRNase.
- Transferrin-targeted EDN showed comparable tumor cell toxicities to the enhanced hRNase.
Conclusions:
- Increasing RI resistance enhances the cytotoxicity of human ribonucleases.
- Transferrin-mediated targeting of RI-resistant ribonucleases offers a promising strategy for developing novel, targeted cancer therapies.
- This approach has the potential to create a new class of recombinant human proteins for cancer treatment.