Endowing human pancreatic ribonuclease with toxicity for cancer cells
P A Leland1, K E Staniszewski, B M Kim
1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Abstract:
Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
Insights
Researchers engineered a human ribonuclease 1 (RNase 1) variant, ERDD RNase 1, to be cytotoxic to human leukemia cells. This modified enzyme shows promise for cancer chemotherapy, demonstrating that few amino acid changes can create a potent anti-cancer agent.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Onconase, an amphibian protein, is in Phase III trials for cancer chemotherapy.
- Human pancreatic ribonuclease 1 (RNase 1) is homologous to Onconase but lacks cytotoxicity.
- Endogenous ribonuclease inhibitor protein typically neutralizes RNase 1 activity in human cells.
Purpose of the Study:
- To engineer a human RNase 1 variant with enhanced cytotoxicity against cancer cells.
- To investigate the impact of specific amino acid substitutions and disulfide bonds on enzyme stability and activity.
- To explore the potential of modified human RNase 1 as a novel cancer chemotherapeutic agent.
Main Methods:
- Site-directed mutagenesis was used to create the ERDD RNase 1 variant (L86E/N88R/G89D/R91D).
- The variant enzyme's conformational stability and ribonucleolytic activity were assessed.
- Binding affinity to the human ribonuclease inhibitor protein was measured.
- Cytotoxicity of ERDD RNase 1 against human leukemia cells was determined.
- The effect of a non-native disulfide bond on enzyme properties and cytotoxicity was evaluated.
Main Results:
- ERDD RNase 1 exhibited similar conformational stability and ribonucleolytic activity to wild-type RNase 1.
- ERDD RNase 1 demonstrated significantly reduced affinity (>10^3-fold) for the endogenous ribonuclease inhibitor protein.
- ERDD RNase 1 displayed potent cytotoxicity against human leukemia cells.
- Introducing a non-native disulfide bond further enhanced conformational stability and cytotoxicity, with an IC(50) value approaching that of Onconase.
Conclusions:
- Specific amino acid substitutions can render human RNase 1 cytotoxic to cancer cells.
- ERDD RNase 1 represents a promising candidate for human cancer chemotherapy.
- Minimal genetic modification of human proteins can yield potent anti-cancer therapeutics, offering new avenues for drug development.
