Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A

Thomas J. Rutkoski; Erin L. Kurten; Julie C. Mitchell; Ronald T. Raines

doi:10.1016/j.jmb.2005.08.007

Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A

Thomas J. Rutkoski, Erin L. Kurten, Julie C. Mitchell, Ronald T. Raines

Research output: Contribution to journal › Article › peer-review

56 Scopus citations

Abstract

Onconase® (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI·RNase A complex having a K_d value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI·RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K_d value of the pRI·RNase A complex by 20×10⁶-fold (to 1.4 μM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 μM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.

Original language	English
Pages (from-to)	41-54
Number of pages	14
Journal	Journal of Molecular Biology
Volume	354
Issue number	1
DOIs	https://doi.org/10.1016/j.jmb.2005.08.007
State	Published - Nov 18 2005
Externally published	Yes

Funding

We are grateful to B. D. Smith for providing a production system for hRI, and to Dr B. G. Miller, Dr M. T. Record Jr, J. E. Lee, S. M. Fuchs, and R. J. Johnson for contributive discussions. This work was supported by grant CA73808 (NIH). T.J.R. was supported by Biotechnology Training grant 08349 (NIH). E.L.K. was supported by a Pfizer Summer Undergraduate Research Fellowship, Wisconsin/Hilldale Undergraduate/Faculty Research Fellowship, University Bookstore Academic Excellence Award, and Trewartha Senior Thesis grant. The University of Wisconsin-Madison Biophysics Instrumentation Facility was established with grants BIR-9512577 (NSF) and RR13790 (NIH). The Keck–UWCCC Small Molecule Screening Facility was established with grants from the W. M. Keck Foundation and University of Wisconsin Comprehensive Cancer Center.

Funders	Funder number
University of Wisconsin Comprehensive Cancer Center
National Science Foundation	RR13790
National Institutes of Health	08349
National Cancer Institute	R01CA073808
W. M. Keck Foundation
Pfizer	BIR-9512577
University of Wisconsin Carbone Cancer Center

Keywords

Cancer
Cytotoxin
Onconase®
Protein-protein interaction
Ribonuclease inhibitor

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.jmb.2005.08.007

Cite this

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title = "Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A",

abstract = "Onconase{\textregistered} (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI·RNase A complex having a Kd value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI·RNase A complex that exhibit a high degree of geometric complementarity. Guided by these {"}knobs{"} and {"}holes{"}, we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the Kd value of the pRI·RNase A complex by 20×106-fold (to 1.4 μM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 μM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.",

keywords = "Cancer, Cytotoxin, Onconase{\textregistered}, Protein-protein interaction, Ribonuclease inhibitor",

author = "Rutkoski, {Thomas J.} and Kurten, {Erin L.} and Mitchell, {Julie C.} and Raines, {Ronald T.}",

year = "2005",

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doi = "10.1016/j.jmb.2005.08.007",

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AU - Rutkoski, Thomas J.

AU - Kurten, Erin L.

AU - Mitchell, Julie C.

AU - Raines, Ronald T.

PY - 2005/11/18

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N2 - Onconase® (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI·RNase A complex having a Kd value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI·RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the Kd value of the pRI·RNase A complex by 20×106-fold (to 1.4 μM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 μM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.

AB - Onconase® (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI·RNase A complex having a Kd value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI·RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the Kd value of the pRI·RNase A complex by 20×106-fold (to 1.4 μM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 μM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.

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KW - Cytotoxin

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KW - Protein-protein interaction

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JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 1

ER -

Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A

Abstract

Funding

Keywords

UN SDGs

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