Creation of zebularine-resistant human cytidine deaminase mutants to enhance the chemoprotection of hematopoietic stem cells
Human cytidine deaminase (hCDA) is a biomedically important enzyme able to inactivate cytidine nucleoside analogs such as the antileukemic agent cytosine arabinoside (AraC) and thereby limit antineoplastic efficacy. Potent inhibitors of hCDA have been developed, e.g. zebularine, that when administer...
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Published in | Protein engineering, design and selection Vol. 29; no. 12; pp. 573 - 582 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.12.2016
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Subjects | |
Online Access | Get full text |
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Summary: | Human cytidine deaminase (hCDA) is a biomedically important enzyme able to inactivate cytidine nucleoside analogs such as the antileukemic agent cytosine arabinoside (AraC) and thereby limit antineoplastic efficacy. Potent inhibitors of hCDA have been developed, e.g. zebularine, that when administered in combination with AraC enhance antineoplastic activity. Tandem hematopoietic stem cell (HSC) transplantation and combination chemotherapy (zebularine and AraC) could exhibit robust antineoplastic potency, but AraC-based chemotherapy regimens lead to pronounced myelosuppression due to relatively low hCDA activity in HSCs, and this approach could exacerbate this effect. To circumvent the pronounced myelosuppression of zebularine and AraC combination therapy while maintaining antineoplastic potency, zebularine-resistant hCDA variants could be used to gene-modify HSCs prior to transplantation. To achieve this, our approach was to isolate hCDA variants through random mutagenesis in conjunction with selection for hCDA activity and resistance to zebularine in an Escherichia coli genetic complementation system. Here, we report the identification of nine novel variants from a pool of 1.6 × 10
transformants that conferred significant zebularine resistance relative to wild-type hCDA2. Several variants revealed significantly higher K
values toward zebularine when compared with wild-type hCDA values and, as such, are candidates for further exploration for gene-modified HSC transplantation approaches. |
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Bibliography: | Edited by Steven Russell Present address: Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. |
ISSN: | 1741-0126 1741-0134 |
DOI: | 10.1093/protein/gzw012 |