title: Creation of zebularine-resistant human cytidine deaminase mutants to enhance the chemoprotection of hematopoietic stem cells.
abstract: 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 × 106transformants that conferred significant zebularine resistance relative to wild-type hCDA2. Several variants revealed significantly higher Kivalues toward zebularine when compared with wild-type hCDA values and, as such, are candidates for further exploration for gene-modified HSC transplantation approaches.
authors.affiliation: School of Molecular Biosciences, Washington State University, PO Box 647520, Pullman, WA 99164-7520, USA.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Ruan
authors.affiliation: School of Molecular Biosciences, Washington State University, PO Box 647520, Pullman, WA 99164-7520, USA.|Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: Songbo
authors.initials: S
authors.lastname: Qiu
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Beard
authors.affiliation: School of Molecular Biosciences, Washington State University, PO Box 647520, Pullman, WA 99164-7520, USA blackm@vetmed.wsu.edu.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Black
mesh_terms: D000595:Amino Acid Sequence; D003561:Cytarabine / Q000494:pharmacology; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering
chemical_list: D003561:Cytarabine; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; D003564:Cytidine Deaminase
keywords: cytidine deaminase; cytosine arabinoside; hematopoietic stem cells; random mutagenesis; zebularine