title: Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target.
abstract: New and improved drugs against tuberculosis are urgently needed as multi-drug-resistant forms of the disease become more prevalent. Mycobacterium tuberculosis cytidylate kinase is an attractive target for screening due to its essentiality and different substrate specificity to the human orthologue. However, we selected the Mycobacterium smegmatis cytidylate kinase for screening because of the availability of high-resolution X-ray crystallographic data defining its structure and the high likelihood of active site structural similarity to the M. tuberculosis orthologue. We report the development and implementation of a high-throughput luciferase-based activity assay and screening of 19,920 compounds derived from small-molecule libraries and an in silico screen predicting likely inhibitors of the cytidylate kinase enzyme. Hit validation included a counterscreen for luciferase inhibitors that would result in false positives in the initial screen. Results of this counterscreen ruled out all of the putative cytidylate kinase inhibitors identified in the initial screening, leaving no compounds as candidates for drug development. Although a negative result, this study indicates that this important drug target may in fact be undruggable and serve as a warning for future investigations.
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA, USA.
authors.forename: Justin K
authors.initials: JK
authors.lastname: Craig
authors.affiliation: Fred Hutchinson Cancer Research Center (Fred Hutch), Genomics Shared Resource, High-Throughput Screening Facility, Seattle, WA, USA.
authors.forename: Jenni K
authors.initials: JK
authors.lastname: Risler
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Loesch
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, USA.
authors.forename: Dwight
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA, USA.
authors.forename: Lynn K
authors.initials: LK
authors.lastname: Barrett
authors.affiliation: Center for Infectious Disease Research (CIDR), Seattle, WA, USA.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Subramanian
authors.affiliation: Department of Biomedical Informatics, University of Buffalo, State University of New York, Buffalo, NY, USA.
authors.forename: Ram
authors.initials: R
authors.lastname: Samudrala
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA, USA Departments of Global Health and Microbiology, University of Washington, Seattle, WA, USA wesley@uw.edu.
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
mesh_terms: D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000627:therapeutic use; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D058990:Molecular Targeted Therapy; D020102:Mycobacterium smegmatis / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000472:pathogenicity; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics; D054852:Small Molecule Libraries / Q000032:analysis; D013379:Substrate Specificity; D014376:Tuberculosis / Q000188:drug therapy* / Q000201:enzymology / Q000382:microbiology
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase
keywords: Mycobacterium tuberculosis; cytidylate kinase; drug development; high-throughput screening; tuberculosis