Journal of Biomolecular Screening 2016, Vol. 21(7) 695–700 © 2016 Society for Laboratory Automation and Screening DOI: 10.1177/1087057116646702 jbx.sagepub.com Original Research Introduction In 2013, 1.5 million people died from tuberculosis (TB), making it the second leading cause of death due to a single infectious organism in the world, behind HIV/AIDS. 1 Also that year, multi-drug-resistant TB (MDR-TB) was esti- mated to have developed in 480,000 people, 1 highlighting the emergence of MDR-TB and the need for new more potent drugs against the disease. Novel drugs against TB can be identified by screening molecular libraries for inhibition of potential protein tar- gets. One such pool of potential targets includes proteins that have been structurally characterized by high-resolution X-ray crystallography. Since these proteins have already been expressed, purified, and crystalized, lead hit com- pounds may be co-crystalized with the target protein pro- viding vital information for the design of compound variants with increased selectivity, binding efficiency, and potency. Unfortunately, many of the proteins that would be good candidates for screening have not been structurally character- ized because they fail somewhere along the gene-to-structure pipeline. Indeed, this applies to approximately 90% of the Mycobacterium tuberculosis (Mtb) proteomes attempted and is a major barrier to drug development. 2 While genetic engineer- ing of problematic targets to introduce truncations, point mutations, and alternative affinity tags can rescue the target for structure determination, this process is labor-intensive and not conducive for implementation in a high-throughput (HT) screening format. An alternative approach is to use homologue proteins from closely related species, that is, orthologues, 646702JBX XX X 10.1177/1087057116646702Journal of Biomolecular ScreeningCraig et al. research-article 2016 1 Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA, USA 2 Fred Hutchinson Cancer Research Center (Fred Hutch), Genomics Shared Resource, High-Throughput Screening Facility, Seattle, WA, USA 3 Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, USA 4 Center for Infectious Disease Research (CIDR), Seattle, WA, USA 5 Department of Biomedical Informatics, University of Buffalo, State University of New York, Buffalo, NY, USA 6 Departments of Global Health and Microbiology, University of Washington, Seattle, WA, USA Received Nov 30, 2015, and in revised form April 4, 2016. Accepted for publication April 5, 2016. Corresponding Author: Wesley C. Van Voorhis, MD, PhD, Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Box 358061, Seattle, WA, USA. Email: wesley@uw.edu Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target Justin K. Craig 1 , Jenni K. Risler 2 , Kimberly A. Loesch 3 , Wen Dong 3 , Dwight Baker 3 , Lynn K. Barrett 1 , Sandhya Subramanian 4 , Ram Samudrala 5 , and Wesley C. Van Voorhis 1,6 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. Keywords tuberculosis, cytidylate kinase, drug development, high-throughput screening, Mycobacterium tuberculosis