Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target

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.

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

ABSTRACTHeterogeneous vancomycin-intermediateStaphylococcus aureus(hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10−6or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes,rpoB,rpoC,walK,pbp4, andpp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) wascmk, which encodes cytidylate kinase, followed closely byrpoB(5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number ofcmkmutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity incmkmutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.