A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors

ACS Infect Dis. 2017 Jun 9;3(6):428-437. doi: 10.1021/acsinfecdis.7b00006. Epub 2017 May 11.


Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

Keywords: CTP synthetase; Mycobacterium tuberculosis; drug discovery; phenotypic screening; pyridine-thiazole; target-based screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Antitubercular Agents / chemistry
  • Antitubercular Agents / pharmacology*
  • Bacterial Proteins / antagonists & inhibitors*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding, Competitive
  • Carbon-Nitrogen Ligases / antagonists & inhibitors*
  • Carbon-Nitrogen Ligases / chemistry
  • Carbon-Nitrogen Ligases / genetics
  • Carbon-Nitrogen Ligases / metabolism
  • Drug Discovery
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Gene Expression
  • High-Throughput Screening Assays
  • Kinetics
  • Lipids / antagonists & inhibitors
  • Lipids / biosynthesis
  • Microbial Sensitivity Tests
  • Models, Molecular
  • Molecular Docking Simulation
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / enzymology
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / growth & development
  • Protein Binding
  • Pyridines / chemistry
  • Pyridines / pharmacology*
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology
  • Structure-Activity Relationship
  • Thiazoles / chemistry
  • Thiazoles / pharmacology*


  • Antitubercular Agents
  • Bacterial Proteins
  • Enzyme Inhibitors
  • Lipids
  • Pyridines
  • Small Molecule Libraries
  • Thiazoles
  • Adenosine Triphosphate
  • Carbon-Nitrogen Ligases
  • CTP synthetase