Rapid Inhibition Profiling Identifies a Keystone Target in the Nucleotide Biosynthesis Pathway

• Published in: ACS chemical biology Vol. 13; no. 12; pp. 3251 - 3258
• Main Authors: Peters, Christine E, Lamsa, Anne, Liu, Roland B, Quach, Diana, Sugie, Joseph, Brumage, Lauren, Pogliano, Joe, Lopez-Garrido, Javier, Pogliano, Kit
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.12.2018
• Subjects: Anti-Bacterial Agents - pharmacology; Bacillus subtilis - cytology; Bacillus subtilis - enzymology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriological Techniques - methods; Carrier Proteins - metabolism; Discriminant Analysis; DNA Replication - physiology; Endopeptidase Clp - metabolism; Enzyme Inhibitors - pharmacology; Escherichia coli Proteins - metabolism; Gene Expression Profiling - methods; Nucleoside-Phosphate Kinase - antagonists & inhibitors; Nucleoside-Phosphate Kinase - genetics; Nucleoside-Phosphate Kinase - metabolism; Recombinant Fusion Proteins; Ribonucleotide Reductases - antagonists & inhibitors; Ribonucleotide Reductases - genetics; Ribonucleotide Reductases - metabolism; Teichoic Acids - antagonists & inhibitors; Transferases (Other Substituted Phosphate Groups) - antagonists & inhibitors; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - metabolism
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.8b00273

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.