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

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.