A Small Molecule Inhibitor of CTP Synthetase Identified by Differential Activity on a Bacillus subtilis Mutant Deficient in Class A Penicillin-Binding Proteins

Abstract

In the course of screening for compounds with differential growth inhibition activity on a mutant of Bacillus subtilis lacking all four class A penicillin-binding proteins (4), we came across an isoquinoline derivative, IQ-1 carboxylic acid (IQC) with relatively high activity on the mutant compared to the wild type strain. Treated cells were slightly elongated and had altered chromosome morphology. Mutants of 4 resistant to IQC were isolated and subjected to whole genome sequencing. Most of the mutants were affected in the gene, pyrG, encoding CTP synthetase (CTPS). Purified wild type CTPS was inhibited in vitro by IQC. Two of the three mutant proteins purified showed decreased sensitivity to IQC in vitro. Finally, inhibition by IQC was rescued by addition of cytidine but not uridine to the growth medium, consistent with the notion that IQC acts by reducing the synthesis of CTP or a related compound. IQC provides a promising new starting point for antibiotic inhibitors of CTPS.

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century

“…The revelation that competence and chromosome segregation rely on a CTP-dependent activity leads to a conjecture where CTP synthesis is the unique node that couples growth, energy and metabolism. This is supported by the observation that CTP synthase (PyrG) plays a key role in growth homeostasis in B. subtilis (Emami et al, 2020), a feature consistent with the conservation of the gene in the synthetic minimal genome of M. mycoides Syn3.0 and in the minimal genomes of cell growing in cytosine derivative-containing media (Breuer et al, 2019;Danchin & Fang, 2016;Hutchison et al, 2016). Strikingly, this role was corroborated after a study of SARS-CoV-2 multiplication aimed at deciphering why the initial evolutionary trend of the virus led to the loss of cytosine residues in its RNA genome (Ou et al, 2020).…”

CTP synthase: the hissing of the cellular serpent

“…These inhibitors suppress RNA synthesis and induce cytotoxicity in proliferating cells (Marquez et al 1988 ) Antibiotic resistance (by Bacillus subtilis ) To develop anti-infectives, PyrG, CTPsyn is targeted by isoquinoline compounds, which have a higher antibiotic effect on B. subtilis mutant lacking all four class A penicillin-binding proteins (PBPs). Since the effect of polymerization of CTPsyn in human and bacteria are completely opposite, it could be a therapeutic target to achieve desired inhibition specificity (Emami et al 2020 ) Respiratory tract infection (RTI) (caused by Streptococcus pneumoniae , Staphylococcus aureus, Haemophilus influenzae ) 2-(3-[3-oxo-1,2-Benzisothiazol-2(3H)-yl]phenylsulfonylamino) benzoic acid (compound G1) could be the potential anti-microbial agent that targets PyrG, CTPsyn. Compound G1 could compete with ATP and/or UTP to bind with PyrG in order to inhibit CTPsyn activity.…”