Exploring the Potent Inhibition of CTP Synthase by Gemcitabine‐5′‐Triphosphate

Abstract

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [K =(3.0±0.1) μm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

“…5c). CTPS kinetic parameters reported for various species vary significantly, but hill coefficients close to 4 have been reported for both activation and inhibition 6,8,[26][27][28] . Remarkably, CTPS2 filaments exhibited an even higher n Hill of 8.3, providing a switchlike response to changes in CTP concentrations (Fig.…”

Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase

“…Cell numbers counted after replace with DMEM increased more quickly in previous culture condition of EBSS + His than that in EBSS alone (EBSS + His: EBSS > 2-fold) (Figure S2F). Treatment with the CTPS inhibitor gemcitabine (McCluskey et al, 2016) substantially reduced cell growth following the addition of DMEM to cells grown in glutamine/serum deprivation and EBSS + His (Figures 2F and S2G). Thus, filament formation positively correlates with cellular proliferation, suggesting that histidine-induced filament formation allows efficient proliferation following nutrient replenishment.…”

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

“…All 4 of these mutations seemed likely to affect general transcription, so we focused on the pyrG mutations. pyrG encodes CTP synthetase (CTPS), an essential enzyme required to make CTP, using UTP, glutamine and ATP as substrates (Mccluskey et al, 2016).…”

“…CTPS has indeed been explored as a potential therapeutic target for a number of infections, especially Mycobacterium tuberculosis (Mori et al, 2015;Esposito et al, 2017;Chiarelli et al, 2018), but also for protozoan infections including trypanosomiasis (Fijolek et al, 2007). Nucleoside analogs acting on CTPS have also been used in human cancer therapies e.g., gemcitabine (Mccluskey et al, 2016); but these tend to have complex off target effects as they can interfere with multiple nucleotide binding proteins and even act as substrates.…”