CTP Synthetase Research Articles

Advances in human glutamine-hydrolyzing synthetases and their therapeutic potential

Bifunctional enzymes, characterized by their dual active sites, enable efficient chemical conversion and substrate channeling using elegant coupling mechanisms to coordinate the two active sites. In humans, several bifunctional enzymes synthesize de novo carbon-nitrogen bonds by hydrolyzing glutamine and ATP in distinct active sites. Notable examples include guanosine monophosphate synthetase, cytidine triphosphate synthetase, phosphoribosylformyl-glycinamidine synthase, asparagine synthetase, and nicotinamide adenine dinucleotide synthetase. A more complex example of multifunctional glutamine-hydrolyzing synthetases in humans is carbamoyl phosphate synthetase. These enzymes are crucial for the biosynthesis of amino acids, nucleic acids, and co-factors, thereby playing pivotal roles in human health. This review delineates recent progress in understanding the structural characteristics, regulatory mechanisms, and disease relevance of glutamine-hydrolyzing synthetases in humans. Insights into their catalysis and activity regulation offer potential pathways for developing novel therapeutics.

To Explore Potential Inhibitors Against Various Enzymatic Targets of Human African Trypanosomiasis.

Synthetic drugs currently prescribed for the treatment of Human African Trypanosomiasis (HAT) are non-specific, toxic, demand extended therapeutic regimes and are of varying efficacy. Along with the challenging demographic and socio-economic hurdles, the everincreasing risk of drug resistance is another major problem to be addressed. Cysteine protease, Heat shock proteins (HSP-90), Trypanothione reductase (TR), Farnesyl diphosphate synthase, Glucose-6-phosphate dehydrogenase, UP-4-galactose epimerase, and Cytidine triphosphate synthetase are potential enzymatic targets for the development of novel inhibitors against HAT which are the main focus of this review. The potential enzymatic targets of Trypanosoma brucei, especially small molecules like cysteine proteases and heat shock proteins are identified as major candidates for the sustenance of the parasite, their proliferation, infection, and spread of the disease. The development of new compounds to combat the disease by thorough ligand modification has been explored in the current review. Extracting these compounds and studying their efficacy, toxicity, and target mechanism extensively, this review has proposed a list of different compounds, including some synthetic and natural compounds along with multi-target inhibitors such as acoziborole, fexinidazole, etc. Potential inhibitors against these enzymatic targets of the T. brucei are important candidates for designing novel therapeutics against HAT. Multi-target inhibitors have also been identified as crucial molecules because of their potential advantage against the development of drug resistance.

A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck

Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h.Graphical

Modularized reconstruction of pyrimidine pathway in Escherichia coli for enhanced pyrimidine nucleosides biosynthesis

Pyrimidine is a fundamental component of genetic material and a precursor for synthesis of various antiviral and antitumor drugs. In this study, we constructed a plasmid-free Escherichia coli strain HD11 as a platform for cytidine production. Compared to strain CR23-p21-p31, a higher titer of cytidine was achieved by overexpressing uridylate kinase from Sulfurovum lithotrophicum and the newly discovered cytidine triphosphate synthetase of Aquifex aeolicus in HD11. To enable efficient cytosine biosynthesis, a modularized metabolic pathway was integrated into HD11. Two different pathways based on the type of precursor were compared, and ribosome binding sites were optimized to enhance cytosine production. For pyrimidine ribonucleotide de novo biosynthesis pathway, an enzyme constrained metabolic model was utilized to guide media optimization. Finally, HD11-pDA20 produced 3.6 g/L cytosine and HD11 produced 8.1 g/L cytidine in fed-batch fermentation. The application of an enzyme-constrained metabolic model in cytidine/cytosine producing strain represents a novel approach to modular engineering.

Differential roles of CTP synthetases CTPS1 and CTPS2 in cell proliferation.

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.

HIF-1α mediates osteoclast-induced disuse osteoporosis via cytoophidia in the femur of mice.

Osteoporosis induced by disuse because of bed rest or the aerospace industry has become one of the most common skeletal disorders. However, mechanisms underlying the disuse osteoporosis remain largely unknown. We validated the tail-suspended model in mice and demonstrated that there is bone loss in the trabecular and cortical bones of the femur. Importantly, we showed that genetical deletion of hypoxia-inducible factor-1α (HIF-1α) in osteoclasts ameliorated osteoclastic bone resorption in the trabecular bone whereas pharmacological treatment with HIF-1α inhibitor protected the hindlimb-unloaded mice from disuse-induced osteoporosis in the trabecular and cortical bones. The HIF-1α knockout RAW264.7 cells and RNA-sequencing proved that HIF-1α is vital for osteoclastogenesis and bone resorption because it regulated the level of inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase (CTPS) via cellular myelocytomatosis (c-Myc) oncogene. The IMPDH and CTPS are vital nucleotide metabolic enzymes which have an important functional role in cell metabolism, and they can assemble into intracellular linear or ring-shaped structures to cope with cell stress. Interestingly, both in vitro and in vivo, the IMPDH and CTPS cytoophidia were found in osteoclasts, and the level of HIF-1α correlated with osteoclastogenesis and bone-resorbing activity. Our data revealed that HIF-1α/c-Myc/cytoophidia signalling might be required for osteoclasts to mediate cell metabolism in disuse-induced osteoporosis. Overall, our results revealed a new role of HIF-1α/c-Myc/cytoophidia in supporting osteoclastogenesis and bone resorption and exposed evidence for its role in the pathogenesis of disuse osteoporosis, which might provide promising therapeutic targets.

Discovery and Optimization of Potent and Orally Available CTP Synthetase Inhibitors for Use in Treatment of Diseases Driven by Aberrant Immune Cell Proliferation.

Herein, we report the discovery of a first-in-class chemotype 2-(alkylsulfonamido)thiazol-4-yl)acetamides that act as pan-selective inhibitors of cytidine 5'-triphosphate synthetase (CTPS1/2), critical enzymes in the de novo pyrimidine synthesis pathway. Weak inhibitors identified from a high-throughput screening of 240K compounds have been optimized to a potent, orally active agent, compound 27, which has shown significant pharmacological responses at 10 mg/kg dose BID in a well-established animal model of inflammation.

STP938, a Selective CTPS1 Inhibitor, Shows Single Agent Activity and Synergy with BCL2 Inhibition in Preclinical Models of Mantle Cell Lymphoma

Background Proliferating tumor cells are dependent on nucleotide synthesis to support DNA replication and RNA synthesis. The homologous CTP synthetases, CTPS1 and CTPS2, play a central role by catalyzing the rate-limiting conversion of UTP to CTP. Interestingly, while most tissues rely on CTPS2, lymphoid cells depend on CTPS1, as observed in rare patients with inherited CTPS1 loss of function manifesting selective B/T cell defects but no non-hematopoietic phenotype (Martin 2014, PMID 24870241). Herein, we investigate inhibition of CTPS1, alone or in combination, as a potential novel therapeutic target in mantle cell lymphoma (MCL). Methods STP938 (Step Pharma) is a highly selective small molecule inhibitor of CTPS1 with >1,300-fold selectivity over CTPS2. In vitro cell proliferation was assessed at 72 hours by CellTiter-Glo. Synergy was quantified by Bliss scores. 3'seq-RNA profiling was performed before and after a 24-hour exposure to STP938; normalized data were subjected to KEGG pathway enrichment analysis. In vivo efficacy was established following subcutaneous transplantation of MCL cells (Z138) into immunodeficient mice (NSG, n=5 per treatment group); treatment was initiated when tumors reached approximately 50 mm3. STP938 was dosed at 30 mg/kg/day subcutaneously days 1-4 of a 7 day cycle; venetoclax at 75 mg/kg/day orally days 2-5 of a 7 day cycle for three cycles. Tumor size was assessed by calipers. Results Analysis of the Achilles CRISPR dataset identified a dependency of human B cell lymphoma cell lines (n=34) on the de novo but not the salvage pyrimidine synthesis pathway. Of note, whilst CRISPR deletion of CTPS1 resulted in a marked proliferation defect, CTPS2 loss had no effect, consistent with the central role for CTPS1 in lymphoid cells established in human genetic studies. Analysis of publicly available data from primary MCL samples (n=122, GSE93291) identified high CTPS1 expression as a predictor of inferior overall survival (P=<0.0001). A selective CTPS1 inhibitor, STP938, was used to evaluate a potential therapeutic role for CTPS1 inhibition. Of 15 MCL cell lines tested, 6 (40%) were highly sensitive to STP938 (IC50 <0.1 μM), 5 (33%) showed intermediate sensitivity (IC50 0.1 - 1 μM) and 4 (27%) were resistant (IC50 >1 μM). Of note, sensitivity to STP938 was independent of known indicators of poor prognosis, such as TP53 mutation, and was independent of resistance to standard of care therapies such as ibrutinib or venetoclax. Pathway analysis of transcriptomic data (n=9 cell lines) identified downregulation of cell cycle pathways in response to STP938 exposure. Directed analysis identified modulation of BCL2-family transcripts, with STP938 inducing increases in BCL2 and BCL2L1 (BCL-XL) expression and a decrease in MCL1 expression (P=0.02, 0.006 and 0.02, respectively; paired t-test). BCL2 increase and MCL1 decrease upon CTPS1 inhibition have been further confirmed at the protein level (24h, 20nM). Given these findings, in vitro combination studies were undertaken with a selective BCL2 inhibitor. STP938 plus venetoclax demonstrated synergy in 5 of 9 MCL cell lines tested (Figure A). Of note, there was no association between synergy and sensitivity to single agent STP938, with two cell lines resistant to single agent STP938 (IC50 3.1 and >10 μM) nonetheless showing synergy with venetoclax. The synergy between STP938 and venetoclax was further investigated in an in vivo model of MCL. Combination therapy was well tolerated. Single agent venetoclax showed no significant inhibition of tumor growth. Single agent STP938 slowed tumor growth to approximately 50% of vehicle control. The combination of STP938 and venetoclax showed strong synergy with more than 80% inhibition of tumor growth for 3 cycles of treatment (Figure B). Conclusions Dependence of tumor cells on elevated rates of nucleotide synthesis has been exploited by agents such as cytarabine. Whilst therapy for hematological cancers is moving away from cytotoxic drugs in favor of targeted agents, inhibition of nucleotide synthesis remains an attractive target. STP938, a highly selective CTPS1 inhibitor, demonstrates single agent activity in vitro and in vivo, and shows strong synergy with the approved BCL2 inhibitor venetoclax. STP938 will enter clinical development for relapsed refractory B and T cell lymphomas in 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Zinc(II) Complex with Pyrazolone-Based Hydrazones is Strongly Effective against Trypanosoma brucei Which Causes African Sleeping Sickness.

Two pyrazolone-based hydrazones H2L′ [in general, H2L′; in detail, H2L1 = 5-methyl-2-phenyl-4-(2-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)-2,4-dihydro-3H-pyrazol-3-one, H2L2 = (Z)-5-methyl-2-phenyl-4-(2-phenyl-1-(2-(pyridin-2-yl)hydrazineyl)ethylidene)-2,4-dihydro-3H-pyrazol-3-one] were reacted with Zn(II) and Cu(II) acceptors affording the complexes [Zn(HL1)2(MeOH)2], [Cu(HL1)2], and [M(HL2)2] (M = Cu or Zn). X-ray and DFT studies showed the free proligands to exist in the N–H,N–H tautomeric form and that in [Zn(HL1)2(MeOH)2], zinc is six-coordinated by the N,O-chelated (HL1) ligand and other two oxygen atoms of coordinated methanol molecules, while [Cu(HL1)2] adopts a square planar geometry with the two (HL1) ligands in anti-conformation. Finally, the [M(HL2)2] complexes are octahedral with the two (HL2) ligands acting as κ-O,N,N-donors in planar conformation. Both the proligands and metal complexes were tested against the parasite Trypanosoma brucei and Balb3T3 cells. The Zn(II) complexes were found to be very powerful, more than the starting proligands, while maintaining a good safety level. In detail, H2L1 and its Zn(II) complex have high selective index (55 and >100, respectively) against T. brucei compared to the mammalian Balb/3T3 reference cells. These results encouraged the researchers to investigate the mechanism of action of these compounds that have no structural relations with the already known drugs used against T. brucei. Interestingly, the analysis of NTP and dNTP pools in T. brucei treated by H2L1 and its Zn(II) complex showed that the drugs had a strong impact on the CTP pools, making it likely that CTP synthetase is the targeted enzyme.

P864: CTPS1 IS A NOVEL THERAPEUTIC TARGET IN MYELOMA - SELECTIVE SMALL MOLECULE INHIBITION DELIVERS SINGLE AGENT ACTIVITY AND SYNERGISES WITH ATR INHIBITION

Background: Recent years have seen major improvements in the treatment of myeloma; however, relapse occurs in the majority of patients. Novel treatment approaches are moving away from cytotoxic chemotherapy towards targeted therapies with defined mechanisms of action. Malignant cells have an increased demand for key cellular components and a reliance on de novo synthesis pathways. CTP synthetase 1 (CTPS1) plays a pivotal role in pyrimidine production, by catalysing the rate limiting step in the de novo synthesis of CTP, which is required for DNA, RNA and phospholipids. Human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation which is complemented by the homologous CTPS2 isoform outside the haemopoietic system. Aims: To evaluate the role of CTPS1 as a novel target in myeloma, elucidate the molecular consequences of CTPS1 inhibition and test rationally designed combination therapy. Methods: STP938 is a potent small molecule CTPS1 inhibitor with >1,300-fold selectivity for CTPS1 over CTPS2. The effects of STP938 on cell proliferation (metabolic activity, tetrazolium indicator), apoptosis (annexin V) and cell cycle (propidium iodide) were assessed in vitro using 12 myeloma cell lines. The role of CTPS1 was further assessed in CRISPR knock-out (KO) experiments. Activation of proteins in the replication stress and DNA damage response pathways was analysed by western blotting. Additive/synergistic effects of combining STP938 with two different ATR inhibitors were assessed on cell line proliferation and enumerated by Bliss score (Synergyfinder 2.0). Results: STP938 showed single agent activity against 6 of 12 cell lines tested, with IC50 values for sensitive lines ranging from 19 to 128 nM (Figure A). STP938 showed cytotoxic activity against sensitive lines, evidenced by induction of apoptosis. KO experiments confirmed that MM cell lines depend on CTPS1 for proliferation. Exposure to STP938 was associated with accumulation of cells in S phase, induction of a replication stress response as evidenced by activation (phosphorylation) of CHEK1, and induction of the DNA damage response pathway as evidenced by activation of CHEK2 (albeit to a lesser extent than activation of CHEK1). Importantly, S phase accumulation, induction of replication stress and induction of DNA damage response was observed in cell lines where STP938 did not produce significant anti-proliferative activity (Figure B). Given the ability of STP938 to activate CHEK1, STP938 was tested in combination with an ATR inhibitor, as ATR is the main upstream activator of CHEK1. The ATR inhibitor ceralasertib demonstrated additive or synergistic activity when combined with STP938 (Figure C). Notably, synergy between STP938 and ATR inhibition, along with induction of apoptosis, was observed in cell lines resistant to single agent STP938. Results were confirmed with a second ATR inhibitor (VE-821, Figure C). Image:Summary/Conclusion: Inhibition of CTPS1 by single agent STP938 showed anti-proliferative activity against 6 of 12 myeloma cell lines, and induced replication stress in all cell lines. Combined STP938 and ATR inhibition showed anti-proliferative activity in all cell lines, including those resistant to single agent STP938. These data suggest a model whereby myeloma cells with high background replication stress are sensitive to killing by single agent STP938, whereas cells with lower background replication stress are sensitised by STP938 to cell death induced by ATR inhibition (Figure D). STP938 will shortly enter clinical development for patients with late stage lymphoid neoplasms.

Pharmacophore mapping of the crucial mediators of dual inhibitor activity of PanK and PyrG in tuberculosis disease

ABSTRACT Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis, responsible for 1.66 million documented deaths in 2020. The increase in reported cases of M. tuberculosis resistance to the main drugs shows the need to develop new and efficient drugs for better TB control. A strategy to overcome the resistance problem could be the polypharmacology approach, whereby researchers develop a single molecule that acts on different targets. Polypharmacology could have features that make it more effective than classical polypharmacy, in which investigators pull various drugs with high affinity for one target together. Based on the effectiveness of multitargeting, this work aimed to use combined in silico techniques to identify potential dual inhibitors for Pantothenate Kinases (PanK) and CTP synthetase PyrG. In silico drug design allows for screening of potential leads, thus decreasing time and resource consumption. This study demonstrates an optimised and proven screening technique to discover a potential small-molecule inhibitor of Mycobacterium tuberculosis PanK and PyrG. The favourable binding free energy values and interaction profiles show ZINC53995076 as a potent dual inhibitor of the Mycobacterium tuberculosis PanK and PyrG.

Nuclear IMPDH Filaments in Human Gliomas.

The analysis of nuclear morphology plays an important role in glioma diagnosis and grading. We previously described intranuclear rods (rods) labeled with the SDL.3D10 monoclonal antibody against class III beta-tubulin (TUBB3) in human ependymomas. In a cohort of adult diffuse gliomas, we identified nuclear rods in 71.1% of IDH mutant lower-grade gliomas and 13.7% of IDH wild-type glioblastomas (GBMs). The presence of nuclear rods was associated with significantly longer postoperative survival in younger (≤65) GBM patients. Consistent with this, nuclear rods were mutually exclusive with Ki67 staining and their prevalence in cell nuclei inversely correlated with the Ki67 proliferation index. In addition, rod-containing nuclei showed a relative depletion of lamin B1, suggesting a possible association with senescence. To gain insight into their functional significance, we addressed their antigenic properties. Using a TUBB3-null mouse model, we demonstrate that the SDL.3D10 antibody does not bind TUBB3 in rods but recognizes an unknown antigen. In the present study, we show that rods show immunoreactivity for the nucleotide synthesizing enzymes inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase. By analogy with the IMPDH filaments that have been described previously, we postulate that rods regulate the activity of nucleotide-synthesizing enzymes in the nucleus by sequestration, with important implications for glioma behavior.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p= 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

Abstract P06: Anticancer agent 3-deazauridine has the potential to be effective against COVID-19 due to its inhibition of replication of RNA viruses

Abstract 3-Deazauridine (3DU) is a nucleoside analogue that was investigated for its antineoplastic activity in cancer patients. 3DU after its phosphorylation in cells to its 5’-triphosphate is a potent inhibitor of CTP synthetase. Inhibition of this enzyme results in a reduction in cellular CTP and inhibition of RNA synthesis. This action of 3DU can also inhibit RNA synthesis since the viral RNA polymerase requires CTP for replication of RNA. 3DU was reported to inhibit the in vitro replication of the RNA influenza virus at concentrations in the range of 40 µM (1) and should also have the potential to inhibit the replication of SARS-CoV-2. In a pilot clinical study, we treated a patient with acute lymphoid leukemia with 3DU at doses ranging from 120 to 360 mg/kg using a continuous intravenous infusion ranging from 40 to 72 hr. (2). The objective of the study was to determine if 3DU could enhance the antileukemic action of the inhibitor of DNA methylation, decitabine, and overcome the problem of drug resistance to this deoxycytidine analogue. Partial responses were observed indicating that pharmacological action of 3DU could accomplish this objective. There were minimal signs of toxicity in this clinical study on 3DU which exhibited plasma concentrations of this analogue in the range that inhibit RNA virus replication. This observation suggests that it would be safe to use 3DU in patients with COVID-19. 3DU has a rapid onset of action with a significant sign of antineoplastic action observable after a 4 h in vitro treatment of human leukemic cells. This pharmacological property rapid action of 3DU may be very important in patients with advanced COVID-19 to inhibit the virus is during its rapid replication phase and prevent progression of the disease to acute lung inflammation. It would be interesting to test this hypothesis in advanced COVID-19 patients at high risk for disease progression. For an initial pilot study I recommend a 36 h infusion of 3DU at a total dose of 120 mg/kg. It is possible that therapy with 3DU may have the potential to rescue patients with advance COVID-19.

Three overlooked key functional classes for building up minimal synthetic cells.

Assembly of minimal genomes revealed many genes encoding unknown functions. Three overlooked functional categories account for some of them. Cells are prone to make errors and age. As a first key function, discrimination between proper and changed entities is indispensable. Discrimination requires management of information, an authentic, yet abstract, currency of reality. For example proteins age, sometimes very fast. The cell must identify, then get rid of old proteins without destroying young ones. Implementing discrimination in cells leads to the second set of functions, usually ignored. Being abstract, information must nevertheless be embodied into material entities, with unavoidable idiosyncratic properties. This brings about novel unmet needs. Hence, the buildup of cells elicits specific but awkward material implementations, ‘kludges’ that become essential under particular settings, while difficult to identify. Finally, a third functional category characterizes the need for growth, with metabolic implementations allowing the cell to put together the growth of its cytoplasm, membranes, and genome, spanning different spatial dimensions. Solving this metabolic quandary, critical for engineering novel synthetic biology chassis, uncovered an unexpected role for CTP synthetase as the coordinator of nonhomothetic growth. Because a significant number of SynBio constructs aim at creating cell factories we expect that they will be attacked by viruses (it is not by chance that the function of the CRISPR system was identified in industrial settings). Substantiating the role of CTP, natural selection has dealt with this hurdle via synthesis of the antimetabolite 3′‐deoxy‐3′,4′‐didehydro‐CTP, recruited for antiviral immunity in all domains of life.

Energy deficiency caused by CTPS downregulation in decidua may contribute to pre-eclampsia by impairing decidualization.

Pre-eclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation. It seriously affects the health of maternity and the fetus. However, the pathogenesis of PE is still unknown. Decidualization deficiency is considered a contributing factor to the development of PE. CTP synthetase (CTPS) which is the rate-limiting enzyme in the CTP de novo biosynthesis, is essential for nucleic acid synthesis and cellular energy metabolism, and often appears as cytoophidium in many cell types. Here, we found that the expression of CTPS was significantly downregulated in decidual tissues of patients with severe PE compared with healthy pregnant women. During in vitro decidualization, changes in CTPS were accompanied by opposite fluctuation of the AMPK signaling pathway. Moreover, the downregulation of CTPS by glutamine analogs or CTPS small interfering RNA inhibited the decidualization process and the AMPK signaling pathway. Investigating the underlying mechanism of action by co-immunoprecipitation coupled with mass spectrometry showed that CTPS interacted with ATP synthase (ATPS) and maintained the content of ATP on Day 3 of decidualization. However, when combined with mitochondrial stress protein STRESS-70 instead of ATPS, the concentration of ATP on Day 6 of induction was reduced. Corresponding to this, CTPS was mainly distributes in the cytoplasm on Day 3 of induction, while it appeared both in the cytoplasm and the nucleus on Day 6 in decidualized cells, which was similar to that in cells before induction. In summary, we believe that CTPS plays an important role in decidualization by participating in energy metabolism. Abnormal expression of CTPS in decidualization would lead to abnormal decidualization and consequently result in the occurrence of PE.

Probing the dual inhibitory mechanisms of novel thiophenecarboxamide derivatives against Mycobacterium tuberculosis PyrG and PanK: an insight from biomolecular modeling study

The growing occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb) strains underscores an urgent need for new antibiotics. The development of more bioactive antibiotics against drug-resistant organisms with a different mode of action could be a game-changer for the cure and eradication of tuberculosis (TB). Pantothenate Kinase (PanK) and CTP synthetase (PyrG) are both essential for RNA, DNA, and Lipids biosynthesis pathways. Given the extensive knowledge on these biosynthesis pathways inhibition of Mtb growth and survival, these enzymes present a fascinating opportunity for anti-mycobacterial drug discovery. Recently, it was experimentally established that the active metabolite 11426026 of compound 7947882 (a prodrug activated by EthA monooxygenase, 5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide) inhibits the activities of PyrG and PanK to indicate novel multitarget therapy aimed at discontinuing Mtb growth. However, the molecular mechanisms of their selective inhibition remain subtle. In this work, molecular dynamics simulations were employed to investigate the inhibitory mechanism as well as the selectivity impact of the active metabolite inhibitor of these enzymes. Computational modeling of the studied protein-ligand systems reveals that the active metabolite can potentially inhibit both PanK and PyrG, thereby creating a pathway as a double target approach in tuberculosis treatment. Communicated by Ramaswamy H. Sarma

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

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 (Δ4), 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 Δ4 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.

Identification Of An Actin Fold Containing Metabolic Enzyme That Undergoes Regulated Polymerization

Cytoskeletal actins are thought to have evolved from actin fold enzymes, such as hexokinase or hsp70, that acquired the ability to polymerize while losing all other enzymatic functions apart from ATP hydrolysis. However, the discovery of metabolic enzymes, such as CTP synthetase, whose enzyme activity is regulated by self‐assembly into filaments has suggested another route to evolving cytoskeletal polymers: a filament forming metabolic enzyme that lost its role in metabolism and acquired novel cellular functions. In order to investigate this possibility, we surveyed all of the actin fold sugar kinases in yeast for their ability to form filaments. This screen identified one metabolic actin, the yeast glucokinase, Glk1p, that was capable of forming filaments in vivo. We have reconstituted Glk1p polymerization in vitro and have found that either ATP/Glucose or glucose‐6‐phosphate is sufficient to trigger filament formation. Consistent with this, we have also found that increasing glucose‐6‐phosphate levels in vivo triggers Glk1p polymerization. Furthermore, the addition of glucose and nonhydrolyzable ATP analogs to Glk1p also causes filaments to form. Thus, ATP hydrolysis is not required for Glk1p polymerization. Interestingly, the addition of ADP causes rapid disassembly of glucose‐6‐phosphate/Glk1p filaments suggesting that the stability of the filament is sensitive to the hydrolysis of ATP. While this is reminiscent of the dynamic behavior of cytoskeletal actins, our studies of Glk1p argue that it forms filaments that are structurally distinct from cytoskeletal actin filaments. Since the early steps of glycolysis consume ATP before generating ATP, we propose that Glk1p polymerization acts as a brake on glycolysis to prevent catastrophic depletion of ATP under certain metabolic conditions. We are currently using our structural and biochemical insights into Glk1p polymerization to test this possibility. Because Glk1p is the first filament forming actin that has more enzymatic functions than simple ATP hydrolysis, our results provide novel insights into both the evolution of the actin cytoskeleton and how the plasticity of the actin fold can support both polymerization and essential enzymatic activities.Support or Funding InformationThis work was supported by a HHMI Collaborative Innovation Award

Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation.

Cytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma.