Application of a HPLC-Q/TOF method with post-column compensation for separation and identification of polar impurities in Cytidine disodium triphosphate for injection

Background: Cytidine Disodium Triphosphate (CTP-2Na) for injection is mainly used for treating nervous system diseases. Currently, there are few studies focused on the separation and identification of polar impurities in CTP-2Na for injection, which is important for ensuring drug safety and efficacy. Objective: The study aimed to establish an HPLC-Q/TOF method for the separation and identification of polar impurities in CTP-2Na for injection. Methods: Chromatographic separation was achieved on a Waters Atlantis T3 column using 5 mM aqueous ammonium acetate solution as the mobile phase in an isocratic elution mode. A postcolumn compensation technology was used to improve the ionization efficiency of impurities in the spray chamber. Results: Three polar impurities (disodium cytidine tetraphosphate, disodium cytidine diphosphate, disodium cytidine monophosphate) were detected in CTP-2Na for injection. The former one is probably the overreaction product during the production of CTP-2Na, the latter two were reported as degradation products. The fragmentation patterns of cytidine phosphate compounds in negative ion mode are summarized. Conclusion: This study provides a good reference for the separation and identification of polar impurities in nucleotide drugs.

The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.

Central Injection of Ghrelin Improves Motor Balance in the Rotarod Test in the Rats: Altering the Expression of Drd1 Gene

Introduction: Motor learning consolidates in adulthood, and its defects begin to appear with aging. Ghrelin, an endogenous peptide, improves memory and learning, targeting dopaminergic circuits. While cytidine diphosphate choline (citicoline) is known as a common drug for enhancing memory and learning in aging, it is not recommended for adults due to its side effects. The current study aimed at investigating if ghrelin treatment would improve motor learning via the expression of a relevant gene. Methods: For this experimental study, adult male Wistar rats were randomly divided into five groups: control group, three groups of ghrelin treatment (0.3, 1.5, and 3 nmol/μL), and one group with citicoline treatment. The injections were done intra-hippocampally. The motor learning rate was determined using the rotarod performance test by measuring the resistance to falling. Then the expression of dopamine receptor type D1 (Drd1) gene in the hippocampus was measured by a real-time polymerase chain reaction (PCR). Results: Ghrelin (3 nmol/μL) and citicoline had similar and significant effects on motor learning improvement (P<0.01). Both drugs significantly increased Drd1 gene expression (P <0.001). Conclusion: Ghrelin, like citicoline, improves motor learning by altering the expression of Drd1 gene in the hippocampus.

Stable Isotopic Tracer Phospholipidomics Reveals Contributions of Key Phospholipid Biosynthetic Pathways to Low Hepatocyte Phosphatidylcholine to Phosphatidylethanolamine Ratio Induced by Free Fatty Acids

There is a strong association between hepatocyte phospholipid homeostasis and non-alcoholic fatty liver disease (NAFLD). The phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) often draws special attention as genetic and dietary disruptions to this ratio can provoke steatohepatitis and other signs of NAFLD. Here we demonstrated that excessive free fatty acid (1:2 mixture of palmitic and oleic acid) alone was able to significantly lower the phosphatidylcholine to phosphatidylethanolamine ratio, along with substantial alterations to phospholipid composition in rat hepatocytes. This involved both a decrease in hepatocyte phosphatidylcholine (less prominent) and an increase in phosphatidylethanolamine, with the latter contributing more to the lowered ratio. Stable isotopic tracer phospholipidomic analysis revealed several previously unidentified changes that were triggered by excessive free fatty acid. Importantly, the enhanced cytidine diphosphate (CDP)-ethanolamine pathway activity appeared to be driven by the increased supply of preferred fatty acid substrates. By contrast, the phosphatidylethanolamine N-methyl transferase (PEMT) pathway was restricted by low endogenous methionine and consequently low S-adenosylmethionine, which resulted in a concomitant decrease in phosphatidylcholine and accumulation of phosphatidylethanolamine. Overall, our study identified several previously unreported links in the relationship between hepatocyte free fatty acid overload, phospholipid homeostasis, and the development of NAFLD.

Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)–choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms

ABSTRACT Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Self-organization of parS centromeres by the ParB CTP hydrolase

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS. ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

Cytidine Diphosphate-Ribitol Analysis for Diagnostics and Treatment Monitoring of Cytidine Diphosphate-l-Ribitol Pyrophosphorylase A Muscular Dystrophy

BACKGROUND Many muscular dystrophies currently remain untreatable. Recently, dietary ribitol has been suggested as a treatment for cytidine diphosphate (CDP)-l-ribitol pyrophosphorylase A (CRPPA, ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) myopathy, by raising CDP-ribitol concentrations. Thus, to facilitate fast diagnosis, treatment development, and treatment monitoring, sensitive detection of CDP-ribitol is required. METHODS An LC-MS method was optimized for CDP-ribitol in human and mice cells and tissues. RESULTS CDP-ribitol, the product of CRPPA, was detected in all major human and mouse tissues. Moreover, CDP-ribitol concentrations were reduced in fibroblasts and skeletal muscle biopsies from patients with CRPPA myopathy, showing that CDP-ribitol could serve as a diagnostic marker to identify patients with CRPPA with severe Walker–Warburg syndrome and mild limb-girdle muscular dystrophy (LGMD) phenotypes. A screen for potentially therapeutic monosaccharides revealed that ribose, in addition to ribitol, restored CDP-ribitol concentrations and the associated O-glycosylation defect of α-dystroglycan. As the effect occurred in a mutation-dependent manner, we established a CDP-ribitol blood test to facilitate diagnosis and predict individualized treatment response. Ex vivo incubation of blood cells with ribose or ribitol restored CDP-ribitol concentrations in a patient with CRPPA LGMD. CONCLUSIONS Sensitive detection of CDP-ribitol with LC-MS allows fast diagnosis of patients with severe and mild CRPPA myopathy. Ribose offers a readily testable dietary therapy for CRPPA myopathy, with possible applicability for patients with FKRP and FKTN myopathy. Evaluation of CDP-ribitol in blood is a promising tool for the evaluation and monitoring of dietary therapies for CRPPA myopathy in a patient-specific manner.

FUNCTIONAL ROLES OF LIPID METABOLITE CDP DIACYLGLYCEROL AND ITS SYNTHASE ENZYMES: RECENT DEVELOPMENTS

Phospholipids are basic building-block molecules for biological membranes. Biosynthesis of phospholipids i.e phosphatidylinositol, phosphatidylglycerol and phosphatidylserine requires a central liponucleotide intermediate named cytidine-diphosphate diacylglycerol (CDP-DAG). The CDP-DAG synthase (CDS) is an integral membrane enzyme catalysing the formation of CDP-DAG, an essential step for phosphoinositide recycling during signal transduction. New roles are being ascribed to the CDP-DAG in signalling and pathophysiological conditions. This pathway may also be the target of novel drugs to be used in neuro-psychiatric conditions. Key words: Phospholipids, Cytidine diphosphate, diacylglycerol, phosphatidic acid, phosphatidylglycerol.