Search Results (162)

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution. Full article

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology. Full article

Fucosylation plays a fundamental role in maintaining cellular functions and biological processes across all animals. As a form of glycosylation, it involves the biochemical addition of fucose, a six-carbon monosaccharide, to biological molecules like lipids, proteins, and glycan chains. This modification is essential for optimizing cellular interactions required for receptor-ligand binding, cell adhesion, immune responses, and development. Disruptions in cellular fucose synthesis or in the mechanisms enabling its transfer to other molecules have been linked to human disease. Inherited defects in the fucosylation pathway are rare, with about thirty patients described. Through genome-wide association studies (GWAS), variants in fucosylation pathway genes have been associated with complex diseases such as glaucoma and stroke, and somatic mutations are often found in cancers. Recent studies have applied targeted genetic animal models to elucidate the mechanisms through which disruptions in fucosylation contribute to disease pathogenesis and progression. Key focus areas include GDP-fucose synthesis through de novo or salvage pathways, GDP-fucose transport into the Golgi and endoplasmic reticulum (ER), and its transfer by fucosyltransferases (FUTs) or protein O-fucosyltransferases (POFUTs) onto acceptor molecules. Loss or gain of function fucosylation gene mutations in animal models such as mice, zebrafish, and invertebrates have provided insights into some fucosylation disease pathogenesis. This review aims to bring together these findings, summarizing key insights from existing animal studies to possibly infer fucosylation disease mechanisms in humans. Full article

Xanthine oxidoreductase (XOR) is the only enzyme responsible for uric acid production and is essential for preventing gout. While XOR inhibitors effectively reduce serum urate levels, they also influence purine salvage and de novo pathways, as well as energy metabolism, raising concerns about metabolic adaptation and rebound effects upon treatment discontinuation. In this review, we outline the fundamental regulatory mechanisms of purine metabolism and summarize the mechanisms of action of XOR inhibitors and their associated metabolic effects with reference to XOR deficiency, type I xanthinuria. Furthermore, we discuss the impact of discontinuing XOR inhibitors and examine their potential for rebound. Full article

CDD plays a pivotal role within the pyrimidine salvage pathway. In this study, a novel, rapid method for the identification of cell lines lacking functional cytidine deaminase was developed. This innovative method utilizes immunocytochemical detection of the product of 5-fluorocytidine deamination, 5-fluorouridine in cellular RNA, enabling the identification of these cells within two hours. The approach employs an anti-bromodeoxyuridine antibody that also specifically binds to 5-fluorouridine and its subsequent detection by a fluorescently labeled antibody. Our results also revealed a strong correlation between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and cytidine deaminase content. On the other hand, no correlation was observed between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and deoxycytidine monophosphate deaminase content. Similarly, no correlation was observed between this ratio and equilibrative nucleoside transporters 1 or 2. Finally, concentrative nucleoside transporters 1, 2, or 3 also do not correlate with the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio. Full article

Despite notable progress in managing B-cell acute lymphoblastic leukemia (B-ALL) over recent decades, particularly in pediatric cohorts where the 5-year overall survival (OS) reaches 90%, outcomes for the 10–15% with relapsed and refractory disease remain unfavorable. This disparity is further accentuated in adults, where individuals over the age of 40 years undergoing aggressive multiagent chemotherapy continue to have lower survival rates. While the adoption of pediatric-inspired treatment protocols has enhanced complete remission (CR) rates among younger adults, 20–30% of these patients experience relapse, resulting in a subsequent 5-year OS rate of 40–50%. For relapsed B-ALL in adults, there is no universally accepted standard salvage therapy, and the median OS is short. The cornerstone of B-ALL treatment continues to be the utilization of combined cytotoxic chemotherapy regimens to maximize early and durable disease control. In this manuscript, we go beyond the multiagent chemotherapy medications developed prior to the 1980s and focus on the incorporation of antibody-based therapy for B-ALL with an eye on existing and upcoming approved indications for blinatumomab, inotuzumab ozogamicin, other monoclonal antibodies, and chimeric antigen receptor (CAR) T cell products in frontline and relapsed/refractory settings. In addition, we discuss emerging investigational therapies that harness the therapeutic vulnerabilities of the disease through targeting apoptosis, modifying epigenetics, and inhibiting the mTOR pathway. Full article

Cholangiocarcinoma-associated mortality has been increasing over the past decade. The sodium-glucose cotransporter 2 inhibitor, canagliflozin, has demonstrated anti-tumor effects against several types of cancers; however, studies examining its potential impact on cholangiocarcinoma are lacking. This study investigated the anti-tumor effects of canagliflozin on cholangiocarcinoma and the effects of nicotinamide adenine dinucleotide (NAD)+ salvage pathway activation and sirtuin 1 on tumor growth. We evaluated cell proliferation and gene expression in several cholangiocarcinoma cell lines and analyzed the effects of canagliflozin on cell proliferation, apoptosis, and migration. Canagliflozin treatment decreased the viability of cholangiocarcinoma cells in a concentration-dependent manner but increased the viability at low concentrations in several cell lines. At high concentrations, canagliflozin arrested the cell cycle checkpoint in the G0/G1 phase. In contrast, at low concentrations, it increased the proportion of cells in the S phase. Canagliflozin also reduced the migratory ability of cholangiocarcinoma cells in a concentration-dependent manner. Canagliflozin treatment upregulated nicotinamide phosphoribosyltransferase (NAMPT), NAD+, and sirtuin 1 in cholangiocarcinoma and activated the NAD+ salvage pathway. The growth-inhibitory effect of canagliflozin was enhanced when combined with an NAMPT inhibitor. Canagliflozin inhibits cholangiocarcinoma cell growth and migration and its anti-tumor effect is enhanced when combined with an NAMPT inhibitor. However, further investigation is required because of its potential tumor growth-promoting effect through the activation of the NAD+ salvage pathway. Full article

Tacrolimus (TAC)-induced chronic nephrotoxicity (TAC nephrotoxicity) is a serious issue for long-term graft survival in kidney transplantation. However, the pathophysiology of TAC nephrotoxicity remains unclear. In this study, we analyzed whole blood samples from mice that developed TAC nephrotoxicity in order to discover its mechanism. Mice were divided into a TAC group and a control group (n = 5 per group). The TAC group received TAC subcutaneously (1 mg/kg/day for 28 days), while the control group received normal saline instead. After the administration period, whole blood was collected and metabolomic analysis was performed, revealing significant changes in 56 metabolites. The major metabolic changes were related to uremic toxins, vascular damage, and NAD⁺. NAD⁺ levels were significantly lower in the TAC group, and ADP-ribose, nicotinamide, and nicotinamide N-oxide, which are degradation products of NAD⁺, were significantly higher, suggesting impairment of the NAD⁺ salvage pathway. NAD⁺ deficiency suggests cellular aging and mitochondrial dysfunction, which may induce vascular damage and chronic kidney disease. Our study demonstrated a correlation between low NAD⁺ levels and the pathophysiology of TAC nephrotoxicity. Full article

Background/Objectives: Cancer cells rely on metabolic reprogramming that is supported by altered mitochondrial redox status and an increased demand for NAD⁺. Over expression of Nampt, the rate-limiting enzyme of the NAD⁺ biosynthesis salvage pathway, is common in breast cancer cells, and more so in triple negative breast cancer (TNBC) cells. Targeting the salvage pathway has been pursued for cancer therapy. However, TNBC cells have heterogeneous responses to Nampt inhibition, which contributes to the diverse outcomes. There is a lack of imaging biomarkers to differentiate among TNBC cells under metabolic stress and identify which are responsive. We aimed to characterize and differentiate among a panel of TNBC cell lines under NAD-deficient stress and identify which subtypes are more dependent on the NAD salvage pathway. Methods: Optical redox imaging (ORI), a label-free live cell imaging microscopy technique was utilized to acquire intrinsic fluorescence intensities of NADH and FAD-containing flavoproteins (Fp) thus the mitochondrial redox ratio Fp/(NADH + Fp) in a panel of TNBC cell lines. Various fluorescence probes were then added to the cultures to image the mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and cell number. Results: Various TNBC subtypes are sensitive to Nampt inhibition in a dose- and time-dependent manner, they have differential mitochondrial redox responses; furthermore, the mitochondrial redox indices linearly correlated with mitochondrial ROS induced by various doses of a Nampt inhibitor. Moreover, the changes in the redox indices correlated with growth inhibition. Additionally, the redox state was found fully recovered after removing the Nampt inhibitor. Conclusions: This study supports the utility of ORI in rapid metabolic phenotyping of TNBC cells under NAD-deficient stress to identify responsive cells and biomarkers of treatment response, facilitating combination therapy strategies. Full article

Distraction osteogenesis (DO) is a process which uses the bone’s natural healing tendencies to repair and lengthen pathologic, missing, or malformed bone. The mechanism of DO mimics the pathway that the body uses in any other fracture repair however the location of the fracture is carefully controlled by a surgical osteotomy. Postoperatively, the bone is allowed to begin its natural healing process, with the lengthener applying constant tension and thus re-initiating the process of healing along the length of the distraction gap. Current clinical indications for DO include limb length discrepancy, congenital bone length deformity, large bone defects, and extremity reconstruction due to hypoplasia or limb salvage procedures. The risks of DO include soft tissue complications, relapse or improper correction, cost or resource-related challenges, and psychosocial stigmas surrounding long treatment durations and the necessity of wearing the distraction lengthening hardware. Future directions for DO include supplements to the bone regeneration process (such as growth factors and/or mechanical stimulation) or improvements to the distractor device itself (changes in material and/or the structure of the device itself). This review aims to offer a comprehensive summary of the indications, underlying biological mechanisms, and practical considerations when implementing the use of distraction osteogenesis in clinical practice. Full article

Entamoeba histolytica causes amebiasis, a significant global health issue, with millions affected annually, especially in developing countries. EhDUF2419, an important protein involved in E. histolytica’s queuine salvage pathway and its interaction network, remains unclear. To explore this, we transfected E. histolytica trophozoites with a plasmid encoding Myc-tagged EhDUF2419 and achieved successful overexpression. Through immunoprecipitation with the Myc antibody followed by mass spectrometry, we identified 335 proteins interacting with Myc-tagged EhDUF2419, including over 100 ribosomal proteins, along with translation initiation and elongation factors, and aminoacyl-tRNA synthetases. Ribosome purification revealed the presence of EhDUF2419 in ribosomal protein-enriched fractions. Treatment with queuosine (Q) significantly reduced the EhDUF2419 protein levels and decreased the Q-modified tRNA in Myc-tagged EhDUF2419 overexpressing trophozoites. This effect, which was Q-dependent, was not observed in strains carrying an empty vector control or overexpressing a truncated form of EhDUF2419 lacking catalytic activity. The reduction in the EhDUF2419 protein levels was regulated by proteasome-mediated degradation, as evidenced by the reduced degradation in the presence of MG132, a proteasome inhibitor. Our study uncovers the novel interaction of EhDUF2419 with ribosomal proteins and its regulation by the proteasome machinery, providing new insights into its role in E. histolytica and potential therapeutic strategies. Full article

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 °C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD⁺) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD⁺ can be synthesized from three different intracellular pathways, but it is the NAD⁺ salvage pathway that generates the largest proportion of NAD⁺. Impaired NAD⁺ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD⁺ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD⁺ metabolism, either by administering NAD⁺ precursor metabolites or small molecules that alter NAD⁺-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD⁺ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models. Full article

Nicotinamide adenine dinucleotide (NAD⁺) is an important cofactor for both metabolic and signaling pathways, with the dysregulation of NAD⁺ levels acting as a driver for diseases such as neurodegeneration, cancers, and metabolic diseases. NAD⁺ plays an essential role in regulating the growth and progression of cancers by controlling important cellular processes including metabolism, transcription, and translation. NAD⁺ regulates several metabolic pathways such as glycolysis, the citric acid (TCA) cycle, oxidative phosphorylation, and fatty acid oxidation by acting as a cofactor for redox reactions. Additionally, NAD⁺ acts as a cofactor for ADP-ribosyl transferases and sirtuins, as well as regulating cellular ADP-ribosylation and deacetylation levels, respectively. The cleavage of NAD⁺ by CD38—an NAD⁺ hydrolase expressed on immune cells—produces the immunosuppressive metabolite adenosine. As a result, metabolizing and maintaining NAD⁺ levels remain crucial for the function of various cells found in the tumor microenvironment, hence its critical role in tissue homeostasis. The NAD⁺ levels in cells are maintained by a balance between NAD⁺ biosynthesis and consumption, with synthesis being controlled by the Preiss–Handler, de novo, and NAD⁺ salvage pathways. The primary source of NAD⁺ synthesis in a variety of cell types is directed by the expression of the enzymes central to the three biosynthesis pathways. In this review, we describe the role of NAD⁺ metabolism and its synthesizing and consuming enzymes’ control of cancer cell growth and immune responses in gynecologic cancers. Additionally, we review the ongoing efforts to therapeutically target the enzymes critical for NAD⁺ homeostasis in gynecologic cancers. Full article

Sphingolipids (SLs) are bioactive signaling molecules essential for various cellular processes, including cell survival, proliferation, migration, and apoptosis. Key SLs such as ceramides, sphingosine, and their phosphorylated forms play critical roles in cellular integrity. Dysregulation of SL levels is implicated in numerous diseases, notably chronic kidney disease (CKD). This review focuses on the role of SLs in CKD, highlighting their potential as biomarkers for early detection and prognosis. SLs maintain renal function by modulating the glomerular filtration barrier, primarily through the activity of podocytes. An imbalance in SLs can lead to podocyte damage, contributing to CKD progression. SL metabolism involves complex enzyme-catalyzed pathways, with ceramide serving as a central molecule in de novo and salvage pathways. Ceramides induce apoptosis and are implicated in oxidative stress and inflammation, while sphingosine-1-phosphate (S1P) promotes cell survival and vascular health. Studies have shown that SL metabolism disorders are linked to CKD progression, diabetic kidney disease, and glomerular diseases. Targeting SL pathways could offer novel therapeutic approaches for CKD. This review synthesizes recent research on SL signaling regulation in kidney diseases, emphasizing the importance of maintaining SL balance for renal health and the potential therapeutic benefits of modulating SL pathways. Full article