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Glucokinase (GCK), a key enzyme in glucose metabolism, plays a central role in glucose sensing and insulin secretion in pancreatic β-cells, as well as glycogen synthesis in the liver. Mutations in the GCK gene have been associated with various monogenic diabetes (MD) disorders, including permanent neonatal diabetes mellitus (PNDM) and maturity-onset diabetes of the young (MODY), highlighting its importance in maintaining glucose homeostasis. Additionally, GCK gain-of-function mutations lead to a rare congenital form of hyperinsulinism known as hyperinsulinemic hypoglycemia (HH), characterized by increased enzymatic activity and increased glucose sensitivity in pancreatic β-cells. This review offers a comprehensive exploration of the critical role played by the GCK gene in diabetes development, shedding light on its expression patterns, regulatory mechanisms, and diverse forms of associated monogenic disorders. Structural and mechanistic insights into GCK's involvement in glucose metabolism are discussed, emphasizing its significance in insulin secretion and glycogen synthesis. Animal models have provided valuable insights into the physiological consequences of GCK mutations, although challenges remain in accurately recapitulating human disease phenotypes. In addition, the potential of human pluripotent stem cell (hPSC) technology in overcoming current model limitations is discussed, offering a promising avenue for studying GCK-related diseases at the molecular level. Ultimately, a deeper understanding of GCK's multifaceted role in glucose metabolism and its dysregulation in disease states holds implications for developing targeted therapeutic interventions for diabetes and related disorders. [ABSTRACT FROM AUTHOR]

Many cancer cells share with yeast a preference for fermentation over respiration, which is associated with overactive glucose uptake and breakdown, a phenomenon called the Warburg effect in cancer cells. The yeast tps1D mutant shows even more pronounced hyperactive glucose uptake and phosphorylation causing glycolysis to stall at GAPDH, initiation of apoptosis through overactivation of Ras and absence of growth on glucose. The goal of the present work was to use the yeast tps1D strain to screen for novel compounds that would preferentially inhibit overactive glucose influx into glycolysis, while maintaining basal glucose catabolism. This is based on the assumption that the overactive glucose catabolism of the tps1D strain might have a similar molecular cause as the Warburg effect in cancer cells. We have isolated Warbicin® A as a compound restoring growth on glucose of the yeast tps1D mutant, showed that it inhibits the proliferation of cancer cells and isolated structural analogs by screening directly for cancer cell inhibition. The Warbicin® compounds are the first drugs that inhibit glucose uptake by both yeast Hxt and mammalian GLUT carriers. Specific concentrations did not evoke any major toxicity in mice but increase the amount of adipose tissue likely due to reduced systemic glucose uptake. Surprisingly, Warbicin® A inhibition of yeast sugar uptake depends on sugar phosphorylation, suggesting transport-associated phosphorylation as a target. In vivo and in vitro evidence confirms physical interaction between yeast Hxt7 and hexokinase. We suggest that reversible transport-associated phosphorylation by hexokinase controls the rate of glucose uptake through hydrolysis of the inhibitory ATP molecule in the cytosolic domain of glucose carriers and that in yeast tps1D cells and cancer cells reversibility is compromised, causing constitutively hyperactive glucose uptake and phosphorylation. Based on their chemical structure and properties, we suggest that Warbicin® compounds replace the inhibitory ATP molecule in the cytosolic domain of the glucose carriers, preventing hexokinase to cause hyperactive glucose uptake and catabolism. [ABSTRACT FROM AUTHOR]

Microsporidia, a group of unicellular eukaryotic parasites, rely intensely on secretory effectors for successful invasion and proliferation within host cells. This review focuses on the identification, characterization, and functional roles of effectors, including secretory proteins and microRNAs. The adhesion proteins like the Ricin‐B‐lectin facilitate initial invasion, which binds to the host cell surface. Once inside, microsporidia deploy a range of effectors to modulate host immune responses, such as serpin proteins, and redirect host cell metabolism to meet the parasite's nutritional needs through hexokinase. Some effectors such as microRNAs, alter the host gene expression to create a more favorable intracellular parasitic environment. In conclusion, the secretory effectors of microsporidia play a pivotal role spanning from host cell invasion to intracellular establishment. In the future, more effectors secreted by microsporidia will be studied, which will not only help to elucidate the molecular mechanism of pathogenic manipulation of the host but also help to provide the potential targets for anti‐parasitic treatments. [ABSTRACT FROM AUTHOR]

Retinopathy of prematurity (ROP) is a retinal disease‐causing retinal neovascularization that can lead to blindness. Oxygen‐induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti‐oxidative and anti‐inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood–retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA‐induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2‐dependent manner, implicating ICA as a potential therapeutic agent for ROP. [ABSTRACT FROM AUTHOR]

Contrary to differentiated cells, cancer cells predominantly convert glucose to lactate even under conditions of adequate oxygen supply ("Warburg effect"). The initial enzyme implicated in this route is hexokinase, which transforms D‐glucose into D‐glucose‐6‐phosphate. We proposed the use of different polyphenols (resveratrol, epigallocatechin gallate, pterostilbene, phloretin) and their derivatives (α‐glucosides and acylated α‐glucosides) to inhibit this enzyme. For this study, we used Saccharomyces cerevisiae hexokinase, whose two isoforms show high resemblance at the active site with human hexokinase HK2. To monitor the reactions, a method of anion‐exchange chromatography coupled with pulsed amperometric detection (HPAEC‐PAD) was developed. Remarkably, most of the assayed compounds inhibited the enzyme more than 50 % in the standard assay. Among them, phloretin 4'‐O‐(6"‐O‐octanoyl)‐α‐D‐glucopyranoside showed the highest inhibition and was studied in depth to determine the inhibition pattern and inhibition constant. The Ki for glucose was calculated to be 22.1±0.4 μM. Computational models of inhibition were carried out with the three molecules displaying the highest inhibition, and correlated adequately with the observed inhibitory effects on the enzyme. The inhibitory effect of several of the assayed polyphenols on hexokinase and their lack of toxicity renders them promising candidates as adjuvant drugs for cancer therapy. [ABSTRACT FROM AUTHOR]

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice. Author summary: About 8%-12% of couples worldwide are infertile, and male infertility is associated with about half of these cases. Common defects of male infertility include low sperm count, reduced sperm motility, abnormal sperm morphology, and unexplained male infertility with normal sperm parameters but infertility. The cause of over 50% of male infertility cases remains unidentified. Research on male reproduction has helped to unravel male infertility. In this study, we used a knockout mouse model to study the function of HK1S in the male mouse reproductive system. We showed that depletion of Hk1s leads to infertility in male mice but with normal sperm morphology and motility. These phenotypes are similar to patients with unexplained male infertility. Furthermore, HK1S is essential for sperm glycolysis. Hk1s deficiency in mice results in decreased glycolysis activity and leads to impaired sperm migration into the oviduct, reduced acrosome reaction, and defective capacitation-associated increase in tyrosine phosphorylation. These results suggested that HK1S is necessary for male fertility in mice. Our study suggests a new avenue for the development of new therapeutic strategies for male infertility. [ABSTRACT FROM AUTHOR]

Background: Glucokinase-maturity onset diabetes of the young (GCK-MODY) is a form of monogenic diabetes that is caused by heterozygous inactivating mutations in GCK gene. The management of GCK-MODY during pregnancy is challenging, as pharmacological treatment dose could not alter glycaemia in GCK-MODY in longitudinal studies and noninvasive antenatal fetal GCK genotyping is unavailable. Case presentation: Here, we report a case of GCK-MODY with pregnancy. The patient was 41 years old. She was diagnosed with GCK-MODY at 10 weeks of gestation. At 22 weeks and 6 days of gestation, she underwent amniocentesis due to her advanced age and genetic testing confirmed that the fetus was a heterozygous mutation carrier. We described the characteristics of blood glucose fluctuation, insulin responsiveness, hypoglycemic risk, fetal development, and pregnancy outcome before and after fetus genetic testing during her entire pregnancy. Until now, there were no studies describing insulin titration and blood glucose fluctuations during pregnancy in patients with GCK-MODY. Conclusion: Our case indicted that insulin therapy during pregnancy is effective in GCK-MODY. Careful dietary modifications, individualized insulin titration and specialized diabetes education can reduce the risk of hypoglycemia and improve gestational outcome in GCK-MODY with pregnancy. [ABSTRACT FROM AUTHOR]

Monogenic diabetes (MD) represents a cluster of different types of diabetes produced by a mutation of a single gene. The commonest type of MD is maturity-onset diabetes of the young (MODY) which has several subtypes, and neonatal diabetes mellitus (NDM). With improved diagnostic facilities, more cases of monogenic diabetes are being described, and pregnancy is being reported in different forms of monogenic diabetes as these patients enter the reproductive age group. The treatment of monogenic diabetes may change once pregnancy sets in. For example, some forms of monogenic diabetes which respond to oral antidiabetic drugs (OHA), particularly the sulfonylurea agents (SUs), may need insulin during pregnancy. However, this depends on the fetal inheritance of mutation from the mother. This review attempts to put together published reports of pregnancy in various types of monogenic diabetes focussing on the most frequently seen forms of MD. [ABSTRACT FROM AUTHOR]

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment. [ABSTRACT FROM AUTHOR]

This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression of biosynthetic enzyme genes, including phosphoglucomutase, hexokinase, phosphomannomutase, polysaccharide synthase, and UDP‐glucose 4‐epimerase genes. The study found higher EPS concentrations in seven recombinant strains compared to the wild‐type C. militaris, indicating that the overexpression of key enzyme genes increased EPS production. Among them, the CM‐pgm‐2 strain exhibited the highest EPS production, reaching a concentration of 3.82 ± 0.26 g/L, which was 1.52 times higher than the amount produced by the wild C. militaris strain. Additionally, the regulatory effects of aromatic amino acids on the QS system of the CM‐pgm‐2 strain were investigated. Under the influence of 45 mg/L tryptophan, the EPS production in CM‐pgm‐2 reached 4.75 ± 0.20 g/L, representing a 1.90‐fold increase compared to wild C. militaris strains. This study provided an effective method for the large‐scale production of EPSs in C. militaris, and opened up new avenues for research into fungal QS mechanisms. [ABSTRACT FROM AUTHOR]

Abstract Background Glucokinase (GK) plays a key role in glucose metabolism. In the liver, GK is regulated by GK regulatory protein (GKRP) with nuclear sequestration at low plasma glucose level. Some GK activators (GKAs) disrupt GK-GKRP interaction which increases hepatic cytoplasmic GK level. Excess hepatic GK activity may exceed the capacity of glycogen synthesis with excess triglyceride formation. It remains uncertain whether hypertriglyceridemia associated with some GKAs in previous clinical trials was due to direct GK activation or impaired GK-GKRP interaction. Methods Using publicly available genome-wide association study summary statistics, we selected independent genetic variants of GCKR and GCK associated with fasting plasma glucose (FPG) as instrumental variables, to mimic the effects of impaired GK-GKRP interaction and direct GK activation, respectively. We applied two-sample Mendelian Randomization (MR) framework to assess their causal associations with lipid-related traits, risks of metabolic dysfunction-associated steatotic liver disease (MASLD) and cardiovascular diseases. We verified these findings in one-sample MR analysis using individual-level statistics from the Hong Kong Diabetes Register (HKDR). Results Genetically-proxied impaired GK-GKRP interaction increased plasma triglycerides, low-density lipoprotein cholesterol and apolipoprotein B levels with increased odds ratio (OR) of 14.6 (95% CI 4.57–46.4) per 1 mmol/L lower FPG for MASLD and OR of 2.92 (95% CI 1.78–4.81) for coronary artery disease (CAD). Genetically-proxied GK activation was associated with decreased risk of CAD (OR 0.69, 95% CI 0.54–0.88) and not with dyslipidemia. One-sample MR validation in HKDR showed consistent results. Conclusions Impaired GK-GKRP interaction, rather than direct GK activation, may worsen lipid profiles and increase risks of MASLD and CAD. Development of future GKAs should avoid interfering with GK-GKRP interaction.

Background: Glucokinase (GK) plays a key role in glucose metabolism. In the liver, GK is regulated by GK regulatory protein (GKRP) with nuclear sequestration at low plasma glucose level. Some GK activators (GKAs) disrupt GK-GKRP interaction which increases hepatic cytoplasmic GK level. Excess hepatic GK activity may exceed the capacity of glycogen synthesis with excess triglyceride formation. It remains uncertain whether hypertriglyceridemia associated with some GKAs in previous clinical trials was due to direct GK activation or impaired GK-GKRP interaction. Methods: Using publicly available genome-wide association study summary statistics, we selected independent genetic variants of GCKR and GCK associated with fasting plasma glucose (FPG) as instrumental variables, to mimic the effects of impaired GK-GKRP interaction and direct GK activation, respectively. We applied two-sample Mendelian Randomization (MR) framework to assess their causal associations with lipid-related traits, risks of metabolic dysfunction-associated steatotic liver disease (MASLD) and cardiovascular diseases. We verified these findings in one-sample MR analysis using individual-level statistics from the Hong Kong Diabetes Register (HKDR). Results: Genetically-proxied impaired GK-GKRP interaction increased plasma triglycerides, low-density lipoprotein cholesterol and apolipoprotein B levels with increased odds ratio (OR) of 14.6 (95% CI 4.57–46.4) per 1 mmol/L lower FPG for MASLD and OR of 2.92 (95% CI 1.78–4.81) for coronary artery disease (CAD). Genetically-proxied GK activation was associated with decreased risk of CAD (OR 0.69, 95% CI 0.54–0.88) and not with dyslipidemia. One-sample MR validation in HKDR showed consistent results. Conclusions: Impaired GK-GKRP interaction, rather than direct GK activation, may worsen lipid profiles and increase risks of MASLD and CAD. Development of future GKAs should avoid interfering with GK-GKRP interaction. [ABSTRACT FROM AUTHOR]

Bioenergetic therapy is emerging as a promising therapeutic approach. However, its therapeutic effectiveness is restricted by metabolic plasticity, as tumor cells switch metabolic phenotypes between glycolysis and oxidative phosphorylation (OXPHOS) to compensate for energy. Herein, Metformin (MET) and BAY‐876 (BAY) co‐loaded CuFe2O4 (CF) nanoplatform (CFMB) is developed to boost energy deprivation by synchronous interventions of glycolysis and OXPHOS for bioenergetic therapy synergetic with chemodynamic/photothermal therapy (CDT/PTT). The MET can simultaneously restrain glycolysis and OXPHOS by inhibiting hexokinase 2 (HK2) activity and damaging mitochondrial function to deprive energy, respectively. Besides, BAY blocks glucose uptake by inhibiting glucose transporter 1 (GLUT1) expression, further potentiating the glycolysis repression and thus achieving much more depletion of tumorigenic energy sources. Interestingly, the upregulated antioxidant glutathione (GSH) in cancer cells triggers CFMB degradation to release Cu+/Fe2+ catalyzing tumor‐overexpressed H2O2 to hydroxyl radical (∙OH), both impairing OXPHOS and achieving GSH‐depletion amplified CDT. Furthermore, upon near‐infrared (NIR) light irradiation, CFMB has a photothermal conversion capacity to kill cancer cells for PTT and improve ∙OH production for enhanced CDT. In vivo experiments have manifested that CFMB remarkably suppressed tumor growth in mice without systemic toxicity. This study provides a new therapeutic modality paradigm to boost bioenergetic‐related therapies. [ABSTRACT FROM AUTHOR]

The nerve growth factor (NGF) participates in cell survival and glucose-stimulated insulin secretion (GSIS) processes in rat adult beta cells. GSIS is a complex process in which metabolic events and ionic channel activity are finely coupled. GLUT2 and glucokinase (GK) play central roles in GSIS by regulating the rate of the glycolytic pathway. The biphasic release of insulin upon glucose stimulation characterizes mature adult beta cells. On the other hand, beta cells obtained from neonatal, suckling, and weaning rats are considered immature because they secrete low levels of insulin and do not increase insulin secretion in response to high glucose. The weaning of rats (at postnatal day 20 in laboratory conditions) involves a dietary transition from maternal milk to standard chow. It is characterized by increased basal plasma glucose levels and insulin levels, which we consider physiological insulin resistance. On the other hand, we have observed that incubating rat beta cells with NGF increases GSIS by increasing calcium currents in neonatal cells. In this work, we studied the effects of NGF on the regulation of cellular distribution and activity of GLUT2 and GK to explore its potential role in the maturation of GSIS in beta cells from P20 rats. Pancreatic islet cells from both adult and P20 rats were isolated and incubated with 5.6 mM or 15.6 mM glucose with and without NGF for 4 hours. Specific immunofluorescence assays were conducted following the incubation period to detect insulin and GLUT2. Additionally, we measured glucose uptake, glucokinase activity, and insulin secretion assays at 5.6 mM or 15.6 mM glucose concentrations. We observed an age-dependent variation in the distribution of GLUT2 in pancreatic beta cells and found that glucose plays a regulatory role in GLUT2 distribution independently of age. Moreover, NGF increases GLUT2 abundance, glucose uptake, and GSIS in P20 beta cells and GK activity in adult beta cells. Our results suggest that besides increasing calcium currents, NGF regulates metabolic components of the GSIS, thereby contributing to the maturation process of pancreatic beta cells. [ABSTRACT FROM AUTHOR]

• In silico screening of 35 phytochemicals from Newbouldia laevis against hexokinase 2. • Insight using molecular docking in terms of binding affinities and interacting residues for predicting molecular mechanism. • Assessing the potency of the lead phytochemicals using molecular dynamic simulations and MMPBSA. • Best drug candidates as hexokinase 2 inhibitors were predicted for further pre-clinical and clinical trials. Newbouldia laevis, also known as the African Border Tree or Fever Tree, is a deciduous tree native to West Africa. The plant is valued for its medicinal properties and is used in traditional medicine for its antimicrobial and anti-inflammatory effects. N. laevis, is a storage tank of phytochemicals with huge health benefits and performances globally for the treatment and management of numerous disease conditions. Limited research exists on the usage of N. laevis for hepatocellular carcinoma (HCC) treatment. This study aims to explore the inhibitory activities of phytochemicals from N. laevis against the hexokinase 2 protein, a target in hepatocarcinoma (HCC) treatment. This study presents a unique in silico approach that includes ligand binding site prediction, molecular docking, molecular dynamics simulation, and Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) methods. A total of 35 phytochemicals with available 3D structures were identified through literature mining. The literature review highlighted the significance of hexokinase 2 protein as a target inhibitor in the treatment of hepatocellular carcinoma (HCC). Molecular docking experiment with the all the identified phytochemicals and hexokinase 2 revealed that all the identified phytochemicals had potential inhibitory activities against the target protein. Moreover, chrysarobin, apigenin and ursolic acid were the best inhibitors with lowest binding energy of −8.9 kcal/mol, −8.7 kcal/mol, and −8.5 kcal/mol, respectively. The docking experiment was validated by comparing the binding affinities with known reference drug Cabozantinib-S-malate (−8.3 kcal/mol). Further, molecular dynamics studies of complexes with the best docking scores and reference drug complexes were described in detail here. The results of 100 ns modeling (RMSD, RMSF, Rg and SASA) show extraordinary stability during the establishment of complexes with apigenin and ursolic acid, as well as favorable binding energy, which was determined theoretically by means of the MM/PBSA method, thereby increase the probability of their acting as promising and likely hexokinase 2 inhibitors. Therefore, the study predicted that apigenin and ursolic acid could be used as a potential inhibitor/antagonist for the hexokinase 2 enzyme. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) is a complicated disease related to metabolism that results from resistance to insulin and sustained hyperglycemia. Traditional antidiabetic drugs cannot meet the demand of different diabetes patients for reaching the glycemic targets; thus, the identification of new antidiabetic drugs is urgently needed for the treatment of T2DM to enhance glycemic control and the prognosis of patients suffering from T2DM. Recently, glucokinase (GK) has attracted much attention and is considered to be an effective antidiabetic agent. Glucokinase activators (GKA) represented by dorzagliatin could activate GK and mimic its function that triggers a counter‐regulatory response to blood glucose changes. Dorzagliatin has shown great potential for glycemic control in diabetic patients in a randomized, double‐blind, placebo‐controlled Phase 3 trial (SEED study) and had a favorable safety profile and was well tolerated (DAWN study). In the SEED study, dorzagliatin significantly reduced glycosylated hemoglobin (HbA1c) by 1.07% and postprandial blood glucose by 2.83 mol/L, showing the great potential of this drug to control blood glucose in diabetic patients, with good safety and good tolerance. An extension of the SEED study, the DREAM study, confirmed that dorzagliatin monotherapy significantly improved 24‐h glucose variability and increased time in range (TIR) to 83.7% over 46 weeks. Finally, the clinical study of dorzagliatin combined with metformin (DAWN study) confirmed that dorzagliatin could significantly reduce HbA1c by 1.02% and postprandial blood glucose by 5.45 mol/L. The current review summarizes the development of GK and GKA, as well as the prospects, trends, applications, and shortcomings of these treatments, especially future directions of clinical studies of dorzagliatin. [ABSTRACT FROM AUTHOR]

Background: Glioma is the main subtype of primary central nervous system (CNS) tumor with high malignancy and poor prognosis under current therapeutic approaches. Glycolysis and suppressive tumor microenvironment (TME) are key markers of glioma with great importance for aggressive features of glioma and inferior clinical outcomes. Hexokinase 3 (HK3) is an important rate-limiting enzyme in glycolysis, but its function in glioma remains unknown. Methods: This study comprehensively assessed the expression distribution and immunological effect of HK3 via pan-cancer analysis based on datasets from Genotype Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and The Cancer Genome Atlas (TCGA). Furthermore, it explored the malignant phenotype and genomic landscape between low-HK3 and high-HK3 expression groups in gliomas from Chinese Glioma Genome Atlas (CGGA) and TCGA. Moreover, data from the TIMER website predicted the relationship between macrophage infiltration and HK3 expression. Also, single-cell sequencing data were used to validate the relationship. Results: For pan-cancer patients, HK3 was expressed in various cancers. The results showed that HK3 was highly expressed in gliomas and positively correlated with tumor-infiltrating immune cells (TIICs), immune checkpoints, immunomodulators, and chemokines. Meanwhile, HK3 expression was highest in normal immune cells and tissues. In gliomas, the expression of HK3 was found to be closely correlated with the malignant clinical characteristics and the infiltration of macrophages. Also, HK3 was proven to be positively associated with macrophage through single-cell sequencing data and immunohistochemistry techniques. Finally, it is predicted that samples with high HK3 expression are often malignant entities and also significant genomic aberrations of driver oncogenes. Conclusions: This is the first comprehensive research to figure out the relationship between HK3 and TME characteristics in gliomas. HK3 is positively associated with macrophage infiltration and can induce the immunosuppressive TME and malignant phenotype of gliomas. [ABSTRACT FROM AUTHOR]

Summary: The hexokinase (HK) enzyme plays a key role in red blood cell energy production. Hereditary non‐spherocytic haemolytic anaemia (HNSHA) caused by HK deficiency is a rare disorder with only 12 different disease‐associated variants identified. Here, we describe the clinical features and genotypes of four previously unreported patients with hexokinase 1 (HK1)‐related HNSHA, yielding two novel truncating HK1 variants. The patients' phenotypes varied from mild chronic haemolytic anaemia to severe infantile‐onset transfusion‐dependent anaemia. Three of the patients had mild haemolytic disease caused by the common HK1 promoter c.‐193A>G variant combined with an intragenic HK1 variant, emphasizing the importance of including this promoter variant in the haemolytic disease gene panels. HK activity was normal in a severely affected patient with a homozygous HK1 c.2599C>T, p.(His867Tyr) variant, but the affinity for ATP was reduced, hampering the HK function. In cases of HNSHA, kinetic studies should be considered in the functional studies of HK. We reviewed the literature of previously published patients to provide better insight into this rare disease and add to the understanding of genotype–phenotype correlation. [ABSTRACT FROM AUTHOR]

As a sensor, glucokinase (GK) controls glucose homeostasis, which progressively declines in patients with diabetes. GK maintains the equilibrium of glucose levels and regulates the homeostatic system set points. Endocrine and hepatic cells can both respond to glucose cooperatively when GK is activated. GK has been under study as a therapeutic target for decades due to the possibility that cellular GK expression and function can be recovered, hence restoring glucose homeostasis in patients with type 2 diabetes. Five therapeutic compounds targeting GK are being investigated globally at the moment. They all have distinctive molecular structures and have been clinically shown to have strong antihyperglycemia effects. The mechanics, classification, and clinical development of GK activators are illustrated in this review. With the recent approval and marketing of the first GK activator (GKA), dorzagliatin, GKA's critical role in treating glucose homeostasis disorder and its long‐term benefits in diabetes will eventually become clear. [ABSTRACT FROM AUTHOR]

The biotechnology of converting methane to single-cell protein (SCP) implies using fast-growing thermotolerant aerobic methanotrophic bacteria. Among the latter, members of the genus Methylococcus received significant research attention and are used in operating commercial plants. Methylococcus capsulatus MIR is a recently discovered member of this genus with the potential to be used for the purpose of SCP production. Like other Methylococcus species, this bacterium stores carbon and energy in the form of glycogen, particularly when grown under nitrogen-limiting conditions. The genome of strain MIR encodes two glycogen synthases, GlgA1 and GlgA2, which are only moderately related to each other. To obtain glycogen-free cell biomass of this methanotroph, glycogen synthase mutants, ΔglgA1, ΔglgA2, and ΔglgA1ΔglgA2, were constructed. The mutant lacking both glycogen synthases exhibited a glycogen-deficient phenotype, whereas the intracellular glycogen content was not reduced in strains defective in either GlgA1 or GlgA2, thus suggesting functional redundancy of these enzymes. Inactivation of the glk gene encoding glucokinase also resulted in a sharp decrease in glycogen content and accumulation of free glucose in cells. Wild-type strain MIR and the mutant strain ΔglgA1ΔglgA2 were also grown in a bioreactor operated in batch and continuous modes. Cell biomass of ΔglgA1ΔglgA2 mutant obtained during batch cultivation displayed high protein content (71% of dry cell weight (DCW) compared to 54% DCW in wild-type strain) as well as a strong reduction in glycogen content (10.8 mg/g DCW compared to 187.5 mg/g DCW in wild-type strain). The difference in protein and glycogen contents in biomass of these strains produced during continuous cultivation was less pronounced, yet biomass characteristics relevant to SCP production were slightly better for ΔglgA1ΔglgA2 mutant. Genome analysis revealed the presence of glgA1-like genes in all methanotrophs of the Gammaproteobacteria and Verrucomicrobia, while only a very few methanotrophic representatives of the Alphaproteobacteria possessed these determinants of glycogen biosynthesis. The glgA2-like genes were present only in genomes of gammaproteobacterial methanotrophs with predominantly halo- and thermotolerant phenotypes. The role of glycogen in terms of energy reserve is discussed. [ABSTRACT FROM AUTHOR]

Demyelinating Charcot–Marie–Tooth 4G (CMT4G) results from a recessive mutation in the 5′UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance. [ABSTRACT FROM AUTHOR]

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure–activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research. [ABSTRACT FROM AUTHOR]

The hexokinase II enzyme is bound to the (VDAC1) channel in the form of a dimer and prevents the release of cell death factors from mitochondria to the cytoplasm. Studies have shown that blocking the binding of hexokinase II enzyme to (VDAC1) led to the initiation of apoptosis in cancer cells. No peptide has been designed so far to inhibit hexokinase II. The aim of this study was to inhibit the dimerization of enzyme subunits in order to inhibition the formation of (VDAC1) and the hexokinase II complex. In this study, the molecular dynamics simulation of the enzyme in monomer and dimer states was investigated in terms of RMSF, RMSD and radius of gyration. The following process involves extracting and designing variable-length peptides from the interacting segments of enzyme monomers. Using molecular dynamics simulation, the stability of the peptide was determined in terms of RMSD. Molecular docking was used to investigate the interaction between the designed peptides. Finally, the inhibitory effect of peptides on subunit association was measured using dynamic light scattering (DLS) technique. Our results showed that the designed peptides, which mimic common amino acids in dimerization, interrupt the bona fide form of the enzyme subunits. The result of this study provides a new way to disrupt the assembly process and thereby decreased the function of the hexokinase II. [ABSTRACT FROM AUTHOR]

ADP-dependent glucokinase (ADPGK) produces glucose-6-phosphate with adenosine diphosphate (ADP) as the phosphate group donor, in contrast to ATP-dependent hexokinases (HKs). Originally found in archaea, ADPGK is involved in glycolysis. However, its biological function in most eukaryotic organisms is still unclear, and the molecular mechanism of action requires further investigation. This paper provides a concise overview of ADPGK's origin, biological function and clinical application. It aims to furnish scientific information for the diagnosis and treatment of human metabolic diseases, neurological disorders, and malignant tumours, and to suggest new strategies for the development of targeted drugs. [ABSTRACT FROM AUTHOR]

Many synthetic glucokinase activators (GKAs), modulating glucokinase (GK), an important therapeutic target in diabetes have failed to clear clinical trials. In this study, an in silico structural similarity search with differing scaffolds of reference GKAs have been used to identify derivatives from natural product databases. Ten molecules with good binding score and similar interactions to that in the co‐crystallized GK as well good activation against recombinant human GK experimentally were identified. Tetrahydropalmatine, an alkaloid present in formulations and drugs from medicinal plants, has not been explored as an antidiabetic agent and no information regarding its mechanism of action or GK activation exists. Tetrahydropalmatine activates GK with EC50 value of 71.7 ± 17.9 μM while lowering the S0.5 (7.1 mM) and increasing Vmax (9.22 μM/min) as compared to control without activator (S0.5 = 10.37 mM; Vmax = 4.8 μM/min). Kinetic data (α and β values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, Kd values of 3.8 μM suggests a good affinity for GK. In HepG2 cell line, the compound potentiated the uptake of glucose and maintained glucose homeostasis by increasing the expression of GK, glycogen synthase, and insulin receptor genes and lowering the expression of glucokinase regulatory protein (GKRP) and glucagon. Tetrahydropalmatine at low concentrations could elicit a good response by reducing expression of GKRP, increasing expression of GK while also activating it. Thus, it could be used alone or in combination as therapeutic drug as it could effectively modulate GK and alter glucose homeostasis. [ABSTRACT FROM AUTHOR]

Despite advances in the management of type 2 diabetes mellitus (T2DM), one-third of patients with diabetes do not achieve the desired glycemic goal. Considering this inadequacy, many agents that activate glucokinase have been investigated over the last two decades but were withdrawn before submission for marketing permission. Dorzagliatin is the first glucokinase activator that has been granted approval for T2DM, only in China. As overstimulation of glucokinase is linked with pathophysiological disturbances such as fatty liver and cardiovascular issues and a loss of therapeutic efficacy with time. This review aims to highlight the benefits of glucokinase activators vis-à-vis the risks associated with chronic enzymatic activation. We discuss the multisystem disturbances expected with chronic activation of the enzyme, the lessons learned with glucokinase activators of the past, the major efficacy and safety findings with dorzagliatin and its pharmacological properties, and the status of other glucokinase activators in the pipeline. The approval of dorzagliatin in China was based on the SEED and the DAWN trials, the major pivotal phase III trials that enrolled patients with T2DM with a mean glycosylated hemoglobin of 8.3–8.4%, and a mean age of 53–54.5 years from multiple sites in China. Patients with uncontrolled diabetes, cardiac diseases, organ dysfunction, and a history of severe hypoglycemia were excluded. Both trials had a randomized double-blind placebo-controlled phase of 24 weeks followed by an open-label phase of 28 weeks with dorzagliatin. Drug-naïve patients with T2DM with a disease duration of 11.7 months were enrolled in the SEED trial while the DAWN trial involved patients with T2DM with a mean duration of 71.5 months and receiving background metformin therapy. Compared with placebo, the decline in glycosylated hemoglobin at 24 weeks was more with dorzagliatin with an estimated treatment difference of − 0.57% in the SEED trial and − 0.66% in the DAWN trial. The desired glycosylated hemoglobin (< 7%) was also attained at more than two times higher rates with dorzagliatin. The glycemic improvement was sustained in the SEED trial but decreased over 52 weeks in the DAWN trial. Hyperlipidemia was observed in 12–14% of patients taking dorzagliatin versus 9–11% of patients receiving a placebo. Additional adverse effects noticed over 52 weeks with dorzagliatin included an elevation in liver enzymes, hyperuricemia, hyperlacticacidemia, renal dysfunction, and cardiovascular disturbances. Considering the statistically significant improvement in glycosylated hemoglobin with dorzagliatin in patients with T2DM, the drug may be given a chance in treatment-naïve patients with a shorter disease history. However, with the waning therapeutic efficacy witnessed in patients with long-standing diabetes, which was also one of the potential concerns with previously tested molecules, extended studies involving patients with chronic and uncontrolled diabetes are needed to comment upon the long-term therapeutic performance of dorzagliatin. Likewise, evidence needs to be generated from other countries, patients with organ dysfunction, a history of severe hypoglycemia, cardiac diseases, and elderly patients before extending the use of dorzagliatin. Apart from monitoring lipid profiles, long-term safety studies of dorzagliatin should involve the assessment of serum uric acid, lactate, renal function, liver function, and cardiovascular parameters. [ABSTRACT FROM AUTHOR]

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.

Diabetes mellitus (DM) is a metabolic disease caused by a deficiency of insulin secretion, insulin resistance, and increased hepatic glucose production. Secang wood (Caesalpinia sappan L.) is known to have antihyperglycemic activity. However, these compounds are not yet known. In silico studies are needed to determine the compounds that act as antidiabetics. This study performed molecular docking of flavonoid compounds in sappan wood against the 1V4S glucokinase receptor. The results showed that all flavonoid compounds of sappan wood were predicted to have antidiabetic activity because they had a lower docking score than metformin, the first-line therapy of type 2 diabetes mellitus. Butein is expected to have the best activity. It has the lowest docking score (-94.4836). Visualization of the docking results shows that butein interacts with the identical amino acid residues as metformin, namely ARG 63 and THR 65, through the formation of hydrogen bonds and Van der Waals interactions. SWISS-ADME web tool predicted that butein has good oral absorption and excretion. The toxicity prediction tool showed a slight contradiction in the mutagenic effect. Based on this research, molecular docking may be able to design new drugs, especially from butein in sappan wood (Caesalpinia sappan L.), as antidiabetic candidates.

In this issue of Molecular Cell , Pilic et al. 1 show that hexokinase, the first enzyme of glycolysis, forms perimitochondrial rings that prevent mitochondrial fragmentation when ATP levels drop. In this issue of Molecular Cell , Pilic et al. show that hexokinase, the first enzyme of glycolysis, forms perimitochondrial rings that prevent mitochondrial fragmentation when ATP levels drop. [ABSTRACT FROM AUTHOR]

Abstract O-GlcNAcylation, an important post-translational modification catalyzed by O-GlcNAc transferase (OGT), plays critical roles in several biological processes. In this study, we present our findings on the function of FpOGT in regulating physiological processes and pathogenicity of Fusarium proliferatum (Fp), the alfalfa root rot fungus. The deletion of FpOGT impaired mycelial growth and altered macroconidia morphology in Fp. Furthermore, ΔFpOGT mutant displayed altered tolerance to various stressors, including cell wall perturbing agents, osmotic stressors, metal ionic stressors, and fungicides. Deletion of FpOGT significantly decreased Fp virulence toward alfalfa. The transcriptome analysis demonstrated that FpOGT plays a regulatory role in glucose metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and hexosamine biosynthesis pathway (HBP), by influencing the expression of relevant genes. The downregulation of the glucokinase gene, FpGCK, was observed in ΔFpOGT, and the disruption of FpGCK led to a decrease in Fp virulence. Additionally, FpOGT affected the expression levels of the FpGCK-AS1 isoform, thereby impacting glucokinase function. The molecular docking analysis elucidated the plausible physical interaction between FpOGT and FpGCK, thereby offering valuable insights into their interrelationship. These findings underscore the indispensable involvement of FpOGT, the sole O-GlcNAc transferase in Fp, in various biological processes and the pathogenicity through its regulation of fundamental metabolic processes. Consequently, this study emphasizes the significance and elucidates the molecular mechanism underlying the role of O-GlcNAc transferase in diverse fundamental biological processes and the pathogenicity of phytopathogenic fungi.

Leontodon hispidulus Boiss is a wild annual plant growing in Egypt. The present study aims for the first time, to evaluate the phytochemical profile of the main secondary metabolites of the optimized ethanolic extract of the plant using Quadrupole Time-of-Flight Liquid chromatography-mass spectrometry and Gas chromatography-mass spectrometry. It also aims to assess the anticancer activity of its different fractions against the prostate carcinoma cell line. Moreover, an in-silico docking study was performed using the Hexokinase-two enzyme. LC-qToF-MS analysis revealed the tentative identification of 36 phenolic compounds including the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin, and daphnetin), and phenolic acids (chlorogenic, caffeic, quinic, P-coumaric, and rosmarinic). GC–MS/MS analysis revealed the presence of 18 compounds where palmitic acid, myristic acid, alpha-amyrin, and beta-amyrin were the major ones. The cytotoxic activity results revealed that methylene chloride and ethyl acetate fractions showed the highest cytotoxic activity against the PC3 cell line, with IC50 values of 19, and 19.6 μg/ml, respectively. Interestingly, the docking study demonstrated that apigenin-7-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4′-O-glucoside, esculin, rosmarinic acid, chlorogenic acid, and α-amyrin exhibited high affinity to the selected target, HEK-2 enzyme. [ABSTRACT FROM AUTHOR]

The pursuit of small molecule inhibitors targeting hexokinase 2 (HK2) has significantly captivated the field of cancer drug discovery. Nevertheless, the creation of selective inhibitors aimed at specific isoforms of hexokinase (HK) remains a formidable challenge. Here, we present a multiple-pharmacophore modeling approach for designing ligands against HK2 with a marked antiproliferative effect on FaDu and Cal27 oral cancer cell lines. Molecular dynamics (MD) simulations showed that the prototype ligand exhibited a higher affinity towards HK2. Complementing this, we put forth a sustainable synthetic pathway: an environmentally conscious, single-step process facilitated through a direct amidation of the ester with an amine under transition-metal-free conditions with an excellent yield in ambient temperature, followed by a column chromatography avoided separation technique of the identified lead bioactive compound (H2) that exhibited cell cycle arrest and apoptosis. We observed that the inhibition of HK2 led to the loss of mitochondrial membrane potential and increased mitophagy as a potential mechanism of anticancer action. The lead H2 also reduced the growth of spheroids. Collectively, these results indicated the proof-of-concept for the prototypical lead towards HK2 inhibition with anticancer potential. [ABSTRACT FROM AUTHOR]

Background: Adopting the WHO protocol for glucose analysis is arguably impractical in the routine clinical setting. Deviations may develop due to a lack of understanding regarding the impact of glycolysis on the accuracy of results. Aim: We sought to assess the stability of glucose in two different blood collection tubes (BCT), BD Vacutainer® FX 'Fl-Ox' and Greiner Vacuette® FC-Mix 'FC-Mix' stored at room temperature (RT:18–22°C) and 4°C over 8.5 days. Method: Each participant provided venous whole blood collected into 51 BCTs; 'Fl-Ox' (n = 26) and 'FC-Mix' (n = 25). One Fl-Ox sample from each participant was handled according to the WHO recommended method. The remaining BCTs were stored at 4°C/RT prior to analyses at designated study timepoints. Glucose was measured using the hexokinase assay on the Cobas® 8000 platform. Results: Participants (n = 8, Male = 2) were aged 24–56 years. Plasma glucose measured in FI-Ox BCTs according to the WHO sample-handling method had a median concentration of 5.73 mmol/L (Range: 5.39–10.37 mmol/L). Glucose decreased by greater than minimal difference (>0.26 mmol/L) in blood collected into Fl-Ox and stored @4°C/RT within 24 h of phlebotomy. FC-Mix BCT maintained glucose <0.26 mmol/L @4°C over a period of 8.5 days and up to 4 days @RT when compared to the WHO recommended method. Conclusion: Glucose in FC-Mix BCT stored @4°C demonstrated the best agreement with results determined using the WHO specifications. When FC-Mix tubes were stored @RT, glucose was stable for 4 days. These findings suggest that the FC-Mix BCT effectively inhibits glycolysis and should be introduced into routine clinical practice. [ABSTRACT FROM AUTHOR]

The hole mutagenesis approach was used to interrogate the importance of F337 in Trypanosoma cruzi glucokinase (Tc GlcK) in order to understand the complete set of binding interactions that are made by d -glucosamine analogue inhibitors containing aromatic tail groups that can extend to the outer part of the active site. An interesting inhibitor of this analogue class includes 2- N -carboxybenzyl-2-deoxy- d -glucosamine (CBZ-GlcN), which exhibits strong Tc GlcK binding with a K i of 710 nM. The residue F337 is found at the outer part of the active site that stems from the second protein subunit of the homodimeric assembly. In this study, F337 was changed to leucine and alanine so as to diminish phenylalanine's side chain size and attenuate intermolecular interactions in this region of the binding cavity. Results from enzyme – inhibitor assays revealed that the phenyl group of F337 made dominant hydrophobic interactions with the phenyl group of CBZ-GlcN as opposed to π – π stacking interactions. Moreover, enzymatic activity assays and X-ray crystallographic experiments indicated that each of these site-directed mutants primarily retained their activity and had high structural similarity of their protein fold. A computed structure model of T. cruzi hexokinase (Tc HxK), which was produced by the artificial intelligence system AlphaFold, was compared to an X-ray crystal structure of Tc GlcK. Our structural analysis revealed that Tc HxK lacked an F337 counterpart residue and probably exists in the monomeric form. We proposed that the d -glucosamine analogue inhibitors that are structurally similar to CBZ-GlcN may not bind as strongly in Tc HxK as they do in Tc GlcK because of absent van der Waals contact from residue side chains. [Display omitted] • The hole mutagenesis approach was applied to Tc GlcK targeting residue F337. • The phenyl group of CBZ-GlcN interacts with F337 by hydrophobic forces, and contributes to the strong active site binding. • Comparison of the AlphaFold Tc HxK CSM lacks the Tc GlcK F337 counterpart residue. • The crystal structure of wt- Tc GlcK in the open conformation has been reported. [ABSTRACT FROM AUTHOR]

Introduction: Glucokinase (GK) plays a pivotal role in maintaining glucose homeostasis; globalagliatin, a newly developed drug, is a GK activator (GKA). This study constitutes a randomized, open-label, two-cycle, two-crossover, single-dose, phase I clinical trial conducted at a single center with healthy Chinese volunteers, aiming to examine the influence of a high-fat meal on the pharmacokinetics (PK) of orally administered globalagliatin. Methods: Twenty-four healthy volunteers were randomly divided into two groups, with a washout period of 16 days between the two cycles. The first cycle involved Group 1 volunteers who were orally administered globalagliatin 80 mg with 240 mL of water while fasting on Day 1. In contrast, Group 2 volunteers began oral administration of globalagliatin 80 mg with 240 mL of water, 30 min after consuming a high-fat meal (where high-fat content contributed to 54% of the total calories; the high-calorie meal amounted to 988.4 kcal). After the washout period, both groups of volunteers entered the second cycle of drug administration, with meals and medication being swapped on Day 17. Each volunteer collected blood samples at the following time points: 0 h (within 1 h before administration), and 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, 48, 72, 96, 120, and 168 h after administration on both trial Day 1 and Day 17. The primary and secondary PK parameters were collected. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the concentration of globalagliatin in collected plasma samples, and the results were analyzed using Phoenix WinNonlin software. Safety evaluation was conducted by detecting or observing various adverse events (AEs) and serious AEs (SAEs). Results: All 24 healthy Chinese volunteers enrolled completed the study and underwent PK analysis. The maximum concentration (Cmax; ng/mL), area under the plasma concentration-time curve (AUC) from time zero to time of the last quantifiable concentration (AUCt; h·ng/mL), and AUC from time zero extrapolated to infinity (AUC∞; h·ng/mL) of fasting administration were 22.35 ± 7.02, 725.74 ± 303.04, and 774.07 ± 343.89, respectively, while the Cmax, AUCt, and AUC∞ administered after a high-fat meal were 28.95 ± 12.60, 964.84 ± 333.99, and 1031.28 ± 392.80, respectively. The geometric mean ratios of Cmax, AUCt, and AUC∞ for high-fat meal/fasting administration of globalagliatin were 124.81%, 135.24%, and 135.42%, respectively, with 90% confidence intervals of 109.97–141.65, 124.24–147.20, and 124.42–147.39, respectively. Compared with the fasting state, healthy volunteers who consumed a high-fat meal showed a 24.8% increase in Cmax, a 35.2% increase in AUCt, and a 35.4% increase in AUC∞. The geometric mean of Tmax was 4.69 h under fasting conditions and 5.93 h in a high-fat state, with a median of 4.98 h. Among the 24 enrolled volunteers, 9 cases (37.5%) had 11 AEs, and 6 cases (25.0%) had 7 adverse drug reactions (ADRs) after medication, all of which were cured or improved without taking any treatment measures. There were no SAEs in this study, no volunteers withdrew from the study due to AEs or ADRs, and no hypoglycemic events occurred during the trial. Conclusion: A high-fat meal increased the Cmax, AUCt, and AUC∞ of globalagliatin compared with fasting conditions in healthy Chinese adult volunteers. Meanwhile, globalagliatin showed favorable safety and tolerability under fasting or high-fat meal conditions. [ABSTRACT FROM AUTHOR]

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells. [ABSTRACT FROM AUTHOR]

Bio-synthesized silver nanoparticles (AgNPs) were successfully obtained using the leaf extract from Ventilago maderaspatana. Extensive analysis was conducted to evaluate the physical and chemical characteristics of the bioderived AgNPs. XRD analysis confirmed their cubic structure, and revealed a well-defined size distribution with average crystallite size of 11.7 nm. FE-SEM and TEM images visually supported the observed size range. The presence of plant-mediated phytochemicals on the surface of AgNPs was confirmed through DLS, FTIR, and TGA/DTA studies. To assess their antidiabetic potential, rats were induced with streptozotocin, resulting in elevated levels of biochemical parameters associated with diabetes. Conversely, serum insulin levels (2.50 ± 0.55) and glucokinase activity (64.50 ± 8.66) decreased. However, treatment with AgNPs demonstrated a dose-dependent reduction in blood glucose, total protein, albumin, and HbA1c levels, effectively restoring them to normal ranges. Moreover, the treatment significantly increased insulin levels (7.55 ± 0.63) and glucokinase activity (121.50 ± 4.60), indicating the antidiabetic potential of V. maderaspatana-mediated AgNPs. Notably, the exitance of phytochemicals, like flavonoids and phenols, on the surface of AgNPs facilitated their ability to neutralize reactive oxygen species (ROS) through electron donation. This property enhanced their overall antidiabetic efficiency. [ABSTRACT FROM AUTHOR]

Schiff bases attract research interest due to their applications in chemical synthesis and medicinal chemistry. In recent years, benitrobenrazide and benserazide containing imine moiety have been synthesized and characterized as promising inhibitors of hexokinase 2 (HK2), an enzyme overexpressed in most cancer cells. Benserazide and benitrobenrazide possess a common structural fragment, a 2,3,4-trihydroxybenzaldehyde moiety connected through a hydrazone or hydrazine linker acylated on an N′ nitrogen atom by serine or a 4-nitrobenzoic acid fragment. To avoid the presence of a toxicophoric nitro group in the benitrobenrazide molecule, we introduced common pharmacophores such as 4-fluorophenyl or 4-aminophenyl substituents. Modification of benserazide requires the introduction of other endogenous amino acids instead of serine. Herein, we report the synthesis of benitrobenrazide and benserazide analogues and preliminary results of inhibitory activity against HK2 evoked by these structural changes. The derivatives contain a fluorine atom or amino group instead of a nitro group in BNB and exhibit the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a persistent, inflammatory autoimmune disease that mostly affects joints. Advanced RA may cause irreversible bone damage, limiting joint mobility. RA patients are at higher risk of secondary complications like myocardial infarctions, strokes, pulmonary dysfunction, neurological abnormalities, and depression. Physical therapy is a common arthritis treatment that can reduce symptoms when combined with medications. In RA, metabolic reprogramming is frequently characterized by increased glycolysis and glutaminolysis, which coincide with increased expression of key enzymes, namely hexokinase 2 (HK2) and glutaminase 1 (GLS1). Methods: Here in this study, 2706 phytochemicals from the Biopurify Phytochemicals subsets of the ZINC database were computationally screened against the active sites of HK2 and GLS1 proteins using Autodock vina. The docking poses and interaction analysis were examined using the Discovery Studio visualizer. Results: The compounds ZINC8234294, ZINC49888756, ZINC95627883, ZINC4096846, ZINC4098366 and ZINC49823084 interacted strongly with the majority of the active site residues of both HK2 and GLS1 proteins. The interactions of these compounds with HK2 and GLS1 were found to be facilitated by various aspects of the interactions, including the Ligand SASA, the total number of favorable and unfavorable interactions, the charge, and the number of hydrophobic and hydrogen bonds formed during the interaction. Conclusion: Targeting HK2 and GLS1, these compounds could serve as potential drug candidates for metabolic reprogramming in RA. Given that the current study is based on in silico analysis, experimental validation is necessary. [ABSTRACT FROM AUTHOR]

Background: Pancreatic adenocarcinoma, recognized for its aggressive behavior and frequent late-stage diagnosis, imposes significant challenges in early detection and prognosis. This study aimed to evaluate the diagnostic and prognostic potential by measuring the expression levels of long non-coding RNA HOTAIR and the glycolytic enzyme hexokinase 2 (HK2) mRNA in both tumorous and adjacent non-tumorous pancreatic tissue samples (n = 25 each) using RT-qPCR. Results Both lncRNA HOTAIR and HK2 expression levels significantly increased in tumorous pancreatic tissues compared to non-tumorous tissue (P = 0.001). However, their levels in stage T2 and T3 showed no statistically significant difference (P = 0.01). lncRNA HOTAIR and HK2 expression levels positively correlated with each other (P = 0.001; r = 0.95); however, no significant associations were found with serum tumor markers CA19-9 and CEA (P = 0.01; r = 005; p = 0.1, r = 0.2). ROC analysis demonstrated the significant abilities of both lncRNA HOTAIR and HK2 expression levels to discriminate between tumorous and non-tumorous pancreatic tissues (AUC = 0.92 and 0.84, respectively) with 96% and 88% sensitivity, and 72% and 40% specificity, respectively, at optimal cut-off values of 1.12 and 0.84 relative expression units. Patients with elevated lncRNA HOTAIR and HK2 expression had shorter median survival (8 and 7 months, respectively), increasing the risk of adverse outcomes or recurrence 4–4.8 times (HR = 4.08, p = 0.07; HR = 4.8, p = 0.01), thus emphasizing their prognostic potential in pancreatic cancer. Conclusion: lncRNA HOTAIR and HK2 expression levels exhibit diagnostic potential in pancreatic tumors. Elevated levels of both markers correlate strongly with adverse outcomes, underscoring their prognostic value. [ABSTRACT FROM AUTHOR]

O-GlcNAcylation, an important post-translational modification catalyzed by O-GlcNAc transferase (OGT), plays critical roles in several biological processes. In this study, we present our findings on the function of FpOGT in regulating physiological processes and pathogenicity of Fusarium proliferatum (Fp), the alfalfa root rot fungus. The deletion of FpOGT impaired mycelial growth and altered macroconidia morphology in Fp. Furthermore, ΔFpOGT mutant displayed altered tolerance to various stressors, including cell wall perturbing agents, osmotic stressors, metal ionic stressors, and fungicides. Deletion of FpOGT significantly decreased Fp virulence toward alfalfa. The transcriptome analysis demonstrated that FpOGT plays a regulatory role in glucose metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and hexosamine biosynthesis pathway (HBP), by influencing the expression of relevant genes. The downregulation of the glucokinase gene, FpGCK, was observed in ΔFpOGT, and the disruption of FpGCK led to a decrease in Fp virulence. Additionally, FpOGT affected the expression levels of the FpGCK-AS1 isoform, thereby impacting glucokinase function. The molecular docking analysis elucidated the plausible physical interaction between FpOGT and FpGCK, thereby offering valuable insights into their interrelationship. These findings underscore the indispensable involvement of FpOGT, the sole O-GlcNAc transferase in Fp, in various biological processes and the pathogenicity through its regulation of fundamental metabolic processes. Consequently, this study emphasizes the significance and elucidates the molecular mechanism underlying the role of O-GlcNAc transferase in diverse fundamental biological processes and the pathogenicity of phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

Background: Genetic metabolic illnesses called Glycogen Storage Disease type I (GSD I) are caused due to abnormalities in the glucose-6-phosphatase enzyme complex involved in the metabolism of glycogen. Objectives: The current study explores the induction and management of GSD I manifestations. Materials and Methods: Chlorogenic Acid (CGA), a hydroxycinnamic acid found in various fruits and abundant in green coffee, can inhibit the enzyme glucose-6-phosphatase complex and metformin can alter the lactic acid levels. Results: The in silico and in vivo approach unveiled, that ß-carotene showed the highest binding affinity with glucokinase, forming 7 hydrogen bonds. Riboflavin exhibited the highest binding affinity with protein phosphatase 1, forming 4 hydrogen bonds. Vitamin C interacted at the active site of the control ligand. Vitamin E also displayed significant binding affinity with glucokinase. Overall, these bioactive micro molecules show promising interactions with their respective target proteins. In vivo results revealed the significant impact of Macronutrients and Micronutrients, particularly when combining Cassava starch with the micronutrients. This combination showed notable improvements in various parameters, including liver glycogen levels, liver weight, fasting blood glucose levels, and liver histology. The findings suggest the potential of this synergistic approach in enhancing liver function and glucose regulation. To conclude this work, it was evident that the combination of Macro and Micronutrients revealed distinct advantages. UCCS and Cassava starch effectively maintained normoglycemia due to their sustained glucose release from complex carbohydrates. Remarkably, Cassava starch, both alone and combined with micronutrients, outperformed UCCS in promoting better outcomes. This highlights the potential for comprehensive research on Cassava starch's effects in GSD subjects, exploring its impact on other manifestations such as lactic acidosis, hypertriglyceridemia, and hyperuricemia. Conclusion: These findings offer promising avenues for further investigation and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Increased glycolysis is a key characteristic of malignant cells that contributes to their high proliferation rates and ability to develop drug resistance. The glycolysis rate-limiting enzyme hexokinase II (HK II) is overexpressed in most tumor cells and significantly affects tumor development. This paper examines the structure of HK II and the specific biological factors that influence its role in tumor development, as well as the potential of HK II inhibitors in antitumor therapy. Furthermore, we identify and discuss the inhibitors of HK II that have been reported in the literature. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the growth performance and carbohydrate metabolism of Pacific whiteleg shrimp after feeding with different cinnamaldehyde concentrations and protein-energy ratios. The study used a completely randomized design with six treatments in triplicates. The treatments were S003213; treatment S053213; treatment S052814; treatment S102814; treatment S052815; and treatment S102815. The study was conducted for 56 days in a 76 L volume aquarium using shrimps with 1.38 ± 0.01 g at 200 individuals/m³ . The results showed that the S053213 treatment was significantly different (P<0.05) compared to other treatments for the specific growth rate (SGR). Hexokinase (hk) and phosphoenolpyruvate carboxykinase (pepck) produced by the S053213 treatment were significantly different (P<0.05) from the S003213 treatment. The S052815 and S102815 treatments produced higher protein retention (PR) and protein efficiency ratio (PER) compared to other treatments (P<0.05) and also produced the same final average weight (FW) as the S003213 treatment. This research shows that vannamei fed by 0.10% supplementation dose of cinnamaldehyde with a decreased feed protein up to 28% and C/P ratio 14 and 15 are able to utilize feed as well as protein 32% without cinnamaldehyde. The addition of cinnamaldehyde with a higher C/P ratio requires a higher dose of cinnamaldehyde than the optimal dose. [ABSTRACT FROM AUTHOR]

Achieving glycemic control and sustaining functional pancreatic β-cell activity remains an unmet medical need in the treatment of type 2 diabetes mellitus (T2DM). Glucokinase activators (GKAs) constitute a class of anti-diabetic drugs designed to regulate blood sugar levels and enhance β-cell function in patients with diabetes. A significant progression in GKA development is underway to address the limitations of earlier generations. Dorzagliatin, a dual-acting GKA, targets both the liver and pancreas and has successfully completed two phase III trials, demonstrating favorable results in diabetes treatment. The hepato-selective GKA, TTP399, emerges as a strong contender, displaying clinically noteworthy outcomes with minimal adverse effects. This paper seeks to review the current literature, delve into the mechanisms of action of these new-generation GKAs, and assess their efficacy and safety in treating T2DM based on published preclinical studies and recent clinical trials. [ABSTRACT FROM AUTHOR]

The popularity of Chenopodium quinoa seeds has increased in recent years due to health benefits with superfood qualities. Studies to date on the health benefits of quinoa have restricted to animal models, and the results provide weak to moderate evidence to support improved plasma lipid profiles. The present research aimed to examine the health-promoting potential of Chenopodium quinoa seed extract against cigarette smoke-induced acute lung injury in Wistar rat. Results showed that body weight was significantly increased while hematology analysis showed non-significant differences in RBC count. The results revealed the serum analysis for leptin, thyroid profiling, cholesterol, vLDL and triglycerides levels were significantly raised in control positive in comparison to all the other treatment groups. While a significant decline in serum GCK level was observed in the control positive. Histopathology of the lung tissue revealed low infiltration of leukocytes and reduced thickening of the hyaline membrane was observed in treatment groups as compared to the control positive. The expression level of Bcl-2, Foxa1 and IGF-1 was significantly down regulated in the treatment groups relative to the control positive. Overall, the results indicated that Chenopodium quinoa seeds extract restores the damaged lung tissue, reduces oxidative stress-dependent inflammatory response, regulates and promotes cell growth. [ABSTRACT FROM AUTHOR]

PB201 is an orally active, partial glucokinase activator targeting both pancreatic and hepatic glucokinase. As the second glucokinase activator studied beyond phase I, PB201 has demonstrated promising glycemic effects as well as favorable pharmacokinetic (PK) and safety profiles in patients with type 2 diabetes mellitus (T2DM). This study aims to develop a population PK/pharmacodynamic (PD) model for PB201 using the pooled data from nine phase I/II clinical trials conducted in non-Chinese healthy volunteers and a T2DM population and to predict the PK/PD profile of PB201 in a Chinese T2DM population. We developed the PK/PD model using the non-linear mixed-effects modeling approach. All runs were performed using the first-order conditional estimation method with interaction. The pharmacokinetics of PB201 were well fitted by a one-compartment model with saturable absorption and linear elimination. The PD effects of PB201 on reducing the fasting plasma glucose and glycosylated hemoglobin levels in the T2DM population were described by indirect response models as stimulating the elimination of fasting plasma glucose, where the production of glycosylated hemoglobin was assumed to be stimulated by fasting plasma glucose. Covariate analyses revealed enhanced absorption of PB201 by food and decreased systemic clearance with ketoconazole co-administration, while no significant covariate was identified for the pharmacodynamics. The population PK model established for non-Chinese populations was shown to be applicable to the Chinese T2DM population as verified by the PK data from the Chinese phase I study. The final population PK/PD model predicted persistent and dose-dependent reductions in fasting plasma glucose and glycosylated hemoglobin levels in the Chinese T2DM population receiving 50/50 mg, 100/50 mg, and 100/100 mg PB201 twice daily for 24 weeks independent of co-administration of metformin. Overall, the proposed population PK/PD model quantitatively characterized the PK/PD properties of PB201 and the impact of covariates on its target populations, which allows the leveraging of extensive data in non-Chinese populations with the limited data in the Chinese T2DM population to successfully supported the waiver of the clinical phase II trial and facilitate the optimal dose regimen design of a pivotal phase III study of PB201 in China. [ABSTRACT FROM AUTHOR]

Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl‐phosphate without enzymes and deacetylated by CobB deacetylase. [ABSTRACT FROM AUTHOR]

Abstract As a sensor, glucokinase (GK) controls glucose homeostasis, which progressively declines in patients with diabetes. GK maintains the equilibrium of glucose levels and regulates the homeostatic system set points. Endocrine and hepatic cells can both respond to glucose cooperatively when GK is activated. GK has been under study as a therapeutic target for decades due to the possibility that cellular GK expression and function can be recovered, hence restoring glucose homeostasis in patients with type 2 diabetes. Five therapeutic compounds targeting GK are being investigated globally at the moment. They all have distinctive molecular structures and have been clinically shown to have strong antihyperglycemia effects. The mechanics, classification, and clinical development of GK activators are illustrated in this review. With the recent approval and marketing of the first GK activator (GKA), dorzagliatin, GKA's critical role in treating glucose homeostasis disorder and its long‐term benefits in diabetes will eventually become clear.