Uridine Diphosphate Research Articles

Evaluation of pathways to the C-glycosyl isoflavone puerarin in roots of kudzu (Pueraria montana lobata).

Kudzu ( Pueraria montana lobata) is used as a traditional medicine in China and Southeast Asia but is a noxious weed in the Southeastern United States. It produces both O‐ and C‐glycosylated isoflavones, with puerarin (C‐glucosyl daidzein) as an important bioactive compound. Currently, the stage of the isoflavone pathway at which the C‐glycosyl unit is added remains unclear, with a recent report of direct C‐glycosylation of daidzein contradicting earlier labeling studies supporting C‐glycosylation at the level of chalcone. We have employed comparative mRNA sequencing of the roots from two Pueraria species, one of which produces puerarin (field collected P. montana lobata) and one of which does not (commercial Pueraria phaseoloides ), to identify candidate uridine diphosphate glycosyltransferase (UGT) enzymes involved in puerarin biosynthesis. Expression of recombinant UGTs in Escherichia coli and candidate C‐glycosyltransferases in Medicago truncatula were used to explore substrate specificities, and gene silencing of UGT and key isoflavone biosynthetic genes in kudzu hairy roots employed to test hypotheses concerning the substrate(s) for C‐glycosylation. Our results confirm UGT71T5 as a C‐glycosyltransferase of isoflavone biosynthesis in kudzu. Enzymatic, isotope labeling, and genetic analyses suggest that puerarin arises both from the direct action of UGT71T5 on daidzein and via a second route in which the C‐glycosidic linkage is introduced to the chalcone isoliquiritigenin.

Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration–response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5′-triphosphate (UTP)- but not adenosine 5′-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5′-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5′-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK.

Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.

Effects of common genetic variants of human uridine diphosphate glucuronosyltransferase subfamilies on irinotecan glucuronidation

The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6–UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects.

Risk Factors for Septic Shock After Irinotecan-Containing Chemotherapy: An Exploratory Case-Control Study.

Irinotecan sometimes causes lethal septic shock but the risk factors remain unclear. This retrospective case-control study explored the potential risk factors for septic shock following irinotecan treatment. All women who received irinotecan-containing chemotherapy for gynecologic malignancies at Shizuoka General Hospital from October 2014 to September 2020 were investigated. The clinical backgrounds and blood test results of those who developed septic shock after irinotecan-containing chemotherapy were compared with those who did not. Odds ratios (ORs) for developing septic shock after receiving irinotecan were calculated with 95% confidence intervals (CIs), using univariable logistic regression analysis. During the study period, 147 women received irinotecan-containing chemotherapy. Three women developed septic shock due to neutropenic enterocolitis after irinotecan treatment, and 144 did not. The three patients with septic shock had recurrent cervical cancer, heterozygous variants in the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene (two patients had *1/*6, one had *1/*28 variants), a history of concurrent chemoradiation therapy, 50-60 Gy of pelvic irradiation, and platinum-combined chemotherapy. A history of pelvic irradiation was identified as a possible risk factor for developing septic shock after irinotecan-containing chemotherapy (OR 63.0, 95% CI 5.71-8635; p<0.001). The OR of UGT1A1 polymorphism for septic shock was 9.09 (95% CI 0.86-1233; p=0.070) in the complete case analysis. Medical personnel involved in cancer therapy should consider the possible risk of septic shock developing due to neutropenic enterocolitis when administering irinotecan-containing chemotherapy in patients with a history of pelvic irradiation.

Mechanisms underlying the methylglyoxal-induced enhancement of uridine diphosphate-mediated contraction in rat femoral artery

Although femoral artery dysfunctions, including aberrant vascular reactivity to vasoactive substances, are common in many chronic disorders, such as diabetes and hypertension, their inducible and/or progressive factors remain unclear. Methylglyoxal (MGO), a highly reactive dicarbonyl compound, has been implicated in the pathogenesis of various chronic disorders. However, its direct correlation with extracellular nucleotides including uridine 5′-diphosphate (UDP) in the femoral artery function is currently unknown. Therefore, we investigated the acute effect of MGO on UDP-induced contraction in the rat femoral artery. MGO (4.2 × 10−4 M for 1 h) enhanced the UDP-induced contraction. This enhancement was not abolished in all conditions, including nitric oxide synthase inhibition, cyclooxygenase inhibition, or endothelial denudation. In the endothelium-denuded arteries, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (10−5 M) suppressed the UDP-induced contraction in both control and MGO-treated groups, while MGO enhanced the p38 MAPK activation regardless of the UDP presence. Moreover, in the endothelium-denuded arteries, the Syk tyrosine kinase inhibitor piceatannol (10−5 M) suppressed the UDP-induced contraction. These results suggest that MGO augments UDP-induced contraction in rat femoral arteries and that this may be partly due to the alterations in the activities of Syk tyrosine kinase and p38 MAPK in the smooth muscle.

Identification of naturally occurring inhibitors in Xian-Ling-Gu-Bao capsule against the glucuronidation of estrogens.

Xian-Ling-Gu-Bao (XLGB) capsule, a well-known traditional Chinese medicine prescription, is widely used for the treatment of osteoporosis. It could significantly increase the levels of estrogen in ovariectomized rats and mice. However, this working mechanism has not been well elucidated. Considering that UDP-glucuronosyltransferase (UGT) enzymes are the important enzymes that inactivate and regulate estrogen activity in vivo, this study aimed to identify the bioactive compounds from XLGB against the glucuronidation of estrogens. First, thirty compounds were considered as candidate bioactive compounds based on our previous studies including pharmacological evaluation, chemical profiles, and metabolic profiles. Second, the characteristics of estrogen glucuronidation by uridine diphosphate glucuronic acid (UDPGA)-supplemented human liver microsomes (HLM), human intestine microsomes (HIM), and expressed UGT enzymes were determined, and the incubation systems of their key UGT enzymes were optimized. Then, inhibitory effects and mechanisms of XLGB and its main compounds toward the key UGT isozymes were further investigated. As a result, estrogen underwent efficient glucuronidation by HLM and HIM. UGT1A10, 1A1, and 2B7 were mainly responsible for the glucuronidation of estrone, β-estradiol, and estriol, respectively. For E1 and E2, UGT1A10 and 1A1 tended to mediate estrogen-3-O-glucuronidation, while UGT2B7 preferred catalyzing estrogen-16-O-glucuronidation. Furthermore, the incubation system for active UGT isoforms was optimized including Tris-HCl buffer, detergents, MgCl2 concentration, β-glucuronidase inhibitors, UDPGA concentration, protein concentration, and incubation time. Based on optimal incubation conditions, eleven, nine, and nine compounds were identified as the potent inhibitors for UGT1A10, 1A1, and 2B7, respectively (IC50 < 4.97 μM and Ki < 3.35 μM). Among them, six compounds (bavachin, isobavachin, isobavachalcone, neobavaisoflavone, corylifol A, and icariside II) simultaneously demonstrated potent inhibitory effects against these three active enzymes. Prenylated flavanols from Epimedium brevicornu Maxim., prenylated flavonoids from Psoralea corylifolia L., and salvianolic acids from Salvia miltiorrhiza Bge. were characterized as the most important and effective compounds. The identification of potent natural inhibitors of XLGB against the glucuronidation of estrogen laid an important foundation for the pharmacodynamic material basis.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine

SummaryN-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.

Synthesis of UDP-glucose with 1,2-trans glycoside in a one-step reaction

Uridine diphosphate (UDP)-glucose with 1,2-trans glycoside was synthesized using 2-chloro-1,3-dimethylimidazolium chloride (DMC) in a one-step reaction. The reaction achieved a high yield by the increase in glucose equivalents and UDP concentration as well as decreased the reaction temperature. The UDP-glucose in a 71% yield was purified by reverse-phase chromatography.

PHARMACOGENETIC ASPECTS OF THE USE OF PERIOPERATIVE DRUGS IN PEDIATRICS. Review

Relevance. It is known that in children the frequency of perioperative critical events due to the introduction of drugs is more than 5%. For example fentanyl, which is used as an adjunct to surgical anesthesia, is a major factor in increasing the number of deaths from opioid overdoses. Among the areas of reducing the frequency of severe adverse reactions to perioperative drugs - taking into account the pharmacogenetic variations of the patient.&#x0D; Objective is to consider current data on pharmacogenetic aspects of pharmacokinetics and pharmacodynamics of perioperative drugs.&#x0D; Methods. Analysis of the data presented in PubMed by keywords "pharmacogenetics", "general anesthetics", "analgesics", "muscle relaxants", "children". Search depth – 7 years (2014-2020), with a retrospective deepening of some positions until 2002.&#x0D; Results. The pharmacogenetic aspects of the pharmacokinetics of perioperative drugs are related to the genes of the enzymes that metabolize them and their transporters. Current data on the prevalence of polymorphic alleles of CYP2C9 genes (provides metabolism of nonsteroidal anti-inflammatory and anticonvulsant drugs) and CYP2D6 (metabolizes opioids, antidepressants, antiemetics) in Europe and, in particular, in Ukraine were presented. Thus, the inactive allele CYP2C19 * 2 was found in 13%, while the allele of increased activity CYP2C19 * 17 - in 25% of the population of Ukraine; allele with lost CYP2D6 function (CYP2D6 * 4) - in 18.6% of Ukrainians. Homozygotes with CYP2C9 * 3 polymorphism metabolize nonsteroidal anti-inflammatory drugs much more slowly than wild-type carriers, which may lead to their accumulation and side effects. The analgesic effect of codeine occurs only after it is metabolized in the liver by CYP2D6 to morphine. Respiratory depression, apnea and death may occur in patients with excessive metabolic rate even after a single dose of codeine; however, was noted the ineffectiveness of ondansetron due to a decrease in its concentration in the blood in thesepatients. Concomitant use of midazolam and fentanyl may prolong the effect of fentanyl by competing metabolism of midazolam by the hepatic enzyme CYP3A4, especially in patients with low metabolism. Plasma butyrylcholinesterase deficiency reduces succinylcholine inactivation in 1 in 1,500 people. Changing the genotype of uridine diphosphate glycosyltransferase causes increased glucuronidation of morphine, which may lead to a decrease in its effectiveness. As an example of pharmacogenetic changes in pharmacodynamics can be considered malignant hyperthermia, which occurs due to exposure to volatile anesthetics and depolarizing muscle relaxants in mutations in the RYR1 gene. Carriers of the minor allele (G) of the 5HT2A rs6313 gene require less propofol and 40% less time to start induction of anesthesia.&#x0D; Conclusions. The efficacy and safety of perioperative drugs are largely related to the pharmacogenetic aspects of their pharmacokinetics, especially mutations in genes of enzymes of the metabolism. Mutations in the genes of volatile anesthetic receptors and depolarizing muscle relaxants when their used cause a risk of malignant hyperthermia. The application of pharmacogenetics is the need to titrate drugs that have significant variability of action depending on the genotype. It is advisable to avoid the use of drugs with a high risk of pharmacogenetic reactions, if other alternative drugs are available.

Activities of Key Enzymes Involved in Starch Accumulation In Sorghum (Sorghum Bicolor L. Moench) Grains With Different Starch Contents

Starch accumulation and the seven metabolic enzyme activities in sorghum grains with different starch contents were investigated under field conditions in Shenyang Agricultural University. The results indicated that the starch accumulation in Tieza17 (high starch content variety) and Liaoza11 (low starch content variety) grains could be fitted with logistic equation. The activities of sucrose synthase (SS), soluble starch synthase (SSS) and uridine diphosphate glucose pyrophosphorylase (UGPase) in Tieza17 were similar to those in Liaoza11. Adenosine diphosphate glucose pyrophosphorylase (AGPase) and granule-bound starch synthase (GBSS) activities in Tieza17 grains were consistently higher than those in Liaoza11 grains. Starch branching enzyme (SBE) and starch debranching enzyme (DBE) activities increased or decreased significantly between the two varieties at different filling stages. It is suggested that the differences in the activities of AGPase, GBSS, SBE and DBE and their tradeoff played an important role in regulating starch accumulation in sorghum grains. Bangladesh J. Bot. 51(2): 217-222, 2022 (June)

An isoform of sucrose synthase involved in sink strength of potato (Solanum tuberosum L.): Molecular cloning, sequence analyses, 3-D structure, crucial motifs and expression

Sucrose synthase (SuSy, EC 2.4.1.13) refers to a glycosyltranferase (GT) that plays a crucial role in sugar metabolism mainly in the sink tissues of plants. Using uridine diphosphate (UDP) or adenosine diphosphate (ADP), SuSy catalyzes the reversible cleavage of sucrose into fructose, and UDP-glucose (UDP-Glc) or ADP-glucose (ADP-Glc), respectively. In plants, SuSy is encoded by relatively small gene families. It exists in multiple forms distributed differentially in the plant tissues suggesting their functional specializations. SuSy activity is regulated by a hierarchy of mechanisms including transcriptional control. Here, we report isolation and characterization of a 2,668-bp cDNA encoding a distinct full-length SuSy4 form, corresponding to SUS I group gene family, from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR using tuber total RNA. The predicted protein consisted of 805 amino acids (designated KC-SuSy). All of the available potato SuSy proteins could be categorized into 3 groups i.e., SUS I−III as reported earlier in other plants. Phylogenetic analysis was carried out using the SuSy sequences from the potato, tomato and Arabidopsis. Multiple sequence alignments revealed segment-wise identity and divergence between the SuSy forms. Using KC-SuSy sequence, secondary structures, crucial domains/motifs and amino acids, and a three-dimensional (3-D) structure were predicted. KC-SuSy was found to be predominantly expressed in tuber. The early stages of tuber development were associated with high levels of SuSy activity. This report would be particularly useful for further studies on the SuSy proteins in potato and other Solanaceae family members.

Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in flowers of safflower (Carthamus tinctorius L.) during colour-transition.

BackgroundSafflower (Carthamus tinctorius L.), well known for its flower, is widely used as a dye and traditional Chinese medicine. Flavonoids, especially flavonoid glycosides, are the main pigments and active components. However, their biosynthesis is largely unknown. Interestingly, the colour of flowers in safflower changed from yellow to red during flower development, while much of the gene and chemical bases during colour transition are unclear.MethodsIn this research, widely targeted metabolomics and transcriptomics were used to elucidate the changes in flavonoid biosynthesis from the gene and chemical points of view in flowers of safflower during colour transition. The screening of differential metabolites depended on fold change and variable importance in project (VIP) value. Differential expressed genes (DEGs) were screened by DESeq2 method. RT-PCR was used to analyse relative expressions of DEGs.ResultsA total of 212 flavonoid metabolites, including hydroxysafflor yellow A, carthamin and anthocyanins, were detected and showed a large difference. The candidate genes of glycosyltransferases and flavonoid hydroxylase that might participate in flavonoid glycoside biosynthesis were screened. Ten candidate genes were screened. Through integrated metabolomics and transcriptome analysis, a uridine diphosphate glucose glycosyltransferase gene, CtUGT9 showed a significant correlation with flavonoid glycosides in safflower. In addition, expression analysis showed that CtUGT9 was mainly expressed in the middle development of flowers and was significantly upregulated under MeJA treatment. Our results indicated that CtUGT9 might play an important role in flavonoid glycoside biosynthesis during colour-transition in safflower.

The function of uridine diphosphate glucose pyrophosphorylase in the lyophilization-stress response of Lactobacillus acidophilus

PurposeUridine diphosphate glucose pyrophosphorylase (UGPase) plays an important role in glucose metabolism, catalyzing the reversible formation and decomposition of UDP-glucose (UDPG). In previous work, we found that UGPase is a key enzyme in lyophilization response for Lactobacillus acidophilus (L. acidophilus). However, its function and regulatory mechanism in the freeze-drying stress response are unknown. Herein, the effect of UGPase on freeze-drying survival rate of Staphylococcus carnosus (S. carnosus) was studied.MethodsIn this work, the genes LBA1719 encoding UGPase of L. acidophilus ATCC4356 were inserted into plasmid pMG-36e to construct the recombinant plasmid pMG-LBA1719 and then overexpressed in S. carnosus; the control group was S. carnosus transformed by pMG-36e. The lyophilization-survival rate of overexpressed S. carnosus was determined, and the differentially expressed genes (DEGs) were analyzed by transcriptome to disclose the mechanism of LBA1719 in regulating the lyophilization-survival rate.ResultsCompared with the control group, the UGPase activities of the overexpressed S. carnosus increased by 35.49%, while the lyophilization-survival rates decreased by 11.17% (p < 0.05). Overexpression of LBA1719 decreased the expression of genes gapA, gapB, and pgiA in carbohydrate metabolism and dapA, dapB, and dapE in amino acid metabolism, significantly changing the physiological characteristics of S. carnosus and decreasing its lyophilization-survival rate.ConclusionIn summary, overexpression of UGPase accelerated the growth rate of S. carnosus and reduced its lyophilization-survival rates. GapA, gapB, pgiA, dapA, dapB, and dapE are vital to lyophilization protection in lactic acid bacteria (LAB). These findings provide new theoretical basis for analyzing the regulatory and molecular mechanisms of lyophilization resistance in LABs.

Abstract 5434: Discovery of SCR-7952, a novel, potent and high selective MAT2A inhibitor shows robust anti-tumor activities and better safety profile

Abstract The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was identified as a synthetic lethal target in methylthioadenosine phosphorylase (MTAP)-deleted cancers. The co-deletion of MTAP and CDKN2A was observed in approximately 15% of all cancers and predicted worse outcomes, which provides MAT2A a potential therapeutic target. AG-270, a first MAT2A inhibitor, is currently evaluated in early clinical study. Given the inhibitory activity on UGT1A1, a bilirubin uridine diphosphate glucuronosyl transferase regulating the homeostasis of bilirubin, treatment of AG-270 led to the bilirubin increase in patients, which limited the further dose escalation. Therefore, to avoid those off-targeted side effects and maximize benefit from the MAT2A targeted therapy, it is necessary to develop a new generation of MAT2A inhibitor with better activity and higher selectivity, especially, removal of UGT1A1 activity. Here, we report SCR-7952 is a novel MAT2A inhibitor with greater potency and higher selectivity. In biochemical assay, SCR-7952 exhibited potent inhibitory activity on MAT2A enzyme with IC50 of 21 nM, which was more potent than AG-270 (IC50, 68 nM). In HCT116 MTAP-/- cells, SCR-7952 strongly inhibited the cellular SAM production with IC50 of 2 nM, which was significantly more potent than AG-270 (IC50, 6 nM). And SCR-7952 potently inhibited HCT116 MTAP-/- cell growth with IC50 of 53 nM, which was almost 6-fold more potent than AG-270 tested in parallel (IC50, 300 nM). Meanwhile, SCR-7952 displayed more than 20-fold selectivity over the HCT116 parent cells on proliferation assay. However, AG-270 only demonstrated 4-fold selectivity in the same assay. Furthermore, inhibition of SCR-7952 on a panel of 47 life-dependent targets was determined at 10 μM, result showed that there is no significant inhibition observed. In addition to MTAP KO cells, SCR-7952 showed potently inhibited cell growth in multiple cell lines with natural MTAP-deficiency. SCR-7952 revealed excellent oral bioavailability and PK properties in pre-clinical species. In the HCT-116 MTAP-/- xenograft model, SCR-7952 demonstrated robust anti-tumor activity and achieved 72% tumor growth inhibition (TGI) at 1 mpk, QD, which was more potent than AG-270 (TGI 56% at 200 mpk, QD). Furthermore, SCR-7952 was well tolerant and no elevation of bilirubin was observed. In contrast, AG-270 at 200mpk resulted in significant increase of bilirubin. In conclusion, the above data demonstrate that SCR-7952 is a more potent and higher selective MAT2A, and will be filed IND in 2022. Citation Format: Feng Zhou, Feng Tang, Zhiyong Yu, Zhen Li, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. Discovery of SCR-7952, a novel, potent and high selective MAT2A inhibitor shows robust anti-tumor activities and better safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5434.

Abstract 2387: The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells

Abstract Metabolic changes are commonly seen in cancer. Tumor cells present a high rate of glucose and glutamine uptake and part of these nutrients supplies the hexosamine biosynthesis pathway (HBP), whose exacerbation has been related to solid tumors progression, including colorectal cancer. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end product of HBP, serves as the donor for several enzymes involved in glycan biosynthesis. Aberrant glycosylation is known to affect cellular and molecular mechanisms related to the malignant phenotype. Beta 1,6-GlcNAc branches on N-glycans (products of the MGAT5 enzyme) are associated with greater stability of transmembrane glycoproteins, as glutamine and glucose transporters, and growth factor receptors. Furthermore, changes in the availability of UDP-GlcNAc can affect the levels of hyaluronic acid (HA) and O-GlcNAc-modified proteins. The interplay between different direct demands for UDP-GlcNAc is not well understood and may help to better understand tumor metabolism. Therefore, we intend to evaluate in colorectal cancer cells how changes in levels of branched beta1,6-GlcNAc N-glycans can affect both the hexosamine pathway and the different processes which demand UDP-GlcNAc (such as O-GlcNAcylation and hyaluronic acid synthesis). For this, two experimental strategies were performed. The first focused on the inhibition of complex N-glycans using swainsonine, and the second focused on the MGAT5 inactivation using CRISPR-Cas9. The levels of UDP-GlcNAc were also manipulated using DON, an inhibitor of the GFAT enzyme. Initially, it was found that both the pharmacological inhibition of complex N-glycan biosynthesis and the specific inactivation of MGAT5 lead to significantly reduced levels of hexosamine pathway intermediates, activated monosaccharides, and proteins modified by O-GlcNAc. Furthermore, it was also observed that MGAT5 inactivation enhances the expression of genes encoding HA synthases (HAS2 and HAS3). Upon detection of metabolites by LC-MS, a multivariate analysis was performed using the statistical method of Partial Least-Squares Discriminant Analysis (PLS-DA), and it was observed that KO MGAT5 cells formed a metabolically distinct population from MOCK cells. Interestingly, after pharmacological inhibition of GFAT, an increase in the levels of beta 1,6-GlcNAc branched N-glycans and a reduction in O-GlcNAcylation were observed. Treatment with DON was also able to reduce HAS2 expression however an increased in HAS3 expression was observed. These data suggest that there is a relationship among different demands for UDP-GlcNAc in tumor cells and a possible regulatory role played by beta 1,6-GlcNAc branched N-glycans in this issue. This work contributes to a better understanding of interconnections between biochemical processes that demand UDP-GlcNAc in the context of colorectal cancer. Citation Format: Érika Elias Ferreira, Isadora de Araújo Oliveira, Adriane Regina Todeschini, Julio Cesar de Freitas-Junior. The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2387.

Abstract 5250: Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer

Abstract Objective: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is a key enzyme in irinotecan metabolism. However, the relationship between UGT1A1 genotype and the safety and efficacy of irinotecan monotherapy in the treatment of Chinese advanced gastric cancer remains unclear. Methods: A total of 110 patients were enrolled. Intravenous irinotecan was administered every 3 weeks. Irinotecan dose was selected according to the polymorphism of UGT1A1 gene, which was divided into 3 groups: UGT1A1*6 wild-type (GG type): 125mg/m2, d1, d8; UGT1A1*6 mutant heterozygosity (GA type) 100mg/m2, d1, d8; UGT1A1*6 homozygosity mutation (AA type) 75mg/m2, d1, d8 or paclitaxel 125mg/m2, d1, d8. UGT1A1 genotypes were determined by direct sequencing or next-generation sequencing of genomic DNA extracted from peripheral blood. Results: Among 110 patients of the subjects, the genotypes of UGT1A1*28 were wild-type in 78 patients (70.91%), mutant heterozygosity in 28 (25.45%) and mutant homozygosity in 4 (3.64%). UGT1A1*6 were GG in 67 cases (60.91%), GA in 35 cases (31.82%), and AA in 8 cases (7.27%). There were two heterozygous mutations in 3 patients, and no homozygous mutation was detected. A total of 10 cycles of irinotecan were administered, with a median of five cycles per patient. There was no significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*28 genotypes (P&amp;gt;0.05), while there was significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*6 genotypes (P&amp;lt;0.05). The dose intensity of irinotecan was different in patients with different subtypes of UGT1A1*6 gene, and the dose intensity of heterozygous mutant patients was lower than that of wild-type patients. However, there were no significant differences in PFS and OS among patients with different subtypes after dose adjustment and program adjustment (P&amp;gt;0.05). Conclusion: UGT1A1*6 gene polymorphism was significantly associated with diarrhea and neutropenia induced by irinotecan. The safety and effectiveness of antitumor therapy might be significantly improved by individualized drug administration according to the results of UGT1A1*6 gene polymorphism. Citation Format: Huifang Lv, Yunduan He, Caiyun Nie, Beibei Chen, Jianzheng Wang, Weifeng Xu, Jing Zhao, Xiaobing Chen, Junling Zhang. Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5250.

CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.