Liver Isoform Research Articles

Evidence for the Quercetin Binding Site of Glycogen Phosphorylase as a Target for Liver-Isoform-Selective Inhibitors against Glioblastoma: Investigation of Flavanols Epigallocatechin Gallate and Epigallocatechin.

Glycogen phosphorylase (GP) is the rate-determining enzyme in glycogenolysis, and its druggability has been extensively studied over the years for the development of therapeutics against type 2 diabetes (T2D) and, more recently, cancer. However, the conservation of binding sites between the liver and muscle isoforms makes the inhibitor selectivity challenging. Using a combination of kinetic, crystallographic, modeling, and cellular studies, we have probed the binding of dietary flavonoids epigallocatechin gallate (EGCG) and epigallocatechin (EGC) to GP isoforms. The structures of rmGPb-EGCG and rmGPb-EGC complexes were determined by X-ray crystallography, showing binding at the quercetin binding site (QBS) in agreement with kinetic studies that revealed both compounds as noncompetitive inhibitors of GP, with EGCG also causing a significant reduction in cell viability and migration of U87-MG glioblastoma cells. Interestingly, EGCG exhibits different binding modes to GP isoforms, revealing QBS as a promising site for GP targeting, offering new opportunities for the design of liver-selective GP inhibitors.

Structural basis for allosteric regulation of human phosphofructokinase-1

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

Hypoxia-preconditioned bone marrow-derived mesenchymal stem cells protect neurons from cardiac arrest-induced pyroptosis.

JOURNAL/nrgr/04.03/01300535-202504000-00027/figure1/v/2024-07-06T104127Z/r/image-tiff Cardiac arrest can lead to severe neurological impairment as a result of inflammation, mitochondrial dysfunction, and post-cardiopulmonary resuscitation neurological damage. Hypoxic preconditioning has been shown to improve migration and survival of bone marrow-derived mesenchymal stem cells and reduce pyroptosis after cardiac arrest, but the specific mechanisms by which hypoxia-preconditioned bone marrow-derived mesenchymal stem cells protect against brain injury after cardiac arrest are unknown. To this end, we established an in vitro co-culture model of bone marrow-derived mesenchymal stem cells and oxygen-glucose deprived primary neurons and found that hypoxic preconditioning enhanced the protective effect of bone marrow stromal stem cells against neuronal pyroptosis, possibly through inhibition of the MAPK and nuclear factor κB pathways. Subsequently, we transplanted hypoxia-preconditioned bone marrow-derived mesenchymal stem cells into the lateral ventricle after the return of spontaneous circulation in an 8-minute cardiac arrest rat model induced by asphyxia. The results showed that hypoxia-preconditioned bone marrow-derived mesenchymal stem cells significantly reduced cardiac arrest-induced neuronal pyroptosis, oxidative stress, and mitochondrial damage, whereas knockdown of the liver isoform of phosphofructokinase in bone marrow-derived mesenchymal stem cells inhibited these effects. To conclude, hypoxia-preconditioned bone marrow-derived mesenchymal stem cells offer a promising therapeutic approach for neuronal injury following cardiac arrest, and their beneficial effects are potentially associated with increased expression of the liver isoform of phosphofructokinase following hypoxic preconditioning.

Sulfone-based human liver pyruvate kinase inhibitors – Design, synthesis and in vitro bioactivity

Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 μM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Site-specific crosslinking reveals Phosphofructokinase-L inhibition drives self-assembly and attenuation of protein interactions

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo, respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic "gatekeeper" enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared to cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Clinical Trials in Hypertrophic Cardiomyopathy Therapy: A Comprehensive Analysis of Trials Registered in Global Clinical Databases.

With the disappointing results associated with the use of cardiac myosin inhibitors in the treatment of hypertrophic cardiomyopathy (HCM), the development of new therapies in clinical trials for HCM has rapidly increased. We assessed the characteristics of therapeutic intervention in HCM registered on ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP). We conducted a cross-sectional, descriptive study of clinical trials for therapeutic intervention in HCM registered on ClinicalTrials.gov and ICTRP. This study analyzed 137 registered trials. Regarding study designs of these trials, 77.37% were purpose of treatment, 59.12% were randomized, 50.36% were parallel assignment, 45.26% were performed with masking, 48.18% recruited less than 50 participants, and 27.74% were Phase 2 trials. In total, 67 trials were new drug trials, of which 35 drugs were tested in these trials, and 13 trials involved treatment with mavacamten. Of these 67 clinical drug trials, 44.78% of trials involved the study of amines, and 16.42% involved 1-ring heterocyclic compounds. Regarding the NCI Thesaurus Tree, 23.81% of trials involved myosin inhibitors, 23.81% of trials involved drugs belonging to agents affecting the cardiovascular system, and 20.63% were involved in testing cation channel blockers. The drug-target network showed that myosin-7, potassium voltage-gated channel subfamily h member 2, beta-1 adrenergic receptor, carnitine o-palmitoyltransferase 1, and liver isoform were the most targeted pathways of the clinical trials analyzed in the drug-target network. The number of clinical trials investigating therapeutic interventions for HCM has increased in recent years. Ultimately, recent HCM therapeutic clinical trials generally did not incorporate either randomized controlled trials or masking and were small studies recruiting fewer than 50 participants. Although recent research has focused on targeting myosin-7, the molecular signaling mechanisms involved in the pathogenesis of HCM have the potential to elucidate novel target pathways.

CONSUMPTION OF BROCCOLI SPROUTS INCREASED THE ACTIVITY OF GLUTATHIONE-DEPENDENT ANTIOXIDANT ENZYMES IN MURINE LIVER

Aim. The purpose of this work was to investigate effects of the consumption of broccoli sprouts on the activity of antioxidant defense enzymes in the liver of. GPx isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Methods. Frozen tissue was homozeniged in lysis buffer, centrifuged and resulted supernatants were used for analysis. Activities of enzymes were determined by using electrophoresis in polyacrylamide gel. After electrophoresis we conducted dyeing of separate gels for detection of superoxide dismutase (SOD), glutathione-S-transferase (GST) or glutathione peroxidase (GPx) isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Results. In the hepatic tissue of all four groups of mice, two isoforms of SOD (SOD1 and SOD2) were detected in gels. The intensity of the bands of both isoforms was not significantly different between groups. Three isoforms of GST (GST1, GST2, GST3) were detected in the liver samples. The activity of GST1 did not significantly differ between the experimental groups. Activities of GST2 and GST3 forms were significantly higher in the group “Broccoli” compared to Control and Cafeteria Diet groups. We identified three isoforms of glutathione peroxidase (GPx1, GPx2, GPx3) in liver samples. The activity of GPx isoform 1 was not significantly different between the experimental groups. The activity of GPx2 was significantly higher in the group of mice that consumed Broccoli and Cafeteria Diet + Broccoli compared to Control. GPx2 activity was significantly higher in the Broccoli group compared to the Cafeteria Diet group. Activity of GPx3 was significantly higher in the Broccoli group compared to the Control and Cafeteria Diet group. Conclusions. Cafeteria diet did not significantly affect the activity of SOD isoforms, but led to redistribution of in the activity of GST and GPx isoforms in murine liver. Feeding with broccoli spouts significantly increased the activity of 2 and 3 isoforms of GST and GPx in murine liver compared to values in control mice and mice fed with cafeteria diet. Combination of Broccoli + Cafereria diet had small activating effects on antioxidant enzyme acivity, compared with cafeteria diet.

Zymomonas mobilis pyruvate kinase: Rise of the unstable zombies.

Pyruvate kinase (PYK) is the final enzyme of glycolysis and is present in all domains of life. Mutations in PYK isozymes have a range of outcomes. Single amino acid changes in the human liver isoform (hLPYK) “tune” the Km for phosphoenolpyruvate by ∼200-fold relative wild-type; very few changes (∼10%) abolished enzyme activity. In contrast, most single substitutions in the distantly-related, Zymomonas mobilis PYK (ZmPYK) either had Km values like wild-type (∼50%) or abolished enzyme activity (∼47%). To investigate the molecular basis for substitution sensitivity, we determined the crystal structure of ZmPYK. Although ZmPYK elutes from a size exclusion column as a dimer, it crystalized as a tetramer, but with a less-extensive protein-protein interface than hLPYK. The ZmPYK structure also showed a phosphate bound in the pocket analogous to hLPYK's fructose-1,6-bisphosphate allosteric site. Thermal denaturation and activity assays suggest that phosphate plays a structural role rather than an allosteric role. By adding phosphate and modifying the protein preparation, the activity for previously-dead ZmPYK variants could be recovered (“zombies”). Kapp-PEP values for ZmPYK zombies fell within a narrow range, near the wild-type values. To assess stability of ZmPYK, we performed thermal denaturation experiments, which was irreversible for all isozymes. ZmPYK had a reproducibly lower Tm-app than hLPYK, and zombie ZmPYK variants had reproducibly lower Tm-app values than wild-type-like ZmPYK variants. Taken together, these results suggest that the amino acid changes of the zombie ZmPYK variants may decrease protein stability. The results leave open the question as to why Kapp-PEP for ZmPYK is less tunable than for hLPYK. Future studies will focus on the interplay of tuneability and allosteric regulation, since hLPYK is allosterically regulated by several effectors, whereas neither phosphate nor other potential effectors have shown allosteric regulation for ZmPYK.

Tuning liver pyruvate kinase activity up or down with a new class of allosteric modulators

The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 μM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Crystal structures revealed the conformations of these modulators, which were used as a reference for designing new modulators.

Expression Profile of Sphingosine Kinase 1 Isoforms in Human Cancer Tissues and Cells: Importance and Clinical Relevance of the Neglected 1b-Isoform.

Overexpression of sphingosine kinase 1 (SphK1) is casually associated with many types of cancer, and inhibitors of SphK1 sensitize tumors to chemotherapy. SphK1 is expressed as two major isoforms, SphK1a and SphK1b. To date, no information has been reported on the SphK1 isoform expression profile and its clinical relevance. The objective is to examine the expression profile of the SphK1a and SPhK1b isoforms in human cancer and noncancer tissues and cell lines and explore their clinical relevance. We used PCR to qualitatively examine the expression profile of these two isoforms in breast, liver, and prostate cancer tissues plus paired adjacent tissues and in 11 cancer and normal cell lines (breast, cervical, bone, prostate, colon, brain, mesothelioma tumor and benign, and human kidney cells). We found that SphK1a was ubiquitously expressed in all cancer cells and tissues tested; in contrast, SphK1b was only expressed in selective cell types in breast, prostate, and lung cancer. Our data suggest that SphK1a is important for generic SphK1/S1P functions, and SphK1b mediates specialized and/or unique pathways in a specific type of tissue and could be a biomarker for cancer. This discovery is important for future SphK1-related cancer research and may have clinical implications in drug development associated with SphK1-directed cancer treatment.

Quantification of Accurate Composition and Total Abundance of Homologous Proteins by Conserved-Plus-Surrogate Peptide Approach: Quantification of UDP Glucuronosyltransferases in Human Tissues.

Characterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucuronosyltransferases (UGTs), is important for predicting the fractional contribution of individual isoforms involved in the metabolism of a drug for applications in physiologically based pharmacokinetic (PBPK) modeling. Conventional targeted proteomics utilizes surrogate peptides, which often results in high technical and interlaboratory variability due to peptide-specific digestion leading to data inconsistencies. To address this problem, we developed a novel conserved-plus-surrogate peptide (CPSP) approach for determining the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), and human liver S9 (HLS9) fraction. The relative percent composition of UGT1A and UGT2B isoforms in the human liver was 35:5:36:11:13 for UGT1A1:1A3:1A4:1A6:1A9 and 20:32:22:21:5 for UGT2B4:2B7:2B10:2B15:2B17. The human kidney and intestine also showed unique compositions of UGT1As and UGT2Bs. The reproducibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2> 0.91). The analysis of the conserved peptides also provided the abundance for individual UGT isoforms included in this investigation as well as the total abundance (pmol/mg protein) of UGT1As and UGT2Bs across tissues, i.e., 268 and 342 (HLM), 21 and 92 (HIM), and 138 and 99 (HKM), respectively. The CPSP approach could be used for applications in the in-vitro-to-in-vivo extrapolation of drug metabolism and PBPK modeling. SIGNIFICANCE STATEMENT: We quantified the absolute compositions and total abundance of UDP glucuronosyltransferases (UGTs) in pooled human liver, intestine, and kidney microsomes using a novel conserved-plus-surrogate peptide (CPSP) approach. The CPSP approach addresses the surrogate peptide-specific variability in the determination of the absolute composition of UGTs. The data presented in this manuscript are applicable for the estimation of the fraction metabolized by individual UGTs towards better in vitro-to-in vivo extrapolation of UGT-mediated drug metabolism.

A Mouse Model of Glycogen Storage Disease Type IX-Beta: A Role for Phkb in Glycogenolysis.

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, β, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (Phkb−/−) mouse model. In this study, we assessed fasting blood glucose and ketone levels, serum metabolite concentrations, glycogen phosphorylase activity, and gene expression of gluconeogenic genes and fibrotic genes. Phkb−/− mice displayed hepatomegaly with lower fasting blood glucose concentrations. Phkb−/− mice showed partial liver glycogen phosphorylase activity and increased sensitivity to pyruvate, indicative of partial glycogenolytic activity and upregulation of gluconeogenesis. Additionally, gene expression analysis demonstrated increased lipid metabolism in Phkb−/− mice. Gene expression analysis and liver histology in the livers of old Phkb−/− mice (>40 weeks) showed minimal profibrogenic features when analyzed with age-matched wild-type (WT) mice. Collectively, the Phkb−/− mouse recapitulates mild clinical features in patients with GSD-IX-beta. Metabolic and molecular analysis confirmed that Phkb−/− mice were capable of sustaining energy homeostasis during prolonged fasting by using partial glycogenolysis, increased gluconeogenesis, and potentially fatty acid oxidation in the liver.

Unsupervised Learning for Feature Representation Using Spatial Distribution of Amino Acids in Aldehyde Dehydrogenase (ALDH2) Protein Sequences

Aldehyde dehydrogenase 2 (ALDH2) enzyme is required for alcohol detoxification. ALDH2 belongs to the aldehyde dehydrogenase family, the most important oxidative pathway of alcohol digestion. Two main liver isoforms of aldehyde dehydrogenase are cytosolic and mitochondrial. Approximately 50% of East Asians have ALDH2 deficiency (inactive mitochondrial isozyme), with lysine (K) for glutamate (E) substitution at position 487 (E487K). ALDH2 deficiency is also known as Alcohol Flushing Syndrome or Asian Glow. For people with an ALDH2 deficiency, their face turns red after drinking alcohol, and they are more susceptible to various diseases than ALDH2-normal people. This study performed a machine learning analysis of ALDH2 sequences of thirteen other species by comparing them with the human ALDH2 sequence. Based on the various quantitative metrics (physicochemical properties, secondary structure, Hurst exponent, Shannon entropy, and fractal dimension), these fourteen species were clustered into four clusters using the unsupervised machine learning (K-means clustering) algorithm. We also analyze these species using hierarchical clustering (agglomerative clustering) and draw the phylogenetic trees. The results show that Homo sapiens is more closely related to the Bos taurus and Sus scrofa species. Our experimental results suggest that the testing for discovering medicines may be done on these species before being tested in humans to alleviate the impacts of ALDH2 deficiency.

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

EP208: GSD IX natural history and novel liver disease severity score: Multicenter international collaboration uncovers longitudinal trends in liver disease severity

GSD IX is a glycogen storage disease which results from a deficiency of the enzyme phosphorylase kinase. While PhK is expressed in both muscle and liver, the muscle and liver isoforms are encoded by different genes, with the liver isozyme serving as the focus of this project. The clinical spectrum of hepatic GSD IX varies widely with genetic subtype (PHKA2, PHKB, PHKG2). Hepatic GSD IX presents early in life with mild to moderate hepatomegaly, growth retardation, fasting hyperketotic hypoglycemia, transaminitis, and elevated plasma lipids.

Characterization of human sulfotransferase 1A1 (SULT1A1) as a new target for structure-based design of drugs to treat cancer

Cytosolic sulfotransferases (SULTs) are a family of enzymes that are involved in the biotransformation and homeostasis of a diverse range of endo- and xenobiotic substrates with hydroxyl or amine groups. Sulfonation is generally considered to be a detoxification pathway as sulfo-conjugated products are more hydrophilic and have facilitated excretion. Sulfotransferase 1A1 (SULT1A1) is considered the major detoxification SULT isoform in the human liver and GI tract due to its broad substrate reactivity.

The druggability of the ATP binding site of glycogen phosphorylase kinase probed by coumarin analogues

Glycogen phosphorylase kinase (PhK) converts by phosphorylation, the inactive glycogen phosphorylase (GPb) into active GPa in the glycogenolytic pathway. It is a complex enzyme comprising of the catalytic (γ) and three regulatory subunits (α, β, δ) forming a hexadecamer with stoichiometry (αβγδ)4. Several studies have indicated PhK as a promising target for the development of antihyperglycemics as its inhibition blocks glycogenolysis in liver and a potential therapeutic target for cancer against pathological angiogenesis and tumor progression. The identification of compounds that inhibit the kinase through their direct binding to its catalytic site is an effective approach to identify bioactive molecules of therapeutic significance. Towards this, the structure of the N-terminal kinase domain (residues 1–298) of the catalytic γ subunit of PhK (PhKγtrnc) has been determined by X-ray crystallography while staurosporine and indirubin analogues have been characterized as potent inhibitors targeting the ATP binding site. In this study, a series of 38 synthetic analogues of naturally occurring coumarins were screened for inhibition of PhKγtrnc, in vitro, using a photometric assay. The IC50 values of the two most potent compounds were determined for PhKγtrnc and the pharmacologically relevant target, human liver isoform (PHKG2A). Their cellular efficacy and toxicity in HepG2 cells were further assessed ex vivo. Docking experiments and the structural comparison with previously described inhibitors reveal the binding mode of the coumarin scaffold at a no hinge region of the ATP site of PhK and the role of a conserved β3-Lys in binding. The experimental findings provide structural insights with implications to the kinase targeting and drug design.

Abstract P148: Glycogen phosphorylase and synthase inhibitors: Novel therapeutic approaches in anaplastic thyroid cancer

Abstract Thyroid cancer (TC) is the most common endocrine cancer. Effective treatment options for papillary (PTC) and follicular (FTC) thyroid cancers afford positive patient prognoses. However, there are no effective, long-lasting treatments for anaplastic thyroid cancer (ATC), which has a median survival of five to six months. While the receptor tyrosine kinase inhibitor sorafenib can extend ATC patient survival to eleven months, tumor reoccurrence and drug resistance often develop. Therefore, there is a need for more effective targeted therapies for ATC. While the cell signaling landscape is well described in ATC, little is known about tumorigenic adaptations in ATC metabolism. Tumors exhibit an increased consumption of glucose compared to normal tissues to fuel tumor progression. Some cancers meet this high glucose requirement by storing and breaking down glycogen. While glycogen has been detected in bovine and canine thyroids, no report thus far has investigated glycogen in normal human thyroids or thyroid cancer. Therefore, our objective was to determine if normal thyroid and TC cells metabolize glycogen and to evaluate pharmacological inhibition of glycogen metabolism in ATC cells. We show for the first time that normal thyroid, PTC, FTC, and ATC cells express glycogen synthase and glycogen phosphorylase brain and liver (PYGB, PYGL) isoforms. We confirmed these observations in patient samples of normal thyroid and TC tissues via immunofluorescence. We revealed that the TC samples expressed high levels of PYGB compared to normal thyroid tissue. Electron microscopy revealed that normal thyroid cells organized glycogen in discrete glycosomes, while ATC cells exhibited smaller, dispersed glycogen packets. Importantly, we demonstrated that the glycogen synthase inhibitor guaiacol depleted glycogen content and reduced ATC cell viability. Conversely, the glycogen phosphorylase inhibitor CP-91,149 (CP) increased glycogen levels and induced apoptosis. Importantly, CP also reduced the number of stem cells in the ATC cell population. We further showed that CP synergized with sorafenib to more effectively inhibit ATC proliferation. CP enhanced glycolysis but inhibited oxidative phosphorylation as revealed using Seahorse. We confirmed an increase in glucose transporter expression using RT-qPCR and glucose import following CP treatment. This possibly represents a futile response to PYGB inhibition. We reasoned that glycogen was preferentially metabolized in ATC to fuel the pentose phosphate pathway to protect against reactive oxygen species (ROS). Indeed, we detected significantly higher levels of ROS in ATC cells treated with CP. We demonstrated that thyroid cells are able to metabolize glycogen and identified a potential biomarker for thyroid cancer (PYGB), which was targeted with CP-91,149. Our work establishes glycogen metabolism as a novel metabolic process in thyroid cells that is associated with TC dedifferentiation and provides insight to the effectiveness of inhibiting glycogen metabolism as a novel therapeutic strategy in ATC. Citation Format: Cole Davidson, Jennifer Tomczak, Eyal Amiel, Frances Carr. Glycogen phosphorylase and synthase inhibitors: Novel therapeutic approaches in anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P148.