Cryptotanshinone strengthens the effect of gefitinib against non-small cell lung cancer through inhibiting transketolase.

Lung cancer is the leading cause of cancer-related mortality and causes more than a million deaths per year. Gefitinib is the first-line agent of advanced lung cancer, however, resistance to gefitinib becomes a major problem in clinical application. Transketolase (TKT) is a key enzyme functioning between the oxidative arm and the non-oxidative arm of the pentose phosphate pathway. In this study, we firstly found that the expression of TKT was remarkably up-regulated in NSCLC cells, while the knockdown of TKT could inhibit cell proliferation and enhance the effect of gefitinib on NSCLC cells, which indicated the role of TKT in treating advanced lung cancer. Cryptotanshinone (CTS) is a natural active compound possessing anti-cancer effect. Here we demonstrated that CTS could strengthen the effect of gefitinib on NSCLC cells via inhibition of TKT in vitro and in vivo. Moreover, Nrf2 was involved in the repression of CTS on TKT expression. Collectively, these findings indicated the role of TKT in lung cancer progression and may provide novel therapeutic strategies to overcome resistance to gefitinib. Furthermore, CTS may serve as a new candidate in adjuvant treatment of advanced lung cancer.

Characterisation of plasmodial transketolases and identification of potential inhibitors: an in silico study.

BACKGROUND: Plasmodial transketolase (PTKT) enzyme is one of the novel pharmacological targets being explored as potential anti-malarial drug target due to its functional role and low sequence identity to the human enzyme. Despite this, features contributing to such have not been exploited for anti-malarial drug design. Additionally, there are no anti-malarial drugs targeting PTKTs whereas the broad activity of these inhibitors against PTKTs from other Plasmodium spp. is yet to be reported. This study characterises different PTKTs [Plasmodium falciparum (PfTKT), Plasmodium vivax (PvTKT), Plasmodium ovale (PoTKT), Plasmodium malariae (PmTKT) and Plasmodium knowlesi (PkTKT) and the human homolog (HsTKT)] to identify key sequence and structural based differences as well as the identification of selective potential inhibitors against PTKTs. METHODS: A sequence-based study was carried out using multiple sequence alignment, phylogenetic tree calculations and motif discovery analysis. Additionally, TKT models of PfTKT, PmTKT, PoTKT, PmTKT and PkTKT were modelled using the Saccharomyces cerevisiae TKT structure as template. Based on the modelled structures, molecular docking using 623 South African natural compounds was done. The stability, conformational changes and detailed interactions of selected compounds were accessed viz all-atom molecular dynamics (MD) simulations and binding free energy (BFE) calculations. RESULTS: Sequence alignment, evolutionary and motif analyses revealed key differences between plasmodial and the human TKTs. High quality homodimeric three-dimensional PTKTs structures were constructed. Molecular docking results identified three compounds (SANC00107, SANC00411 and SANC00620) which selectively bind in the active site of all PTKTs with the lowest (better) binding affinity ≤ - 8.5 kcal/mol. MD simulations of ligand-bound systems showed stable fluctuations upon ligand binding. In all systems, ligands bind stably throughout the simulation and form crucial interactions with key active site residues. Simulations of selected compounds in complex with human TKT showed that ligands exited their binding sites at different time steps. BFE of protein-ligand complexes showed key residues involved in binding. CONCLUSIONS: This study highlights significant differences between plasmodial and human TKTs and may provide valuable information for the development of novel anti-malarial inhibitors. Identified compounds may provide a starting point in the rational design of PTKT inhibitors and analogues based on these scaffolds.

Structural basis for antibiotic action of the B1 antivitamin 2'-methoxy-thiamine.

The natural antivitamin 2'-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2'-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.

Structure-Based Discovery and Synthesis of Potential Transketolase Inhibitors.

Transketolase (TKL) plays a key role in plant photosynthesis and has been predicted to be a potent herbicide target. Homology modeling and molecular dynamics simulation were used to construct a target protein model. A target-based virtual screening was developed to discover novel potential transketolase inhibitors. Based on the receptor transketolase 1 and a target-based virtual screening combined with structural similarity, six new compounds were selected from the ZINC database. Among the structural leads, a new compound ZINC12007063 was identified as a novel inhibitor of weeds. Two novel series of carboxylic amide derivatives were synthesized, and their structures were rationally identified by NMR and HRMS. Biological evaluation of the herbicidal and antifungal activities indicated that the compounds 4u and 8h were the most potent herbicidal agents, and they also showed potent fungicidal activity with a relatively broad-spectrum. ZINC12007063 was identified as a lead compound of potential transketolase inhibitors, 4u and 8h which has the herbicidal and antifungal activities were synthesized based on ZINC12007063. This study lays a foundation for the discovery of new pesticides.

Innovative Electrochemical Screening Allows Transketolase Inhibitors to Be Identified.

Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC50 of 63 µM and an inhibition constant ( KI) of 3.4 µM. The electrochemical assay was also used to propose an inhibition mechanism.

Nrf2-driven TERT regulates pentose phosphate pathway in glioblastoma.

Given the involvement of telomerase activation and dysregulated metabolism in glioma progression, the connection between these two critical players was investigated. Pharmacological inhibition of human Telomerase reverse transcriptase (hTERT) by Costunolide induced glioma cell apoptosis in a reactive oxygen species (ROS)-dependent manner. Costunolide induced an ROS-dependent increase in p53 abrogated telomerase activity. Costunolide decreased Nrf2 level; and ectopic Nrf2 expression decreased Costunolide-induced ROS generation. While TERT knock-down abrogated Nrf2 levels, overexpression of Nrf2 increased TERT expression. Inhibition of hTERT either by Costunolide, or by siRNA or dominant-negative hTERT (DN-hTERT) abrogated (i) expression of Glucose-6-phosphate dehydrogenase (G6PD) and Transketolase (TKT) - two major nodes in the pentose phosphate (PPP) pathway; and (ii) phosphorylation of glycogen synthase (GS). hTERT knock-down decreased TKT activity and increased glycogen accumulation. Interestingly, siRNA-mediated knock-down of TKT elevated glycogen accumulation. Coherent with the in vitro findings, Costunolide reduced tumor burden in heterotypic xenograft glioma mouse model. Costunolide-treated tumors exhibited diminished TKT activity, heightened glycogen accumulation, and increased senescence. Importantly, glioblastoma multiforme (GBM) patient tumors bearing TERT promoter mutations (C228T and C250T) known to be associated with increased telomerase activity; exhibited elevated Nrf2 and TKT expression and decreased glycogen accumulation. Taken together, our findings highlight the previously unknown (i) role of telomerase in the regulation of PPP and glycogen accumulation and (ii) the involvement of Nrf2-TERT loop in maintaining oxidative defense responses in glioma cells.

The uremic toxin oxythiamine causes functional thiamine deficiency in end-stage renal disease by inhibiting transketolase activity.

Decreased transketolase activity is an unexplained characteristic of patients with end-stage renal disease and is linked to impaired metabolic and immune function. Here we describe the discovery of a link to impaired functional activity of thiamine pyrophosphate cofactor through the presence, accumulation, and pyrophosphorylation of the thiamine antimetabolite oxythiamine in renal failure. Plasma oxythiamine was significantly increased by 4-fold in patients receiving continuous ambulatory peritoneal dialysis and 15-fold in patients receiving hemodialysis immediately before the dialysis session (healthy individuals, 0.18 [0.11-0.22] nM); continuous ambulatory peritoneal dialysis patients, 0.64 [0.48-0.94] nM; and hemodialysis patients (2.73 [1.52-5.76] nM). Oxythiamine was converted to the transketolase inhibitor oxythiamine pyrophosphate. The red blood cell oxythiamine pyrophosphate concentration was significantly increased by 4-fold in hemodialysis (healthy individuals, 15.9 nM and hemodialysis patients, 66.1 nM). This accounted for the significant concomitant 41% loss of transketolase activity (mU/mg hemoglobin) from 0.410 in healthy individuals to 0.240 in hemodialysis patients. This may be corrected by displacement with excess thiamine pyrophosphate and explain lifting of decreased transketolase activity by high-dose thiamine supplementation in previous studies. Oxythiamine is likely of dietary origin through cooking of acidic thiamine-containing foods. Experimentally, trace levels of oxythiamine were not formed from thiamine degradation under physiologic conditions but rather under acidic conditions at 100(°)C. Thus, monitoring of the plasma oxythiamine concentration in renal failure and implementation of high-dose thiamine supplements to counter it may help improve the clinical outcome of patients with renal failure.

Transketolase counteracts oxidative stress to drive cancer development.

Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH-Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.

Mechanical insights of oxythiamine compound as potent inhibitor for human transketolase-like protein 1 (TKTL1 protein).

Transketolase is a connecting link between glycolytic and pentose phosphate pathway, which is considered as the rate-limiting step due to synthesis of large number of ATP molecule and it can be proposed as a plausible target facilitating the growth of cancerous cells suggesting its potential role in cancer. Oxythiamine, an antimetabolite has been proved to be an efficient anticancerous compound in vitro, but its structural elucidation of the inhibitory mechanism has not yet been done against the human transketolase-like 1 protein (TKTL1). The three-dimensional (3D) structure of TKTL1 protein was modeled and subjected for refinement, stability and validation. Based on the reported homologs of transketolase (TKT), the active site residues His46, Ser49, Ser52, Ser53, Ile56, Leu82, Lys84, Leu123, Ser125, Glu128, Asp154, His160, Thr216 and Lys218 were identified and considered for molecular-modeling studies. Docking studies reveal the H-bond interactions with residues Ser49 and Lys218 that could play a major role in the activity of TKTL1. Molecular dynamics (MD) simulation study was performed to reveal the comparative stability of both native and complex forms of TKTL1. MD trajectory at 30 ns, confirm the role of active site residues Ser49, Lys84, Glu128, His160 and Lys218 in suppressing the activity of TKTL1. Glu128 is observed to be the most important residue for deprotonation state of the aminopyrimidine moiety and preferred to be the site of inhibitory action. Thus, the proposed mechanism of inhibition through in silico studies would pave the way for structure-oriented drug designing against cancer.

Substrate inhibition of transketolase.

We studied the influence of the acceptor substrate of transketolase on the activity of the enzyme in the presence of reductants. Ribose-5-phosphate in the presence of cyanoborohydride decreased the transketolase catalytic activity. The inhibition is caused by the loss of catalytic function of the coenzyme-thiamine diphosphate. Similar inhibitory effect was observed in the presence of NADPH. This could indicate its possible regulatory role not only towards transketolase, but also towards the pentose phosphate pathway of carbohydrate metabolism overall, taking into account the fact that it inhibits not only transketolase but also another enzyme of the pentose phosphate pathway--glucose 6-phosphate dehydrogenase [Eggleston L.V., Krebs H.A. Regulation of the pentose phosphate cycle, Biochem. J. 138 (1974) 425-435].

Sugiol inhibits STAT3 activity via regulation of transketolase and ROS-mediated ERK activation in DU145 prostate carcinoma cells.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in various human cancers and has been used as a therapeutic target for tumors. This study screened natural products to identify compounds that inhibit STAT3 activity using a STAT3-dependent luciferase reporter system. Sugiol was identified as a compound that decreased luciferase activity in a dose-dependent manner. Sugiol specifically inhibited STAT3 phosphorylation at Tyr-705 in DU145 prostate cells, and this inhibition was independent of the STAT3 upstream kinase. Sugiol induced cell cycle arrest and decreased the expression levels of STAT3 target genes, such as cyclin D1, cyclin A, and survivin. Notably, we observed that sugiol interacted with transketolase, an enzyme in central metabolism, and increased ROS levels leading to the activation of ERK, which inhibits STAT3 activity. The protein phosphatase MEG2 was also responsible for sugiol-induced STAT3 dephosphorylation. The inhibitory effect of sugiol on cell growth was confirmed using the DU145 mouse xenograft model. We propose that sugiol inhibits STAT3 activity through a mechanism that involves the inhibition of transketolase, which leads to increased ROS levels and MEG2 activation in DU145 cells. Therefore, sugiol is the first compound regulating STAT3 activity via modulation of cancer metabolic pathway and we suggest the use of sugiol as an inhibitor of the STAT3 pathway for the treatment of human solid tumors with activated STAT3.

An efficient amperometric transketolase assay: towards inhibitor screening.

This paper describes an innovative amperometric biosensor for the in vitro determination of activity of transketolase from Escherichia coli (TKec) using commercially available TK substrates, namely d-fructose-6-phosphate a physiological donor and glycolaldehyde the best non-phosphorylated acceptor. A galactose oxidase (GAOx) biosensor, based on the immobilization of this enzyme within laponite clay, allows amperometric detection of L-erythrulose released upon TK-catalyzed reaction. A calibration curve has been established from 0.01 to 0.1 U ml(-1) TKec concentration in solution. These data are comparable to that obtained by a fluorometric method. In order to ensure a higher sensitivity and re-usability of the system, an original bienzymatic sensing system was further developed based on apoenzyme TKec and GAOx separately immobilized on the electrode surface. The inner sensing layer contains GAOx@laponite and the outer layer TKec@layered double hydroxide biohybrid. The biosensor response was validated by the determination of KD(app) for thiamine diphosphate, the TK cofactor and the inhibition action of two commercially available products, pyrophosphate, a TK cofactor analog and d-arabinose-5-phosphate, a substrate analog.

Oral cancer cells may rewire alternative metabolic pathways to survive from siRNA silencing of metabolic enzymes.

BACKGROUND: Cancer cells may undergo metabolic adaptations that support their growth as well as drug resistance properties. The purpose of this study is to test if oral cancer cells can overcome the metabolic defects introduced by using small interfering RNA (siRNA) to knock down their expression of important metabolic enzymes. METHODS: UM1 and UM2 oral cancer cells were transfected with siRNA to transketolase (TKT) or siRNA to adenylate kinase (AK2), and Western blotting was used to confirm the knockdown. Cellular uptake of glucose and glutamine and production of lactate were compared between the cancer cells with either TKT or AK2 knockdown and those transfected with control siRNA. Statistical analysis was performed with student T-test. RESULTS: Despite the defect in the pentose phosphate pathway caused by siRNA knockdown of TKT, the survived UM1 or UM2 cells utilized more glucose and glutamine and secreted a significantly higher amount of lactate than the cells transferred with control siRNA. We also demonstrated that siRNA knockdown of AK2 constrained the proliferation of UM1 and UM2 cells but similarly led to an increased uptake of glucose/glutamine and production of lactate by the UM1 or UM2 cells survived from siRNA silencing of AK2. CONCLUSIONS: Our results indicate that the metabolic defects introduced by siRNA silencing of metabolic enzymes TKT or AK2 may be compensated by alternative feedback metabolic mechanisms, suggesting that cancer cells may overcome single defective pathways through secondary metabolic network adaptations. The highly robust nature of oral cancer cell metabolism implies that a systematic medical approach targeting multiple metabolic pathways may be needed to accomplish the continued improvement of cancer treatment.

Partial inhibition and bilevel optimization in flux balance analysis.

MOTIVATION: Within Flux Balance Analysis, the investigation of complex subtasks, such as finding the optimal perturbation of the network or finding an optimal combination of drugs, often requires to set up a bilevel optimization problem. In order to keep the linearity and convexity of these nested optimization problems, an ON/OFF description of the effect of the perturbation (i.e. Boolean variable) is normally used. This restriction may not be realistic when one wants, for instance, to describe the partial inhibition of a reaction induced by a drug. RESULTS: In this paper we present a formulation of the bilevel optimization which overcomes the oversimplified ON/OFF modeling while preserving the linear nature of the problem. A case study is considered: the search of the best multi-drug treatment which modulates an objective reaction and has the minimal perturbation on the whole network. The drug inhibition is described and modulated through a convex combination of a fixed number of Boolean variables. The results obtained from the application of the algorithm to the core metabolism of E.coli highlight the possibility of finding a broader spectrum of drug combinations compared to a simple ON/OFF modeling. CONCLUSIONS: The method we have presented is capable of treating partial inhibition inside a bilevel optimization, without loosing the linearity property, and with reasonable computational performances also on large metabolic networks. The more fine-graded representation of the perturbation allows to enlarge the repertoire of synergistic combination of drugs for tasks such as selective perturbation of cellular metabolism. This may encourage the use of the approach also for other cases in which a more realistic modeling is required.

Epicatechin gallate impairs colon cancer cell metabolic productivity.

Green tea and grape phenolics inhibit cancer growth and modulate cellular metabolism. Targeting the tumor metabolic profile is a novel therapeutic approach to inhibit cancer cell proliferation. Therefore, we treated human colon adenocarcinoma HT29 cells with the phenolic compound epicatechin gallate (ECG), one of the main catechins in green tea and the most important catechin in grape extracts, and evaluated its antiproliferation effects. ECG reduced tumor viability and induced apoptosis, necrosis, and S phase arrest in HT29 cells. Later, biochemical determinations combined with mass isotopomer distribution analysis using [1,2-(13)C2]-D-glucose as a tracer were used to characterize the metabolic network of HT29 cells in response to different concentrations of ECG. Glucose consumption was importantly decreased after ECG treatment. Moreover, metabolization of [1,2-(13)C2]-D-glucose indicated that the de novo synthesis of fatty acids and the pentose phosphate pathway were reduced in ECG-treated cells. Interestingly, ECG inhibited the activity of transketolase and glucose-6-phosphate dehydrogenase, the key enzymes of the pentose phosphate pathway. Our data point to ECG as a promising chemotherapeutic agent for the treatment of colon cancer.

Diphenyl urea derivatives as inhibitors of transketolase: a structure-based virtual screening.

Transketolase is an enzyme involved in a critical step of the non-oxidative branch of the pentose phosphate pathway whose inhibition could lead to new anticancer drugs. Here, we report new human transketolase inhibitors, based on the phenyl urea scaffold, found by applying structure-based virtual screening. These inhibitors are designed to cover a hot spot in the dimerization interface of the homodimer of the enzyme, providing for the first time compounds with a suggested novel binding mode not based on mimicking the thiamine pyrophosphate cofactor.

Transketolase-like protein 1 confers resistance to serum withdrawal in vitro.

Transketolase-like protein 1 (TKTL1) is a member of the family of transketolase enzymes of which the founder member transketolase (TKT) is known to play a central role in the non-oxidative part of the pentose phosphate pathway. According to several publications TKTL1 is the only family member, whose expression is substantially de-regulated in a variety of solid tumours. Over-expression of TKTL1 correlates with poor prognosis of cancer patients and TKTL1 itself represents a potential therapeutic target owing to its possible involvement in the regulation of the proliferation and metabolism of cancer cells. We show that exogenously expressed TKTL1 provides HEK293 cells with moderate growth advantages under standard culture conditions, while protecting cells from growth factor withdrawal-induced apoptosis. Importantly, we identified TKTL1 with the JFC12T10 antibody as a 65kDa protein, which was however absent in most tumour cell lines tested. Primary head and neck squamous cell carcinomas of various localisations were characterised by a focal pattern with single cells strongly expressing TKTL1, rather than by a homogeneous expression pattern within the tumour mass.

Inhibition of transketolase by hexacyanoferrate(III).

The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.

Silencing of TKTL1 by siRNA inhibits proliferation of human gastric cancer cells in vitro and in vivo.

Tumorigenesis requires energy production via aerobic glycolysis (Warburg effect) in malignant tumors. Recent research has demonstrated that the pentose phosphate pathway (PPP) is augmented in some tumors, especially the non-oxidative part of the PPP which is controlled by transketolase (TKT) enzyme reactions. One TKT isoform, transketolase-like protein 1 (TKTL1), is specifically upregulated in different human cancers, and its overexpression predicts poor patient survival. To further define the function of in malignant progression, we employed the small interference RNA (siRNA) technique to knockdown gene expression of TKTL1 in the gastric cancer cell line AGS. We used TKTL1 siRNA to observe the effect of reduced TKTL1 expression on gastric cancer tumorigenesis in a nude mice xenograft model and on proliferation in vitro. Our results showed that the expression of double stranded RNA led to the efficient and specific inhibition of endogenous TKTL1 expression in AGS cells. In addition, the TKT activity was significantly deceased in the TKTL1 siRNA-treated AGS cells. TKTL1 suppression resulted in delayed cell proliferation in vitro. Furthermore, loss of TKTL1 inhibited the growth of AGS tumor xenografts. Altogether, our findings indicate that the specific inhibition of TKTL1 may be important therapeutically.

Is transketolase like 1 a target for the treatment of differentiated thyroid carcinoma? A study on thyroid cancer cell lines.

Radioactive iodine-refractory [(18)F] fluorodeoxy-glucose-positron emission tomography-positive thyroid carcinomas represent especially aggressive tumors. Targeting glucose metabolism by the transketolase isoenzyme transketolase like 1 (TKTL-1) which is over-expressed in various neoplasms, may be effective. The correlation of TKTL-1 expression and the response to oxythiamine as the currently best-characterized inhibitor of transketolases was studied in differentiated thyroid cancer cell lines. We determined TKTL-1 expression, proliferation, glucose uptake and GLUT-1 expression in non-treated thyroid cells and recorded the effect of oxythiamine on iodide uptake and on thymidine uptake. TKTL 1 was highest expressed in cell lines derived from more invasive tumors but the expression level was not strongly correlated to proliferation rate, to GLUT-1 expression or to the response to oxythiamine. Oxythiamine showed only a weak effect in the TKTL-1 expressing cell lines. Over-expression of TKTL-1 is not an indicator for responsiveness to oxythiamine. More specific inhibitors should be tested.