The PKM2 inhibitor shikonin enhances piceatannol-induced apoptosis of glyoxalase I-dependent cancer cells.

Glyoxalase I (GLO I), a major enzyme involved in the detoxification of the anaerobic glycolytic byproduct methylglyoxal, is highly expressed in various tumors, and is regarded as a promising target for cancer therapy. We recently reported that piceatannol potently inhibits human GLO I and induces the death of GLO I-dependent cancer cells. Pyruvate kinase M2 (PKM2) is also a potential therapeutic target for cancer treatment, so we evaluated the combined anticancer efficacy of piceatannol plus low-dose shikonin, a potent and specific plant-derived PKM2 inhibitor, in two GLO I-dependent cancer cell lines, HL-60 human myeloid leukemia cells and NCI-H522 human non-small-cell lung cancer cells. Combined treatment with piceatannol and low-dose shikonin for 48 h synergistically reduced cell viability, enhanced apoptosis rate, and increased extracellular methylglyoxal accumulation compared to single-agent treatment, but did not alter PKM1, PKM2, or GLO I protein expression. Taken together, these results indicate that concomitant use of low-dose shikonin potentiates piceatannol-induced apoptosis of GLO I-dependent cancer cells by augmenting methylglyoxal accumulation.

Synaptic mitochondria glycation contributes to mitochondrial stress and cognitive dysfunction.

Mitochondrial and synaptic dysfunction are pathological features of brain aging and cognitive decline. Synaptic mitochondria are vital for meeting the high energy demands of synaptic transmission. However, little is known about the link between age-related metabolic changes and the integrity of synaptic mitochondria. To this end, we investigate the mechanisms of advanced glycation endproducts (AGEs)-mediated mitochondrial and synaptic stress and evaluate the strategies to eliminate these toxic metabolites. Using aged brain and novel transgenic mice overexpressing neuronal glyoxalase 1 (GLO1), we comprehensively analyzed alterations in accumulation/buildup of AGEs and related metabolites in synaptic mitochondria and the association of AGE levels with mitochondrial function. We demonstrate for the first time that synaptic mitochondria are an early and major target of AGEs and the related toxic metabolite methylglyoxal (MG), a precursor of AGEs. MG/AGEs-insulted synaptic mitochondria exhibit deterioration of mitochondrial and synaptic function. Such accumulation of MG/AGEs positively correlated with mitochondrial perturbation and oxidative stress in aging brain. Importantly, clearance of AGEs-related metabolites by enhancing neuronal GLO1, a key enzyme for detoxification/of AGEs, reduces synaptic mitochondrial AGEs accumulation and improves mitochondrial and cognitive function in aging and AGE-challenged mice. Furthermore, we evaluated the direct effect of AGEs on synaptic function in hippocampal neurons in live brain slices as an ex-vivo model and in vitro cultured hippocampal neurons by recording long-term potentiation (LTP) and measuring spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs). Neuronal GLO1 rescues deficits in AGEs-induced synaptic plasticity and transmission by fully recovery of decline in LTP or frequency of mEPSC. These studies explore crosstalk between synaptic mitochondrial dysfunction and age-related metabolic changes relevant to brain aging and cognitive decline. Synaptic mitochondria are particularly susceptible to AGEs-induced damage, highlighting the central importance of synaptic mitochondrial dysfunction in synaptic degeneration in age-related cognitive decline. Thus, augmenting GLO1 function to scavenge toxic metabolites represents a therapeutic approach to reduce age-related AGEs accumulation and to improve mitochondrial function and learning and memory.

Computational fragment-based drug design of potential Glo-I inhibitors.

In this study, a fragment-based drug design approach, particularly de novo drug design, was implemented utilising three different crystal structures in order to discover new privileged scaffolds against glyoxalase-I enzyme as anticancer agents. The fragments were evoluted to indicate potential inhibitors with high receptor affinities. The resulting compounds were served as a benchmark for choosing similar compounds from the ASINEX® database by applying different computational ligand-based drug design techniques. Afterwards, the selection of potential hits was further aided by various structure-based approaches. Then, 14 compounds were purchased, and tested in vitro against Glo-I enzyme. Of the tested 14 hits, the biological screening results showed humble activities where the percentage of Glo-I inhibition ranged from 0-18.70 %. Compound 19 and compound 28, whose percentage of inhibitions are 18.70 and 15.80%, respectively, can be considered as hits that need further optimisation in order to be converted into lead-like compounds.

ABI3- and PIF1-mediated regulation of GIG1 enhances seed germination by detoxification of methylglyoxal in Arabidopsis.

Methylglyoxal (MG) is a toxic by-product of the glycolysis pathway in most living organisms and was previously shown to inhibit seed germination. MG is detoxified by glyoxalase I and II family proteins in plants. MG is abundantly produced during early embryogenesis in Arabidopsis seeds. However, the mechanism that alleviates the toxic effect of MG in maturing seeds is poorly understood. In this study, by T-DNA mutant population screening, we found that mutations in a glyoxalase I gene (named GERMINATION-IMPAIRED GLYOXALASE 1, GIG1) led to significantly impaired germination compared with wild-type seeds. Transformation of full-length GIG1 cDNA under the constitutively active cauliflower mosaic virus 35S promoter in the gig1 background completely recovered the seed germination phenotype. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that GIG1 is uniquely expressed in seeds and is upregulated by abscisic acid (ABA) and downregulated by gibberellic acid (GA) during seed germination. An ABA signaling component, ABI3, directly activated GIG1 in maturing seeds. In addition, PHYTOCHROME INTERACTING FACTOR 1 (PIF1) also plays cooperatively with ABI3 in the regulation of GIG1 expression in the early stage of imbibed seeds. Furthermore, GIG1 expression is stably silenced by epigenetic repressors such as polycomb repressor complexes. Altogether, our results indicate that light and ABA signaling cooperate to enhance seed germination by the upregulation of GIG1 to detoxify MG in maturing seeds.

Glyoxalase I-4 functions downstream of NAC72 to modulate downy mildew resistance in grapevine.

Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α-ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI-4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain-containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI-4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI-4 via the CACGTG element, leading to inhibition of VvGLYI-4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI-4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI-4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG-associated ROS homeostasis through a salicylic acid-mediated defense pathway.

Nutraceutical Prevention of Diabetic Complications-Focus on Dicarbonyl and Oxidative Stress.

Oxidative and dicarbonyl stress, driven by excess accumulation of glycolytic intermediates in cells that are highly permeable to glucose in the absence of effective insulin activity, appear to be the chief mediators of the complications of diabetes. The most pathogenically significant dicarbonyl stress reflects spontaneous dephosphorylation of glycolytic triose phosphates, giving rise to highly reactive methylglyoxal. This compound can be converted to harmless lactate by the sequential activity of glyoxalase I and II, employing glutathione as a catalyst. The transcription of glyoxalase I, rate-limiting for this process, is promoted by Nrf2, which can be activated by nutraceutical phase 2 inducers such as lipoic acid and sulforaphane. In cells exposed to hyperglycemia, glycine somehow up-regulates Nrf2 activity. Zinc can likewise promote glyoxalase I transcription, via activation of the metal-responsive transcription factor (MTF) that binds to the glyoxalase promoter. Induction of glyoxalase I and metallothionein may explain the protective impact of zinc in rodent models of diabetic complications. With respect to the contribution of oxidative stress to diabetic complications, promoters of mitophagy and mitochondrial biogenesis, UCP2 inducers, inhibitors of NAPDH oxidase, recouplers of eNOS, glutathione precursors, membrane oxidant scavengers, Nrf2 activators, and correction of diabetic thiamine deficiency should help to quell this.

Nordihydroguaiaretic acid inhibits glyoxalase I, and causes the accumulation of methylglyoxal followed by cell-growth inhibition.

BACKGROUND: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. METHODS: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. RESULTS: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. CONCLUSION: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition.

Lead optimization and biological evaluation of diazenylbenzenesulfonamides inhibitors against glyoxalase-I enzyme as potential anticancer agents.

In a previous report, we described the discovery of (E)-5-((8-hydroxyquinolin-5-yl)diazenyl)-2-methylbenzenesulfonamide as a potent inhibitor of GLO-I enzyme with IC50 of 1.28 ± 0.12 µM. Herein, lead optimization of this compound was achieved through targeting the central zinc atom and hydrophilic amino acid residues in the active site of the enzyme. Among the synthesized compounds, compound TS010 showed the most potent inhibitory activity with IC50 of 0.57 ± 0.04 µM. Compound TS013 also showed comparable activity to that of the lead compound with IC50 of 1.14 ± 0.03 µM. Molecular docking studies disclosed the binding mode of the compounds inside the active side of GLO-I enzyme.

Addition of hydrophobic side chains improve the apoptosis inducibility of the human glyoxalase I inhibitor, TLSC702.

Glyoxalase I (GLO I) is a known therapeutic target in cancer. Even though TLSC702, a GLO I inhibitor that we discovered, induces apoptosis in tumor cells, exceptionally higher doses are required compared with those needed to inhibit GLO I activity in vitro. In this work, structure-activity optimization studies were conducted on four sections of the TLSC702 molecule to determine the partial structural features necessary for the inhibition of GLO I. Herein, we found that the carboxy group in TLSC702 was critical for binding with the divalent zinc at the active site of GLO I. In contrast, the side chain substituents in the meta- and para- positions of the benzene ring had little influence on the in vitro inhibition of GLO I. The CLogP values of the TLSC702 derivatives showed a positive correlation with the antiproliferative effects on NCI-H522 cells. Thus, two derivatives of TLSC702, which displayed either high or low lipophilicity due to the types of substituents at the phenyl position, were selected. Even though both derivatives showed comparable inhibitory effects as that of their parent compound, the derivative with the high CLogP value was distinctly more antiproliferative than TLSC702. In contrast, the derivative with the low CLogP value did not decrease cell viability in NCI-H522 and HL-60 cells. These findings suggested that structural improvements, such as the addition of hydrophobic moieties to the phenyl group, enhanced the ability of TLSC702 to induce apoptosis by increasing cell membrane permeability.

Methylglyoxal-glyoxalase system as a possible selection module for raising marker-safe plants in rice.

Methylglyoxal (MG) is ubiquitously produced in all living organisms as a byproduct of glycolysis, higher levels of which are cytotoxic, leading to oxidative stress and apoptosis in the living systems. Though its generation is spontaneous but its detoxification involves glyoxalase pathway genes. Based on this understanding, the present study describes the possible role of MG as a novel non-antibiotic-based selection agent in rice. Further, by metabolizing MG, the glyoxalase pathway genes viz. glyoxalase I (GLYI) and glyoxalase II (GLYII), may serve as selection markers. Therefore, herein, transgenic rice harboring GLYI-GLYII genes (as selection markers) were developed and the effect of MG as a selection agent was assessed. The 3 mM MG concentration was observed as optimum for the selection of transformed calli, allowing efficient callus induction and proliferation along with high regeneration frequency (55 ± 2%) of the transgenic calli. Since the transformed calli exhibited constitutively higher activity of GLYI and GLYII enzymes compared to the wild type calli, the rise in MG levels was restricted even upon exogenous addition of MG during the selection process, resulting in efficient selection of the transformed calli. Therefore, MG-based selection method is a useful and efficient system for selection of transformed plants without significantly compromising the transformation efficiency. Further, this MG-based selection system is bio-safe and can pave way towards better public acceptance of transgenic plants.

Design, synthesis and biological evaluation of novel glyoxalase I inhibitors possessing diazenylbenzenesulfonamide moiety as potential anticancer agents.

The enzyme glyoxalase-I (Glo-I) is an essential therapeutic target in cancer treatment. Significant efforts have been made to discover competitive inhibitors of Glo-I as potential anticancer agents. Herein, we report the synthesis of a series of diazenylbenzenesulfonamide derivatives, their in vitro evaluation against Glo-I and the resulting structure-activity relationships. Among the compounds tested, compounds 9h and 9j exhibited the highest activity with IC50 1.28 µM and 1.13 µM, respectively. Docking studies to explore the binding mode of the compounds identified key moieties that may contribute to the observed activities. The active compounds will serve as suitable leads for further chemical optimization.

Discovery of a nanomolar inhibitor of the human glyoxalase-I enzyme using structure-based poly-pharmacophore modelling and molecular docking.

The glyoxalase-I (GLO-I) enzyme, which is the initial enzyme of the glyoxalase system that is responsible for the detoxification of cytotoxic α-ketoaldehydes, such as methylglyoxal, has been approved as a valid target in cancer therapy. Overexpression of GLO-I has been observed in several types of carcinomas, including breast, colorectal, prostate, and bladder cancer. In this work we aimed to identify potential GLO-I inhibitors via employing different structure-based drug design techniques including structure-based poly-pharmacophore modelling, virtual screening, and molecular docking. Poly-pharmacophore modelling was applied in this study in order to thoroughly explore the binding site of the target enzyme, thereby, revealing hits that could bind in a nonconventional way which can pave the way for designing more potent and selective ligands with novel chemotypes. The modelling phase has resulted in the selection of 31 compounds that were biologically evaluated against human GLO-I enzyme. Among the tested set, seven compounds showed excellent inhibitory activities with IC50 values ranging from 0.34 to 30.57 µM. The most active compound (ST018515) showed an IC50 of 0.34 ± 0.03 µM, which, compared to reported GLO-I inhibitors, can be considered a potent inhibitor, making it a good candidate for further optimization towards designing more potent GLO-I inhibitors.

FLOURY ENDOSPERM15 encodes a glyoxalase I involved in compound granule formation and starch synthesis in rice endosperm.

KEY MESSAGE: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.

Multi-Armed 1,2,3-Selenadiazole and 1,2,3-Thiadiazole Benzene Derivatives as Novel Glyoxalase-I Inhibitors.

Glyoxalase-I (Glo-I) enzyme was established to be a valid target for anticancer drug design. It performs the essential detoxification step of harmful byproducts, especially methylglyoxal. A robust computer-aided drug design approach was used to design and validate a series of compounds with selenium or sulfur based heterorings. A series of in-house multi-armed 1,2,3-selenadiazole and 1,2,3-thiadiazole benzene derivatives were tested for their Glo-I inhibitory activity. Results showed that these compounds bind Glo-I active sites competitively with strong potential to inhibit this enzyme with IC50 values in micro-molar concentration. Docking poses revealed that these compounds interact with the zinc atom at the bottom of the active site, which plays an essential role in its viability.

A nuclear-localized rice glyoxalase I enzyme, OsGLYI-8, functions in the detoxification of methylglyoxal in the nucleus.

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni2+ or Zn2+ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast-localized GLYI enzyme, OsGLYI-8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI-8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI-8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady-state kinetics with a low-affinity and a high-affinity substrate-binding component. Loss of AtGLYI-2, the closest Arabidopsis ortholog of OsGLYI-8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI-2 or OsGLYI-8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus.

Expression of macrophage migration inhibitory factor in footpad skin lesions with diabetic neuropathy.

Background Diabetic neuropathy originating in distal lower extremities is associated with pain early in the disease course, overwhelming in the feet. However, the pathogenesis of diabetic neuropathy remains unclear. Macrophage migration inhibitory factor has been implicated in the onset of neuropathic pain and the development of diabetes. Objective of this study was to observe pain syndromes elicited in the footpad of diabetic neuropathy rat model and to assess the contributory role of migration inhibitory factor in the pathogenesis of diabetic neuropathy. Methods Diabetic neuropathy was made in Sprague Dawley rats by streptozotocin. Pain threshold was evaluated using von Frey monofilaments for 24 weeks. On comparable experiment time after streptozotocin injection, all footpads were prepared for following procedures; glutathione assay, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining, immunohistochemistry staining, real-time reverse transcription polymerase chain reaction, and Western blot. Additionally, human HaCaT skin keratinocytes were treated with methylglyoxal, transfected with migration inhibitory factor/control small interfering RNA, and prepared for real-time reverse transcription polymerase chain reaction and Western blot. Results As compared to sham group, pain threshold was significantly reduced in diabetic neuropathy group, and glutathione was decreased in footpad skin, simultaneously, cell death was increased. Over-expression of migration inhibitory factor, accompanied by low expression of glyoxalase-I and intraepidermal nerve fibers, was shown on the footpad skin lesions of diabetic neuropathy. But, there was no significance in expression of neurotransmitters and inflammatory mediators such as transient receptor potential vanilloid 1, mas-related G protein coupled receptor D, nuclear factor kappa B, tumor necrosis factor-alpha, and interleukin-6 between diabetic neuropathy group and sham group. Intriguingly, small interfering RNA-transfected knockdown of the migration inhibitory factor gene in methylglyoxal-treated skin keratinocytes increased expression of glyoxalase-I and intraepidermal nerve fibers in comparison with control small interfering RNA-transfected cells, which was decreased by induction of methylglyoxal. Conclusions Our findings suggest that migration inhibitory factor can aggravate diabetic neuropathy by suppressing glyoxalase-I and intraepidermal nerve fibers on the footpad skin lesions and provoke pain. Taken together, migration inhibitory factor might offer a pharmacological approach to alleviate pain syndromes in diabetic neuropathy.

Interdependence of GLO I and PKM2 in the Metabolic shift to escape apoptosis in GLO I-dependent cancer cells.

Many cancer cells undergo metabolic reprogramming known as the Warburg effect, which is characterized by a greater dependence on glycolysis for ATP generation, even under normoxic conditions. Glyoxalase I (GLO I) is a rate-limiting enzyme involved in the detoxification of cytotoxic methylglyoxal formed in glycolysis and which is known to be highly expressed in many cancer cells. Thus, specific inhibitors of GLO I are expected to be effective anticancer drugs. We previously discovered a novel GLO I inhibitor named TLSC702. Although the strong inhibitory activity of TLSC702 was observed in the in vitro enzyme assay, higher concentrations were required to induce apoptosis at the cellular level. One of the proposed reasons for this difference is that cancer cells alter the energy metabolism leading them to become more dependent on mitochondrial respiration than glycolysis (Metabolic shift) to avoid apoptosis induction. Thus, we assumed that combination of TLSC702 with shikonin-a specific inhibitor of pyruvate kinase M2 (PKM2) that acts as a driver of TCA cycle by supplying pyruvate and which is known to be specifically expressed in cancer cells-would have anticancer effects. We herein show the anticancer effects of combination treatment with TLSC702 and shikonin, and a possible anticancer mechanism.

Computational and experimental exploration of the structure-activity relationships of flavonoids as potent glyoxalase-I inhibitors.

Hit, Lead & Candidate Discovery Glyoxalase-I (Glo-I) enzyme has emerged as a potential target for cancer treatment. Several classes of natural products including coumarins and flavonoids have shown remarkable Glo-I inhibitory activity. In the present study, computational and experimental approaches were used to explore the structure-activity relationships of a panel of 24 flavonoids as inhibitors of the Glo-1 enzyme. Scutellarein with an IC50 value of 2.04 µM was identified as the most potent inhibitor among the series studied. Di- or tri-hydroxylation of the benzene rings A and B accompanied with a C2/C3 double bond in ring C were identified as essential structural features for enzyme inhibition. Moreover, the ketol system showed a minor role in the inhibitory power of these compounds. The structure-activity relationships revealed in this study had deepened our understanding of the Glo-I inhibitory activities of flavonoids and opened the door for further exploration of this promising compound class.

Expression analysis of glyoxalase I gene among patients of diabetic retinopathy.

OBJECTIVES: To study expression of glyoxalase I in patients of diabetic retinopathy. METHODS: This cross-sectional comparative study was conducted at Centre for Research in Experimental and Applied Medicine (CREAM), Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi in collaboration with Armed Forces Institute of Ophthalmology (AFIO) from January 2015 to November 2015. Sampling technique was non- probability purposive sampling. Total 60 subjects were enrolled in two groups. Group-I comprised 30 patients of diabetic retinopathy and Group-II of 30 normal healthy controls. Clinical and demographic data was collected and fasting venous blood samples (2 ml) were drawn. RNA was extracted and subjected to cDNA synthesis. Expression analysis for glyoxalase I was carried out and relative quantification done by double delta Ct method. RESULTS: Mean age of the patients was 61.30 ±7.06 years and mean age of controls was 59.60 ± 6.43 years. There were 17 (56.7%) males and 13 (43.3%) females in Group-I while Group-II comprised 14 (46.7%) males and 16 (53.3%) females. There was down regulation of glyoxalase I among patients of diabetic retinopathy in comparison with controls when relative gene expression was calculated. CONCLUSION: Down regulation of glyoxalase I in patients of diabetic retinopathy suggests it to be a contributory factor in the development of disease.

Piceatannol, a natural trans-stilbene compound, inhibits human glyoxalase I.

Human glyoxalase I (GLO I), a rate-limiting enzyme for detoxification of methylglyoxal (MG), a by-product of glycolysis, is known to be a potential therapeutic target for cancer. Here, we searched new scaffolds from natural compounds for designing novel GLO I inhibitors and found trans-stilbene scaffold. We examined the inhibitory abilities to human GLO I of commercially available trans-stilbene compounds. Among them, piceatannol was found to have the most potent inhibitory activity against human GLO I. Piceatannol could inhibit the proliferation of human lung cancer NCI-H522 cells, which are dependent on GLO I for survival, in a dose- and time-dependent manner. In addition, piceatannol more significantly inhibited the proliferation of NCI-H522 cells than that of NCI-H460 cells, which are less dependent on GLO I. Importantly, overexpression of GLO I in NCI-H522 cells resulted in less sensitive to the antiproliferative activity of piceatannol. Taken together, this is the first report demonstrating that piceatannol inhibits GLO I activity and the GLO I-dependent proliferation of cancer cells. Furthermore, we determined a pharmacophore for novel inhibitors of human GLO I by computational simulation analyses of the binding mode of piceatannol to the enzyme hot spot in the active site. We suggest that piceatannol is a possible lead compound for the development of novel GLO I inhibitory anticancer drugs.