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.     

miR-137 inhibits melanoma cell proliferation through downregulation of GLO1

Late-stage melanoma is refractory to current therapies. MicroRNAs (miRNAs) can modulate many physiological and pathological processes of melanoma. Studies have demonstrated that miR-137 acts as a tumor suppressor by inhibiting the proliferation of melanoma cells through targeting multiple mRNAs. The glyoxalase system member glyoxalase 1 (GLO1) is the principal scavenging enzyme of methylglyoxal (MG), a toxic byproduct of glycolysis. Using ³⁵S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD), we found that miR-137 downregulated the expression of GLO1 in melanoma cells. Bioinformatics analysis predicted that GLO1 is a direct target of miR-137. This was validated by dual luciferase reporter assay. Quantitative RT-PCR (qRT-PCR) and western blot analysis indicated that miR-137 could decrease endogenous GLO1 expression. Furthermore, siRNA targeting of GLO1 mimicked inhibition of melanoma cell proliferation caused by miR-137 overexpression. Re-expression of GLO1 was able to restore miR-137-mediated suppression of melanoma cell proliferation. Therefore, these results suggest that miR-137 inhibits the proliferation of melanoma cells by targeting GLO1.     

A new variant glyoxalase I allele that is readily detectable in stimulated lymphocytes and lymphoblastoid cell lines but not in circulating lymphocytes or erythrocytes

We describe an allele of the human glyoxalase GLO locus that encodes an enzymatically inactive form of the protein, which would not have been detected if only circulating erythrocytes and lymphocytes had been studied. The new allele is named GLO*3 and its protein product, GLO 3. Circulating blood cells of GLO*2/GLO*3 heterozygotes have just one electrophoretic band that migrates as the normal 2-2 dimer. Lymphoblastoid cell lines and phytohemagglutinin-stimulated lymphocytes from the same individuals have two electrophoretic bands, one with the mobility of the 2-2 dimer and one with the mobility of the 2-1 dimer that is present in GLO*2/GLO*1 heterozygotes, but a band with the mobility of the 1-1 dimer is not present. Therefore, the GLO*3 allele encodes a monomer that has the electrophoretic mobility of GLO 1 but is enzymatically inactive unless it is combined with normal monomers in 2-3 and 1-3 heterodimers. The failure to detect the GLO 3 protein in red cells and unstimulated lymphocytes is attributed to a relatively great instability or small rate of production in those cells. Consistent with this interpretation is the reduction of GLO activity in red cells of GLO*2/GLO*3 and GLO*1/GLO*3 heterozygotes to 65% or less of that in normal homozygotes and heterozygotes, while the activity of GLO*3 heterozygous lymphoblastoid cells is about 80% of normal. In contrast, the GLO activity of lymphoblastoid cells that had one copy of the GLO locus deleted by γ-irradiation was 50%–60% of normal. Our observations indicate that certain kinds of mutant alleles of the GLO locus, and perhaps other loci, may not be detected in electrophoretic surveys on circulating blood cells only. The segregation of alleles that are not expressed in circulating red and white blood cells could confuse attempts to determine parentage, as they might have in the family described here. The observations also demonstrate the feasibility of mapping human genes by using ionizing radiation to create partial chromosome deletions in cultured cells.     

The benzimidazole based drugs show good activity against T. gondii but poor activity against its proposed enoyl reductase enzyme target

The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD⁺ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD⁺ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC₅₀ for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.     

The mimics of Nε-acyl-lysine derived from cysteine as sirtuin inhibitors

Sirtuin inhibitors as physiological research tools and therapeutic potentials have caught many attentions in last decades. The mimics of acyl lysine have been approved to be a very efficient strategy for development of mechanism-based sirtuin inhibitors. In current study, a novel scaffold of l-S-(3-carboxamidopropyl) cysteine (l-CAPC) has been exploited for design and synthesis of sirtuin inhibitors. As a result, the mimics of N^ε-acyl-lysine derived from cysteine including small molecules (5a–m) and peptides (9a–m) have been synthesized. Among these, the peptides 9g and 9h were found to be the most inhibitory potency and selectivity against SIRT2.     

Inhibition by active site directed covalent modification of human glyoxalase I

The glyoxalase pathway is responsible for conversion of cytotoxic methylglyoxal (MG) to d-lactate. MG toxicity arises from its ability to form advanced glycation end products (AGEs) on proteins, lipids and DNA. Studies have shown that inhibitors of glyoxalase I (GLO1), the first enzyme of this pathway, have chemotherapeutic effects both in vitro and in vivo, presumably by increasing intracellular MG concentrations leading to apoptosis and cell death. Here, we present the first molecular inhibitor, 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB), able to covalently bind to the free sulfhydryl group of Cys60 in the hydrophobic binding pocket adjacent to the enzyme active site and partially inactivate the enzyme. Our data suggests that partial inactivation of homodimeric GLO1 is due to the modification at only one of the enzymatic active sites. Although this molecule may have limited use pharmacologically, it may serve as an important template for the development of new GLO1 inhibitors that may combine this strategy with ones already reported for high affinity GLO1 inhibitors, potentially improving potency and specificity.     

Glyoxalase-I is a novel prognosis factor associated with gastric cancer progression

Glyoxalase I (GLO1), a methylglyoxal detoxification enzyme, is implicated in the progression of human malignancies. The role of GLO1 in gastric cancer development or progression is currently unclear. The expression of GLO1 was determined in primary gastric cancer specimens using quantitative polymerase chain reaction, immunohistochemistry (IHC), and western blotting analyses. GLO1 expression was higher in gastric cancer tissues, compared with that in adjacent noncancerous tissues. Elevated expression of GLO1 was significantly associated with gastric wall invasion, lymph node metastasis, and pathological stage, suggesting a novel role of GLO1 in gastric cancer development and progression. The 5-year survival rate of the lower GLO1 expression groups was significantly greater than that of the higher expression groups (log rank P = 0.0373) in IHC experiments. Over-expression of GLO1 in gastric cancer cell lines increases cell proliferation, migration and invasiveness. Conversely, down-regulation of GLO1 with shRNA led to a marked reduction in the migration and invasion abilities. Our data strongly suggest that high expression of GLO1 in gastric cancer enhances the metastasis ability of tumor cells in vitro and in vivo, and support its efficacy as a potential marker for the detection and prognosis of gastric cancer.     

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Methylglyoxal,the foe and friend of glyoxalase and Trx/TrxR systems in HT22 nerve cells

Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products (AGEs) which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. Here, we examined the effects of MGO on immortalized mouse hippocampal HT22 nerve cells. The endpoints analyzed were MGO and thiol status, the glyoxalase system, comprising glyoxalase 1 and 2 (GLO1/2), and the cytosolic and mitochondrial Trx/TrxR systems, as well as nuclear Nrf2 and its target genes. We found that nuclear Nrf2 is induced by MGO treatment in HT22 cells, as corroborated by induction of the Nrf2-controlled target genes and proteins glutamate cysteine ligase and heme oxygenase 1. Nrf2 knockdown prevented MGO-dependent induction of glutamate cysteine ligase and heme oxygenase 1. The cystine/glutamate antiporter, system x_c^–, which is also controlled by Nrf2, was also induced. The increased cystine import (system x_c^–) activity and GCL expression promoted GSH synthesis, leading to increased levels of GSH. The data indicate that MGO can act as both a foe and a friend of the glyoxalase and the Trx/TrxR systems. At low concentrations of MGO (0.3 mM), GLO2 is strongly induced, but at high MGO (0.75 mM) concentrations, GLO1 is inhibited and GLO2 is downregulated. The cytosolic Trx/TrxR system is impaired by MGO, where Trx is downregulated yet TrxR is induced, but strong MGO-dependent glycation may explain the loss in TrxR activity. We propose that Nrf2 can be the unifying element to explain the observed upregulation of GSH, GCL, HO1, TrxR1, Trx2, TrxR2, and system x_c^– system activity.     

Repurposing human PDE4 inhibitors for neglected tropical diseases: Design,synthesis and evaluation of cilomilast analogues as Trypanosoma brucei PDEB1 inhibitors

A medicinal chemistry exploration of the human phosphodiesterase 4 (hPDE4) inhibitor cilomilast (1) was undertaken in order to identify inhibitors of phosphodiesterase B1 of Trypanosoma brucei (TbrPDEB1). T. brucei is the parasite which causes African sleeping sickness, a neglected tropical disease that affects thousands each year, and TbrPDEB1 has been shown to be an essential target of therapeutic relevance. Noting that 1 is a weak inhibitor of TbrPDEB1, we report the design and synthesis of analogs of this compound, culminating in 12b, a sub-micromolar inhibitor of TbrPDEB1 that shows modest inhibition of T. brucei proliferation.     

Glyoxalase 1 (GLO) in the chicken: Genetic variation and lack of linkage to the MHC

Electrophoretic variation of glyoxalase 1 (GLO) has been detected in chicken red-cell lysates. Three phenotypes are shown to be inherited through a diallelic system, just as in humans and mice. The chicken GLO phenotypes differ from their mammalian counterparts in that one of the homozygotes is devoid of GLO activity. The heterozygote produces two bands, while the other homozygote yields a single band of GLO activity with mobility equal to the faster of these two bands. In noninbred White Leghorn birds, the GLO ^*2 allele occurred significantly more often in birds homozygous for the B ^*1 allele at the chicken MHC than in those homozygous for B ^*19, suggesting that the products of these loci may have population associations in the chicken. Absence of close linkage between the GLO and B loci was, however, demonstrated by appropriate test crosses.     

One-step partial synthesis of (±)-asperteretone B and related hPTP1B1–400 inhibitors from butyrolactone I

Protein tyrosine phosphatase 1B (PTP1B) is a validated target for developing antiobesity, antidiabetic and anticancer drugs. Over the past years, several inhibitors of PTP1B have been discovered; however, none has been approved by the drug regulatory agencies. Interestingly, the research programs focused on discovering PTP1B inhibitors typically use truncated structures of the protein (PTP1B_1-300, 1–300 amino acids), leading to the loss of valuable information about the inhibition and selectivity of ligands and repeatedly misleading the optimization of putative drug leads. Up to date, only six inhibitors of the full-length protein (hPTP1B_1-400), with affinity constants ranging from 1.3 × 10⁴ to 3.3 × 10⁶ M⁻¹, have been reported. Towards the discovery of new ligands of the full-length human PTP1B (hPTP1B_1-400) from natural sources, herein we describe the isolation of a γ-lactone (1, butyrolactone I) from the fungus Aspergillus terreus, as well as the semisynthesis, inhibitory properties (in vitro and in silico), and the structure-activity relationship of a set of butyrolactone derivatives (1 and 2, and 6–12) as hPTP1B_1-400 inhibitors, as well as the affinity constant (k_a = 2.2 × 10⁵ M⁻¹) of the 1-hPTP1B₁–₄₀₀ complex, which was determined by fluorescence quenching experiments, after the inner filter effect correction.     

Identification of glyoxalase 1 polymorphisms associated with enzyme activity

The glyoxalase system and its main enzyme, glyoxalase 1 (GLO1), protect cells from advanced glycation end products (AGEs), such as methylglyoxal (MG) and other reactive dicarbonyls, the formation of which is increased in diabetes patients as a result of excessive glycolysis. MG is partly responsible for harmful protein alterations in living cells, notably in neurons, leading to their dysfunction, and recent studies have shown a negative correlation between GLO1 expression and tissue damage. Neuronal dysfunction is a common diabetes complication due to elevated blood sugar levels, leading to high levels of AGEs. The aim of our study was to determine whether single nucleotide polymorphisms (SNPs) in the GLO1 gene influence activity of the enzyme. In total, 125 healthy controls, 101 type 1 diabetes, and 100 type 2 diabetes patients were genotyped for three common SNPs, rs2736654 (A111E), rs1130534 (G124G), and rs1049346 (5′-UTR), in GLO1. GLO1 activity was determined in whole blood lysates for all participants of the study.     

CXCR4 inhibitors selectively eliminate CXCR4-expressing human acute myeloid leukemia cells in NOG mouse model

The chemokine receptor CXCR4 favors the interaction of acute myeloid leukemia (AML) cells with their niche but the extent to which it participates in pathogenesis is unclear. Here, we show that CXCR4 expression at the surface of leukemic cells allowed distinguishing CXCR4^high from CXCR4^neg/low AML patients. When high levels of CXCR4 are expressed at the surface of AML cells, blocking the receptor function with small molecule inhibitors could promote leukemic cell death and reduce NOD/Shi-scid/IL-2Rγ^null (NOG) leukemia-initiating cells (LICs). Conversely, these drugs had no efficacy when AML cells do not express CXCR4 or when they do not respond to chemokine CXC motif ligand 12 (CXCL12). Functional analysis showed a greater mobilization of leukemic cells and LICs in response to drugs, suggesting that they target the interaction between leukemic cells and their supportive bone marrow microenvironment. In addition, increased apoptosis of leukemic cells in vitro and in vivo was observed. CXCR4 expression level on AML blast cells and their migratory response to CXCL12 are therefore predictive of the response to the inhibitors and could be used as biomarkers to select patients that could potentially benefit from the drugs.     

Bcl-2 inhibitors as anti-cancer therapeutics: The impact of and on calcium signaling

Bcl-2-protein family members are essential regulators of apoptosis. Anti-apoptotic Bcl-2 proteins ensure cell survival via different mechanisms, including via binding of pro-apoptotic Bcl-2-family members and the modulation of intracellular Ca²⁺-transport systems. Many cancer cells upregulate these proteins to overcome the consequences of ongoing oncogenic stress. Bcl-2 inhibition leading to cell death, therefore emerged as a novel cancer therapy. Different Bcl-2 inhibitors have already been developed including the hydrophobic cleft-targeting BH3 mimetics, which antagonize Bcl-2’s ability to scaffold and neutralize pro-apoptotic Bcl-2-family members. As such, the BH3 mimetics have progressed into clinical studies as precision medicines. Furthermore, new inhibitors that target Bcl-2’s BH4 domain have been developed as promising anti-cancer tools. Given Bcl-2’s role in Ca²⁺ signaling, these drugs and tools can impact Ca²⁺ signaling. In addition to this, some Bcl-2 inhibitors may have “off-target” effects that cause Ca²⁺-signaling dysregulation not only in cancer cells but also in healthy cells, resulting in adverse effects. In this review, we aim to provide an up-to-date overview of the involvement of intracellular Ca²⁺ signaling in the working mechanism and “off-target” effects of the different Bcl-2-antagonizing small molecules and peptides.     

Competitive neutrophil elastase inhibitory isoflavones from the roots of Flemingia philippinensis

Flemingia philippinensis has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (1–16), including the most potent and abundant prenyl isoflavones (1–9), from the F. philippinensis plant. These prenyl isoflavones (2, 3, 5, 7, and 9) competitively inhibited NE, with IC₅₀ values of 1.3–12.0 μM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound 3 had an IC₅₀ = 1.3 μM, k₃ = 0.04172 μM⁻¹ min⁻¹, k₄ = 0.0064 min⁻¹, and K_i^app = 0.1534 μM. The K_ik/K_iv ratios (18.5 ∼ 24.6) for compound 3 were consistent with typical competitive inhibitors. The prenyl functionality of isoflavones significantly affected inhibitory potencies and mechanistic behavior by shifting the competitive mode to a noncompetitive one. The remaining flavonoids (10–16) were confirmed as mixed type I inhibitors that preferred to bind free enzyme rather than the enzyme-substrate complex. Fluorescence quenching analyses indicated that the inhibitory potency (IC₅₀) closely followed the binding affinity (K_SV).     

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H₂O₂-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H₂O₂-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H₂O₂-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.     

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)^P(OPh)₂ displaying K_i and k₂/K_i values of 0.4 µM and 28 265 M^?1s^?1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.