Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A_2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A₁, A_2A, and A_2B ARs. Cell proliferation was measured by [³H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A₁), BAY60-6583 (A_2B), and IB-MECA (A₃), and the antagonists PSB-36 (A₁), MSX-2 (A_2A), and PSB-10 (A₃) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A_2A), and the antagonists preladenant (SCH-420814, A_2A), PSB-1115 (A_2B), and PSB-603 (A_2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC₅₀ value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.     

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer''s disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC₅₀ that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer''s disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer''s therapeutics.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

Discovery of 1,2,4-thiadiazolidine-3,5-dione analogs that exhibit unusual and selective rapid cell death kinetics against acute myelogenous leukemia cells in culture

4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was previously identified as an antileukemic agent exhibiting no evident toxicity toward normal hematopoietic cells. An SAR study has been carried out to examine the effect of varying the C-2 and C-4- substituents on the thiadiazolidinone ring of TDZD-8 on antileukemic activity. These studies resulted in the identification of more druglike analogs that exhibited comparable potency to TDZD-8 in killing acute myelogenous leukemia (AML) cells in culture. Surprisingly, the cell death kinetics induced by several of these novel analogs on MV-411 cells were extremely fast, with commitment to death occurring within 30 min. At a concentration of 10 μM, 3f (LD₅₀ = 3.5 μM) completely eradicated cell viability of MV-411 cells within 2 h, while analog 3e (LD₅₀ = 2.0 μM) decimated cell viability within 30 min at a concentration of 10 μM and effectively abolished cell viability at 5 μM within 1-2 h.     

Myrtucommulone from <Emphasis Type="Italic">Myrtus communis</Emphasis> induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9

Myrtucommulone (MC) is a unique, nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis). Here, we addressed the potential of MC to induce apoptosis of cancer cells. MC potently induced cell death of different cancer cell lines (EC₅₀ 3–8 μM) with characteristics of apoptosis, visualized by the activation of caspase-3, -8 and -9, cleavage of poly(ADP-ribose)polymerase (PARP), release of nucleosomes into the cytosol, and DNA fragmentation. MC was much less cytotoxic for non-transformed human peripheral blood mononuclear cells (PBMC) or foreskin fibroblasts (EC₅₀ cell death = 20–50 μM), and MC up to 30 μM hardly caused processing of PARP, caspase-3, -8 and -9 in human PBMC. MC-induced apoptosis was mediated by the intrinsic rather than the extrinsic death pathway. Thus, MC caused loss of the mitochondrial membrane potential in MM6 cells and evoked release of cytochrome c from mitochondria. Interestingly, Jurkat cells deficient in caspase-9 were resistant to MC-induced cell death and no processing of PARP or caspase-8 was evident. In cell lines deficient in either CD95 (Fas, APO-1) signalling, FADD or caspase-8, MC was still able to potently induce cell death and PARP cleavage. Conclusively, MC induces apoptosis in cancer cell lines, with marginal cytotoxicity for non-transformed cells, via the mitochondrial cytochrome c/Apaf-1/caspase-9 pathway. I. Tretiakova and D. Blaesius contributed equally to this work.     

Hyperthermia causes bovine mammary epithelial cell death by a mitochondrial-induced pathway

The effects of mild hyperthermia on bovine mammary epithelial cells exposed to 40 °C for 1 h were studied. The results showed that cell viability, ultrastructural features as well as mitochondrial function were significantly influenced by the mild heat treatment (40 °C). There was a considerate decrease in cell viability accompanied by cell loss resulting from apoptosis and necrosis followed by G₂/M arrest. Cell death followed the typical cascade, namely decrease in the ratio of Bcl-2/Bax and mitochondrial membrane potential (ΔΨ_m), mitochondrial swelling and caspase-3 activities dramatically increased; DNA was also damaged. In conclusion, hyperthermia depresses cell viability and induces bovine mammary cell apoptosis and necrosis through the mitochondrial-triggered cell death pathway.     

Innovative functional cAMP assay for studying G protein-coupled receptors: application to the pharmacological characterization of GPR17

In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC₅₀ or IC₅₀ values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [³⁵S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G_αi.     

Synthesis, characterization and deepening in the comprehension of the biological action mechanisms of a new nickel complex with antiproliferative activity

Thiosemicarbazones are versatile organic compounds that present considerable pharmaceutical interest because of a wide range of properties. In our laboratory we synthesised some new metal-complexes with thiosemicarbazones derived from natural aldehydes which showed peculiar biological activities. In particular, a nickel complex [Ni(S-tcitr)₂] (S-tcitr = S-citronellalthiosemicarbazonate) was observed to induce an antiproliferative effect on U937, a human histiocytic lymphoma cell line, at low concentrations (IC₅₀ = 14.4 μM). Therefore, we decided to study the interactions of this molecule with various cellular components and to characterise the induced apoptotic pathway. Results showed that [Ni(S-tcitr)₂] causes programmed cell death via down-regulation of Bcl-2, alteration of mitochondrial membrane potential and caspase-3 activity, regardless of p53 function. The metal complex is not active on G₀ cells (i.e. fresh leukocytes) but is able to induce perturbation of the cell cycle on stimulated lymphocytes and U937 cells, in which a G₂/M block was detected. It reaches the nucleus where it induces, at low concentrations (2.5-5.0 μM), DNA damage, which could be partially ascribed to oxidative stress. [Ni(S-tcitr)₂] is moreover able to strongly reduce the telomerase activity. Although the biological target of this metal complex is still unknown, the reported data suggest that [Ni(S-tcitr)₂] could be a good model for the synthesis of new metal thiosemicarbazones with specific biological activity.     

F281, synthetic agonist of the sigma-2 receptor,induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.     

Synthesis and biological evaluation of cinnamido linked benzophenone hybrids as tubulin polymerization inhibitors and apoptosis inducing agents

A new class of hybrid molecules containing cinnamide subunit linked to benzophenone as inhibitors of tubulin polymerization were synthesized and evaluated for their anticancer potential. These hybrids exhibit anticancer activity with IC₅₀ values ranging from 0.06 to 16.3 μM. Compounds 4f and 4g possessing fluoro and trifluoromethyl on the cinnamido subunit showed significant cytotoxic activity with IC₅₀ values 0.06 and 0.09 μM against HeLa cell line, respectively. These compounds showed cell cycle arrest at G2/M phase of the cell cycle and inhibited tubulin polymerization followed by activation of caspase-3 activity and apoptotic cell death. Further in vitro tubulin polymerization assay showed that the level of tubulin inhibition was comparable to that of 2a for the compounds 4f and 4g. Moreover, Hoechst 33258 staining and DNA fragmentation assay suggested that these compounds induce cell death by apoptosis. Overall, the current study demonstrates that the synthesis of benzophenone linked cinnamide subunit conjugates as promising anticancer agents with G2/M arrest and apoptotic-inducing ability via targeting tubulin.     

P2X(7) receptor activation causes phosphatidylserine exposure in human erythrocytes

Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X₇ receptor, an ATP-gated cation channel. The two most potent P2X₇ agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP > ATP > 2MeSATP > ATPγS; however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC₅₀ of ∼75 μM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X₇ receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca²⁺. These results show that P2X₇ activation by extracellular ATP can induce PS exposure in erythrocytes.     

Discovery of natural estrogen receptor modulators with structure-based virtual screening

Eleven compounds were identified as estrogen receptor modulators from an in-house natural product database (NPD) by structure-based virtual screening for ERα and ERβ. Among them, 3 compounds were confirmed as ER agonists and 8 compounds were confirmed as ER antagonists by yeast two-hybrid (Y2H) assay, with EC₅₀ values ranging from several micromolar to 100 micromolar. In this study, a novel series of cycloartane triterpenoids isolated from Schisandra glaucescens Diels was found to have ER antagonistic effect, the most potent antagonist of which exhibited activity with EC₅₀ value of 2.55 and 4.68 μM for ERα and ERβ, respectively. Moreover, the types of modulation and subtype selectivity were also investigated through molecular docking simulation.     

Purification and neuroprotective effects of polysaccharides from Opuntia Milpa Alta in cultured cortical neurons

Opuntia is a traditional plant from China with medicinal applications. In this experiment, polysaccharides from Opuntia Milpa Alta (MAPs) were analyzed using gas chromatograph-mass spectrometer (GC-MS) method and result showed that MAPs contained mannose (6.37%), rhamnose (14.94%), xylose (1.99%), arabinose (24.07%), galactose (38.25%), ribose (2.63%) and glucose (11.48%). The neuroprotective effects of MAPs were evaluated at the mechanistic level in vitro models of cerebral ischemic injury. In vitro oxygen/glucose deprivation (OGD) model, MAPs (0.5 μg/ml, 5 μg/ml, 50 μg/ml) effectively increased cell viability by methyl thiazolyl tetrazolium (MTT) assay, inhibited cell cytotoxicity by lactate dehydrogenase (LDH) assay, reduced neuronal cell death, suppressed the production of intracellular reactive oxygen species (ROS), decreased of intracellular free Ca²⁺ concentrations ([Ca²⁺]_i), and reduced extracellular glutamate level. Therefore, MAPs might prevent intracellular calcium overload and decreased glutamate excitotoxicity, both of which can cause neuronal injury and death in vitro models of cerebral ischemic injury.     

Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro

Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5-110 μM) and hydrogen peroxide (5-90 μM). The effect of G8CG alone or in combination with H₂O₂ was determined. G8CG at concentrations >20 μM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5-10 μM) G8CG showed antioxidant properties against H₂O₂-induced DNA damage with no associated toxicity.     

P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells

Background

The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor.

Methods

Immunofluorescence labelling and a fixed-time ATP-induced ethidium⁺ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA.

Results

RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium⁺ uptake into these cells with an EC₅₀ of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca²⁺ and Mg²⁺. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium⁺ uptake.

Conclusions

P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.

General significance

RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.     

Pyridazolate-bridged dicopper (II) SOD mimics with enhanced antiproliferative activities against estrogen and androgen independent cancer cell lines

The superoxide dismutase (SOD) mimicking potential of three pyridazolato-bridged copper complexes has been investigated by NBT assay. The lowest IC₅₀ value observed for the SOD activity (3.90 × 10⁻⁷ M) for [Cu₂{bis(3,6 pyrazol-1-yl)pyridazine}Cl₄OH] · Cl (3) correlates well with its more facile Cu²⁺/Cu⁺ redox couple. These compounds exhibit remarkable antiproliferative activities against estrogen independent breast (BT-20) and androgen independent prostate cancer (PC-3) cell lines, respectively. The concentration of compound 3 that inhibits 50% of cell growth (IC₅₀) after 96 h of treatment using MTT cell proliferation assay was found to be 1.73 μM in BT20 and 1.42 μM in PC3 cell line. Furthermore, compound 3 does have the ability to induce apoptosis in both cell lines after the treatment for 48 h. This effect is probably through generation of intracellular oxidative stress and induction of intrinsic apoptotic pathway.     

4-(3-Aryloxyaryl)quinoline sulfones are potent liver X receptor agonists

A series of 4-(3-aryloxyaryl)quinolines with sulfone substituents on the terminal aryl ring (7) was prepared as LXR agonists. High affinity LXR ligands with excellent agonist potency and efficacy in functional assays of LXR activity were identified. In general, these sulfone agonists were equal to or superior to previously described alcohol and amide analogs in terms of affinity, functional potency, and microsomal stability. Many of the sulfones had LXRβ binding IC₅₀ values <10 nM while the most potent compounds in an ABCA1 mRNA induction assay in J774 mouse cells had EC₅₀ values <10 nM and were as efficacious as T0901317.     

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells

Aims

Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H₂O₂)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated.

Main methods

Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting.

Key findings

Maysin pretreatment reduced the cytotoxic effect of H₂O₂ on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H₂O₂-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5–50 μg/ml) for 2 h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H₂O₂ (200 μM)-insulted cells.

Significance

These results suggest that CS maysin has neuroprotective effects against oxidative stress (H₂O₂)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells.     

Linalool decreases HepG2 viability by inhibiting mitochondrial complexes I and II, increasing reactive oxygen species and decreasing ATP and GSH levels

Coriander is used as an appetizer, a common food seasoning in Mediterranean dishes, and a remedy for many ailments. In this study we tested the biochemical effect of its essential oil components, in particular linalool, its main component. The oil extract was prepared by hydro-distillation of coriander seeds. The various components were identified by gas chromatography coupled to mass spectroscopy. The effect of the various oil components on the viability of different cell lines (HepG2, Caco2, NIH3t3, MCF7 and Hek293) was examined using MTT assay. Linalool was the most potent and HepG2 cells the most sensitive. A 50% and 100% decrease in the viability of HepG2 was obtained at 0.4 μM and 2 μM linalool, respectively. Whereas none of the other components exerted a significant effect at concentrations lower than 50 μM, myrcene and nerolidol, the structural analogues of linalool, were more potent at 100 μM than the other components decreasing HepG2 viability to 26%. The biochemical effect of linalool on mitochondria isolated from HepG2 showed a concentration-dependent inhibition in complexes I and II activities of the respiratory chain, and a time-dependent decrease in ATP level. In addition, a time-dependent decrease in glutathione (GSH) level and in the reduction of nitroblue tetrazolium was obtained, indicating increase in reactive oxygen species (ROS) generation. Pretreatment with the antioxidants: N-acetyl cysteine (2 mM), Trolox (100 μM) and different flavonoids (50 μM) was partially protective against the linalool-induced cell death; the most effective response was that of rutin and apigenin which restored 91% of HepG2 viability. We hereby report a decrease in cell viability of HepG2 cells by linalool and identify the mitochondria as one possible target for its site of action, inhibiting complexes I and II and decreasing ATP. In addition linalool increased ROS generation and decreased GSH level.     

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC₅₀ = 0.74–4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC₅₀ = 29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.