Aiming Sun

EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism

In this study, we show that the novel synthetic curcumin analog, EF24, induces cell cycle arrest and apoptosis by means of a redox-dependent mechanism in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Cell cycle analysis demonstrated that EF24 causes a G2/M arrest in both cell lines, and that this cell cycle arrest is followed by the induction of apoptosis as evidenced by caspase-3 activation, phosphatidylserine externalization and an increased number of cells with a sub-G1 DNA fraction. In addition, we demonstrate that EF24 induces a depolarization of the mitochondrial membrane potential, suggesting that the compound may also induce apoptosis by altering mitochondrial function. EF24, like curcumin, serves as a Michael acceptor reacting with glutathione (GSH) and thioredoxin 1. Reaction of EF24 with these agents in vivo significantly reduced intracellular GSH as well as oxidized GSH in both the wild-type and Bcl-xL overexpressing HT29 human colon cancer cells. We therefore propose that the anticancer effect of a novel curcumin analog, EF24, is mediated in part by redox-mediated induction of apoptosis.

Inhibition of IκB Kinase-Nuclear Factor-κB Signaling Pathway by 3,5-Bis(2-flurobenzylidene)piperidin-4-one (EF24), a Novel Monoketone Analog of Curcumin

The nuclear factor-κB (NF-κB) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-κB pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-κB signaling pathway through direct action on IκB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-κB, with an IC50 value of 1.3 μM compared with curcumin, with an IC50 value of 13 μM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-α-induced IκB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF-α-induced NF-κB signaling by EF24 extends the therapeutic application of EF24 to other NF-κB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.

Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases.

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.

Sphingoid bases and de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action

The sphingoid base backbones of sphingolipids (sphingosines, sphinganines, 4-hydroxysphinganines and others) are highly bioactive species directly and-in most cases-as their metabolites, the N-acyl-sphingoid bases (ceramides) and sphingoid base 1-phosphates. The complexity of these compounds affords many opportunities to prepare synthetic analogs for studies of sphingolipid metabolism and the functions of the sphingoid bases and metabolites. Described in this review are methods for the preparation of libraries of sphingoid bases, including a series of 1-deoxy-analogs, as well as information about their metabolism and biological activities. Findings with these compounds have uncovered some of the complications of working with compounds that mimic a naturally occurring biomodulator-such as that they are sometimes metabolized by enzymes that handle the endogenous compounds and the products may have potent (and unexpected) biological activities. Through studying such compounds, there is now a greater understanding of the metabolism and mechanism(s) of action of naturally occurring sphingoid bases as well as of these analogs.

Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties

Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogs with enhanced activity on NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analog whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1-100 μM) or vehicle (DMSO 1%) for 1h. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1 μg/mL). EF31 (IC(50)~5 μM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC(50)~35 μM) and curcumin (IC(50) >50 μM). In addition, EF31 exhibited greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB, EF31 (IC(50)~1.92 μM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC(50)~131 μM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analog for further therapeutic development.

Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor

Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE2) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE2 by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE2 on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE2 on EP2 receptors 4- to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE2 receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE2 released in a cell-injury setting is neuroprotective.

Ebselen and Congeners Inhibit NADPH Oxidase 2-Dependent Superoxide Generation by Interrupting the Binding of Regulatory Subunits

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.

Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

Nonnucleoside Inhibitor of Measles Virus RNA-Dependent RNA Polymerase Complex Activity

ABSTRACT Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex.

Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity.