An-Hui Gao

Synthesis and Biological Evaluation of C7-Demethyl Largazole Analogues

by Luesch and co-workers. It consists of a strained 16-membered macrocycle that incorporates a 4-methylthiazoline linearly fused to a thiazole and an ester of a 3-hydroxy-7-mercaptohept-4-enoic acid unit. Part of the latter moiety constitutes the side chain, which was shown to be essential for the potent histone deacetylase (HDAC) inhibitory, and consequently antiproliferative activity of this compound. Largazole also exhibits high HDAC1 selectivity. Recently, inhibition of HDAC activity was clinically validated as a novel therapeutic strategy for cancer treatment. To date, several structurally diverse small-molecule HDAC inhibitors (HDACi) have been reported, which include aryl hydroxamates, benzamides, short-chain fatty acids, electrophilic ketones, and macrocyclic peptides. All of these HDACi share a threemotif pharmacophoric structure that comprises a zinc-binding domain, a linker domain, and a surface recognition domain. Among these HDACi, the macrocyclic peptides possess excellent HDAC inhibition activity and selectivity because their recognition domain moieties are the most complex. One promising macrocyclic peptide compound (FK228) is part of a phase II clinical study. Since its discovery, the 16-membered macrocyclic depsipeptide largazole (1) has received increasing attention because of its high structural similarity to FK228 and its similarly high HDAC inhibitory activity and selectivity. Several total syntheses and structure–activity relationship (SAR) studies of largazole (1) have been reported recently. 8–12] In the present study, we used the LibDock program and the crystal structure of a histone deacetylase-like protein (HDLP) to guide our structural optimization approach to largazole (1). To simplify the 16-membered macrocycle, we eliminated the C7-methyl group to give the 2,4’-bithiazole analogue 2 (Figure 1). In addition to analogue 2, the C17-epimer analogue 3 was also prepared. Using the HDLP, we performed a molecular docking analysis on the thiol groups of compounds 1, 2, and 3, as it was reported that the thiol group of 1 is the reactive species. The results of our docking experiment suggest that the macrocycle made contact at the pocket entrance and at an adjacent surface groove, thus capping the pocket (Figure 2). Additionally, the aliphatic chain inserted into the HDLP pocket and made contact with the tubelike hydrophobic portion of the pocket, while the thiol group reached the polar region at the bottom of the pocket, where it coordinated with the zinc ion (Figure 3). The docking results revealed that the orientation of the macrocycle is similar in largazole and in the C7-demethyl analogues 2 and 3 ; thus, we hypothesized that the C7-methyl group is not necessary for the HDAC inhibitory and antiproliferative activities of largazole. Figure 1. C7-demethyl largazole analogues.

Novel CHOP activator LGH00168 induces necroptosis in A549 human lung cancer cells via ROS-mediated ER stress and NF-κB inhibition

Aim:C/EBP homologous protein (CHOP) is a transcription factor that is activated at multiple levels during ER stress and plays an important role in ER stress-induced apoptosis. In this study we identified a novel CHOP activator, and further investigated its potential to be a therapeutic agent for human lung cancer.Methods:HEK293-CHOP-luc reporter cells were used in high-throughput screening (HTS) to identify CHOP activators. The cytotoxicity against cancer cells in vitro was measured with MTT assay. The anticancer effects were further examined in A549 human non-small cell lung cancer xenograft mice. The mechanisms underlying CHOP activation were analyzed using luciferase assays, and the anticancer mechanisms were elucidated in A549 cells.Results:From chemical libraries of 50 000 compounds, LGH00168 was identified as a CHOP activator, which showed cytotoxic activities against a panel of 9 cancer cell lines with an average IC50 value of 3.26 μmol/L. Moreover, administration of LGH00168 significantly suppressed tumor growth in A549 xenograft bearing mice. LGH00168 activated CHOP promoter via AARE1 and AP1 elements, increased DR5 expression, decreased Bcl-2 expression, and inhibited the NF-κB pathway. Treatment of A549 cells with LGH00168 (10 μmol/L) did not induce apoptosis, but lead to RIP1-dependent necroptosis, accompanied by cell swelling, plasma membrane rupture, lysosomal membrane permeabilization, MMP collapse and caspase 8 inhibition. Furthermore, LGH00168 (10 and 20 μmol/L) dose-dependently induced mito-ROS production in A549 cells, which was reversed by the ROS scavenger N-acetyl-L-cysteine (NAC, 10 mmol/L). Moreover, NAC significantly diminished LGH00168-induced CHOP activation, NF-κB inhibition and necroptosis in A549 cells.Conclusion:LGH00168 is a CHOP activator that inhibits A549 cell growth in vitro and lung tumor growth in vivo.

Berberine combined with 2-deoxy-d-glucose synergistically enhances cancer cell proliferation inhibition via energy depletion and unfolded protein response disruption

BACKGROUND Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy-d-glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth. METHODS The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression. RESULTS BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds. CONCLUSIONS Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR. GENERAL SIGNIFICANCE Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process.

High-throughput screening using pseudotyped lentiviral particles: A strategy for the identification of HIV-1 inhibitors in a cell-based assay

Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

Curcusone D, a novel ubiquitin-proteasome pathway inhibitor via ROS-induced DUB inhibition, is synergistic with bortezomib against multiple myeloma cell growth.

BACKGROUND Ubiquitin-proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM). METHODS Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H2DCFDA. RESULTS Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells. CONCLUSIONS Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib. GENERAL SIGNIFICANCE The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.

Discovery and optimization of 2,4-diaminoquinazoline derivatives as a new class of potent dengue virus inhibitors.

The results of a high-throughput screening assay using the DENV-2 replicon showed that the 2,4-diaminoquinazoline derivative 4a has a high dengue virus inhibitory activity (EC(50) = 0.15 μM). A series of 2,4-diaminoquinazoline derivatives based on 4a as a lead compound were synthesized and subjected to structure-antidengue activity relationship studies. Among the series of 2,4-diaminoquinazoline derivative probed, 4o was observed to display both the highest antiviral potency (EC(50) = 2.8 nM, SI > 1000) and an excellent pharmacokinetic profile.

Design, synthesis and biological evaluation of bisthiazole-based trifluoromethyl ketone derivatives as potent HDAC inhibitors with improved cellular efficacy.

Histone deacetylases (HDACs) are a class of epigenetic modulators with complex functions in histone post-translational modifications and are well known targets for antineoplastic drugs. We have previously developed a series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors. In the present work, a new series of bisthiazole-based compounds with different zinc binding groups (ZBGs) have been designed and synthesized. Among them is compound 7, containing a trifluoromethyl ketone as the ZBG, which displays potent inhibitory activity towards human HDACs and improved antiproliferative activity in several cancer cell lines.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

BACKGROUND Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. METHODS We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. RESULTS Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. CONCLUSIONS AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. GENERAL SIGNIFICANCE These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.

Disruption of the unfolded protein response (UPR) by lead compound selectively suppresses cancer cell growth

Identifying chemotherapy candidates with high selectivity against cancer cells is a major challenge in cancer treatment. Tumor microenvironments cause chronic endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) as an adaptive response. Here, one novel small-molecule compound, 17#, was discovered as a potent pan-UPR inhibitor. It exhibited good selection for growth inhibition when cancer cells were cultured in 2-deoxy-D-glucose (2DG), mimicking an in vitro glucose-deprived status. Additionally, 17# alone could mildly suppress the growth of HeLa tumor xenografts, and a synergistic anti-cancer effect was observed when 17# was combined with 2DG. A mechanistic study showed that 17#-induced selective anti-cancer effects were highly dependent on UPR inhibition, and overexpressing GRP78 or XBP1s reversed the 17#-induced growth inhibition and cell cycle arrest, partially by delaying the downregulation of the cell cycle regulator cyclin B1. Furthermore, 17# improved the sensitivity of anti-cancer drugs such as doxorubicin or etoposide. Our study presents evidence that disrupting the UPR has selective therapeutic potential and may enhance drug sensitivity.

3-(7-Azaindolyl)-4-indolylmaleimides as a novel class of mutant isocitrate dehydrogenase-1 inhibitors: Design, synthesis, and biological evaluation

A series of 3‐(7‐azainodyl)‐4‐indolylmaleimides was designed, synthesized, and evaluated for their isocitrate dehydrogenase 1 (IDH1)/R132H inhibitory activities. Many compounds such as 11a, 11c, 11e, 11g, and 11s exhibited favorable inhibitory effects on IDH1/R132H and were highly selective against the wild‐type IDH1. Evaluation of the biological activities at the cellular level showed that compounds 11a, 11c, 11e, 11g, and 11s could effectively suppress the production of 2‐hydroxyglutaric acid in U87MG cells expressing IDH1/R132H. Preliminary structure–activity relationship (SAR) and molecular modeling studies were discussed based on the experimental data obtained. These findings may provide new insights into the development of novel IDH1/R132H inhibitors.