Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma.

HDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%-35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies.

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting the UPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

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.

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.

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.

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.

Diffuse large B-cell lymphoma with low 18F-fluorodeoxyglucose avidity features silent B-cell receptor signaling.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive lymphomas exhibiting increased glucose uptake. However, some DLBCLs featuring relatively low 18F-fluorodeoxyglucose (18F-FDG) uptake denoted by the maximum standardized uptake value (SUVmax) on PET/CT have been identified. The biologic correlates of such a heterogeneity have remained largely unknown. Herein, we immunohistochemically detected and found low FDG-avid DLBCL cases featuring lower expression of some key molecules involved in B-cell receptor (BCR) signaling (pSYK) and glucose metabolism (GLUT1 and HK2). Besides, BCR-deficient DLBCL xenografts were found displaying lower SUVmax and expressions of pSYK, GLUT1, and HK2. Further immunoblotting demonstrated expressions of GLUT1 and HK2 in BCR-dependent DLBCLs could be down-regulated by a chemical SYK inhibition, whereas the inhibitory effects were not observed in BCR-deficient tumors. These findings suggest low FDG-avid DLBCLs display a silent BCR signaling and PET/CT might be utilized to tailor the BCR signaling-inhibitory treatment.

Actinomycetes for marine drug discovery isolated from mangrove soils and plants in China.

The mangrove ecosystem is a largely unexplored source for actinomycetes with the potential to produce biologically active secondary metabolites. Consequently, we set out to isolate, characterize and screen actinomycetes from soil and plant material collected from eight mangrove sites in China. Over 2,000 actinomycetes were isolated and of these approximately 20%, 5%, and 10% inhibited the growth of Human Colon Tumor 116 cells, Candida albicans and Staphylococcus aureus, respectively, while 3% inhibited protein tyrosine phosphatase 1B (PTP1B), a protein related to diabetes. In addition, nine isolates inhibited aurora kinase A, an anti-cancer related protein, and three inhibited caspase 3, a protein related to neurodegenerative diseases. Representative bioactive isolates were characterized using genotypic and phenotypic procedures and classified to thirteen genera, notably to the genera Micromonospora and Streptomyces. Actinomycetes showing cytotoxic activity were assigned to seven genera whereas only Micromonospora and Streptomyces strains showed anti-PTP1B activity. We conclude that actinomycetes isolated from mangrove habitats are a potentially rich source for the discovery of anti-infection and anti-tumor compounds, and of agents for treating neurodegenerative diseases and diabetes.

Covalent docking modelling-based discovery of tripeptidyl epoxyketone proteasome inhibitors composed of aliphatic-heterocycles.

The potential of specific proteasome inhibitors to act as anti-cancer agents has attracted intensive investigations. The proteasome can be covalently inhibited by epoxyketone derivatives via a two-step reaction. Several computational approaches have been developed to mimic the covalent binding event. Compound 1 composed of a six-membered heterocyclic ring was designed by using covalent docking. With a possible different binding mode from the clinical compound Carfilzomib, it occupied the S5 pocket of 20S proteasome and showed favorable inhibitory activity. Subsequently optimization and evaluation were taken place. Among these compounds, 11h demonstrated extraordinary in vitro inhibitory activity and selectivity, and good in vivo proteasome inhibitory activity, a favorable pharmacokinetic profile and xenograft tumor inhibition. The possible binding pattern of compound 11h against proteasome was further fully explored via calculations, providing a theoretical basis for finding potent proteasome inhibitors.

Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK.

Berberine is a plant alkaloid used in traditional Chinese medicine and has been reported to have antihyperglycemic activity in NIDDM patients. However, the molecular basis for this action is yet to be elucidated. Here we investigate the effects and signaling pathways of berberine on L6 rat skeletal muscles. Our study demonstrates that berberine stimulates glucose uptake in a time- and dose-dependent manner. Intriguingly, berberine-stimulated glucose uptake does not vary as insulin concentration increases, and could not be blocked by the PI 3-kinase inhibitor wortmannin. Berberine only weakly stimulates the phosphorylation of Akt/PKB, a key molecule in the insulin signaling pathway, but strongly promotes the phosphorylation of AMPK and p38 MAPK. The effects of berberine are not a result of pro-oxidant action, but a consequence of an increased cellular AMP:ATP ratio. Moreover, berberine-stimulated glucose uptake is inhibited by the AMPK inhibitor Compound C and the p38 MAPK inhibitor SB202190. Inhibition of AMPK reduces p38 MAPK phosphorylation, suggesting that AMPK lies upstream of p38 MAPK. These results suggest that berberine circumvents insulin signaling pathways and stimulates glucose uptake through the AMP-AMPK-p38 MAPK pathway, which may account for the antihyperglycemic effects of this drug.

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.

Fluevirosines A-C: a biogenesis inspired example in the discovery of new bioactive scaffolds from Flueggea virosa.

Biogenesis inspired chemical investigation of a Chinese folk medicine, Flueggea virosa, returned three unprecedented C,C-linked trimeric Securinega alkaloids, fluevirosines A-C (1-3). Their absolute structures were characterized on the basis of spectroscopic data and computational analysis. Compounds 2 and 3 showed inhibition against the splicing of XBP1 mRNA.

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.