Oleanane-type saponins and prosapogenins from Albizia julibrissin and their cytotoxic activities.

Two undescribed oleanane-type saponins, julibrosides K-L, along with three undescribed oleanane-type prosapogenins, julibrosides M-O, were isolated from the stem bark of Albizia julibrissin Durazz. and the mild alkaline hydrolysate of the total saponin, respectively. Their structures were established by extensive analysis of 1D and 2D NMR experiments (COSY, TOCSY, HSQC, HMBC, and HSQC-TOCSY) and mass spectrometry. Furthermore, the cytotoxic activities of the isolated compounds against BGC-823, A549, HCT-116, and HepG2 cell lines were evaluated, and julibroside L showed significant cytotoxic activities against the four cancer cell lines with IC50 values of 5.77, 4.80, 4.26, and 4.93 µM, respectively.

Small-molecule compounds targeting the STAT3 DNA-binding domain suppress survival of cisplatin-resistant human ovarian cancer cells by inducing apoptosis.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in oncogenic occurrence and transformation by regulating the expression of diverse downstream target genes important for tumor growth, metastasis, angiogenesis and immune evasion. Feasibility of targeting the DNA-binding domain (DBD) of STAT3 has been proven previously. With the aid of 3D shape- and electrostatic-based drug design, we identified a new STAT3 inhibitor, LC28, and its five analogs, based on the pharmacophore of a known STAT3 DBD inhibitor. Microscale thermophoresis assay shows that these compounds inhibits STAT3 binding to DNA with a Ki value of 0.74-8.87 µM. Furthermore, LC28 and its analogs suppress survival of cisplatin-resistant ovarian cancer cells by inhibiting STAT3 signaling and inducing apoptosis. Therefore, these compounds may serve as candidate compounds for further modification and development as anticancer therapeutics targeting the DBD of human STAT3 for treatment of cisplatin-resistant ovarian cancer.

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain.

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds 10b, 13b, and 19. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340.

Synthesis and biological evaluation of curcumin derivatives modified with α-amino boronic acid as proteasome inhibitors.

Curcumin is a well-known pharmacophore and some of its derivatives are shown to target 20S proteasome recently. In this report, we designed and synthesized two series of curcumin derivatives modified with different α-amino boronic acids as potent proteasome inhibitors. The synthesized compounds were evaluated for their cytotoxic activities against HCT116 cells, and the results showed that all of them exhibited excellent cell growth inhibitory activity comparing with curcumin, with the IC50 values varying from 0.17 µM to 1.63 µM. Compound II-2F with free boronic acid was assayed for its proteasome inhibitory activity and the results indicated that II-2F exhibited more potent inhibitory activity against ChT-L with high subunit selectivity than any other reported curcumin derivatives.

Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells.

Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

Cytotoxic oleanane triterpenoid saponins from Albizia julibrissin.

Bioassay-guided fractionation of the ethanolic extract of the stem bark of Albizia julibrissin led to the isolation of ten new oleanane-type triterpenoid saponins, julibrosides J37-J46 (1-10), along with six known analogues (11-16). In addition, 11 prosapogenins (17-27) were prepared by mild or strong alkaline hydrolysis of the total saponin. The structures of 1-27 were determined by spectroscopic and chemical means, and their cytotoxicities against four human cancer cell lines, BGC-823, A549, HCT-116, and HepG2 were evaluated. Compounds 5-16 exhibited significant inhibitory activity with IC50 values ranging from 2.59 to 9.30µM, and 8 turned out to be the most active compound with all IC50 values <5µM. A preliminary structure-activity relationship of these saponins clearly indicated that the outer monoterpenoid moiety (MT') is a crucial substituent for cytotoxicity, and the linkage sites of the MT' unit greatly influenced the activity. It could also be inferred that the existence of 16-OH of the aglycone almost has no effect on cytotoxicity and the N-acetyl-glucosamine moiety at C-3 seems to enhance activity.

Synthesis and biological activity of peptide proline-boronic acids as proteasome inhibitors.

On the basis of the application of proline-boronic acid as pharmacophore in the kinase inhibitors and our previous research results, using proline-boronic acid as warhead, two series of peptide proline-boronic acids, dipeptide proline-boronic acids (I) and tripeptide proline-boronic acids (II), were designed and synthesized. All the synthesized compounds were first evaluated for their biological activity against MGC803 cell, and then, the best compound II-7 was selected to test its anti-tumor spectrum on six human tumor cell lines and proteasome inhibition against three subunits. The results indicated that series II have much better biological activities than series I. The compound II-7 exhibited not only excellent biological activities with IC50 values of nM level in both cell and proteasome models, but also much better subunit selectivity. Thus, proline-boronic acid as warhead is reasonable in the design of proteasome inhibitors.

Urea-containing peptide boronic acids as potent proteasome inhibitors.

A novel class of urea-containing peptide boronic acids as proteasome inhibitors was designed by introducing a urea scaffold to replace an amido bond. Compounds were synthesized and their antitumor activities were evaluated. After two rounds of optimizations, the compound I-14 was found to be a potent proteasome inhibitor. Compared with Bortezomib, I-14 showed higher potency against the chymotrypsin-like activity of human 20S proteasome (IC50 < 1 pM), similar potency against four different cancer cell lines (IC50 < 10 nM), and better pharmacokinetic profile. Furthermore, I-14 significantly inhibited tumor growth in Bel7404 mouse xenograft model. The excellent proteasome inhibition by I-14 was rationalized through docking and molecular dynamics studies.

Diasteltoxins A-C, Asteltoxin-Based Dimers from a Mutant of the Sponge-Associated Emericella variecolor Fungus.

Three novel asteltoxin-bearing dimers namely diasteltoxins A-C (1-3) along with asteltoxin were isolated from a mutated strain of a sponge-derived fungus Emericella variecolor XSA-07-2. Their structures were determined by extensive spectroscopic analyses including the computed electronic circular dichroism (ECD) data for the configurational assignment. The biogenetic formation of the dimers through [2 + 2] cycloaddition of asteltoxin was postulated. Diasteltoxins 1-3 exerted inhibitory effects against the tumor cell lines H1299 and MCF7 and exhibited significant inhibition against thioredoxin reductase (TrxR).

Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells.

Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome.

Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer agents: Synthesis, structure-activity relationship analysis and biological evaluation.

A series of new analogs based on the structure of lead compound 10 were designed, synthesized and evaluated for their in vitro anti-cancer activities against four selected human cancer cell lines (HL-60, Bel-7402, SK-BR-3 and MDA-MB-468). Several synthesized compounds exhibited improved anti-cancer activities comparing with lead compound 10. Among them, 1,3,4-oxadiazole analogs 17o showed highest bioactivity with IC50 values of 1.23, 0.58 and 4.29 µM against Bel-7402, SK-BR-3 and MDA-MB-468 cells, respectively. It is noteworthy that 17o has potent anti-proliferation activity toward a panel of cancer cells with relatively less cytotoxicity to nonmalignant cells. The further mechanistic study showed that it induced apoptosis and cell cycle arrest through disrupting spindle assembly in mitotic progression, indicating these synthesized dithiocarbamates represented a novel series of anti-cancer compounds targeting mitosis.

Anticancer Effect of a Novel Proteasome Inhibitor, YSY01A, via G2/M Arrest in PC-3M Cells in vitro and in vivo.

YSY01A is a new tripeptideboronic acid and an analog of PS341. However, YSY01A's antitumor effects and mechanism have not yet been elucidated. This study demonstrates that YSY01A inhibited proteasome activity by combining with the chymotrypsin-like (CT-L) site (ß5i/ß5), the post-glutamyl peptide hydrolase (PGPH) site (ß1i/ß1) and the trypsin-like (T-L) site (ß2i/ß2) in special fluorgonic substrates and proteasome probe tests. We explored the anticancer effect using methyl thiazolyltetrazolium (MTT) or sulforhodamine B (SRB), and PC-3M cells were sensitive to YSY01A among the four cancer cell types tested. The YSY01A antiproliferative effect was stronger than that of PS341. In vivo, YSY01A (1.25, 2.25, and 3.25 mg/kg) inhibited PC-3M cell xenograft tumor growth, and the tumor volume inhibition rate was approximately 40% to 60%. YSY01A arrested PC-3M cells in the G2/M phase of the cell cycle by flow cytometry (FCM). Many proteins related to the cell cycle were analyzed using western blot, and YSY01A was shown to increase p21, p27, cyclinB1, P-cdc2 (tyr15) and wee1 protein expression in both cells and tumor tissue in a concentration-dependent manner. YSY01A, a proteasome inhibitor, exerts anticancer effects on PC-3M cells in vitro and in vivo. The mechanism of the YSY01A-mediated antitumor effect is that the cell cycle is arrested at the G2/M stage. This study suggests that YSY01A may be a novel therapeutic agent for prostate cancer.

YSY01A, a Novel Proteasome Inhibitor, Induces Cell Cycle Arrest on G2 Phase in MCF-7 Cells via ERα and PI3K/Akt Pathways.

Given that the proteasome is essential for multiple cellular processes by degrading diverse regulatory proteins, inhibition of the proteasome has emerged as an attractive target for anti-cancer therapy. YSY01A is a novel small molecule compound targeting the proteasome. The compound was found to suppress viability of MCF-7 cells and cause limited cell membrane damage as determined by sulforhodamine B assay (SRB) and CytoTox 96(®) non-radioactive cytotoxicity assay. High-content screening (HCS) further shows that YSY01A treatment induces cell cycle arrest on G2 phase within 24 hrs. Label-free quantitative proteomics (LFQP), which allows extensive comparison of cellular responses following YSY01A treatment, suggests that various regulatory proteins including cell cycle associated proteins and PI3K/Akt pathway may be affected. Furthermore, YSY01A increases p-CDC-2, p-FOXO3a, p53, p21(Cip1) and p27(Kip1) but decreases p-Akt, p-ERα as confirmed by Western blotting. Therefore, YSY01A represents a potential therapeutic for breast cancer MCF-7 by inducing G2 phase arrest via ERα and PI3K/Akt pathways.

Evaluation of the effect of TM208 on the activity of five cytochrome P450 enzymes using on-line solid-phase extraction HPLC-DAD: a cocktail approach.

A rapid, simple, and sensitive on-line solid-phase extraction HPLC-DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05-50.0 µg/ml for dapsone and omeprazole, 0.1-50.0 µg/ml for caffeine and 0.2-50.0 µg/ml for metoprolol and chlorzoxazone. The linearity (R(2)) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8-109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.

NO inhibitory guaianolide-derived terpenoids from Artemisia argyi.

An unusual dimeric guaianolide, artemilinin A (1) and a sesquiterpene-monoterpene lactone, isoartemisolide (2), were isolated from the leaves of Artemisia argyi. Their structures were elucidated on the basis of extensive spectroscopic analysis (IR, HR-ESIMS, 1D- and 2D-NMR), and the absolute configurations were determined by CD spectra and quantum chemical ECD calculation. Furthermore, in in vitro assay, compound 2 exhibited pronounced inhibition on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells with an IC50 value of 4.00µM.

A specific peptide ligand-modified lipid nanoparticle carrier for the inhibition of tumor metastasis growth.

Tumor metastasis accounts for 90% of cancer-associated deaths and is almost inaccessible by chemotherapy, surgical operation or radiotherapy. Here, a tumor metastasis-specific nanocarrier system has been constructed by modification of stealth lipid nanoparticles with a specific peptide ligand. Highly metastatic breast cancer MDA-MB-231 that stably expressed luciferase (MDA-MB-231/Luc) was used as tumor cell model. The nanocarrier was very specific for highly metastatic cancer cells in vitro and could specifically target to cancer metastases foci following systemic administration in vivo by both fluorescence imaging and bioluminescence imaging compared to a passive-targeted system. It greatly facilitated the efficacy of doxorubicin loaded in inhibiting tumor metastasis growth and prolonging the survival time of mice. Importantly, this system was also found to prevent the initiation and progression of tumor metastasis. The tumor metastasis-targeted nanocarriers hold great potential in the treatment of cancer metastasis foci and even for the prevention of tumor metastasis. This study may also provide new strategy in the development of nanomedicine for diagnosis and therapy of tumor metastasis.

Structural modifications of a 3-methoxy-2-aminopyridine compound to reduce potential for mutagenicity and time-dependent drug-drug interaction.

(S)-1-((4-(3-(6-Amino-5-methoxypyridin-3-yl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)propan-2-ol, 1, was recently identified as a potent inhibitor of the oncogenic kinase bRAF. Compounds containing 3-methoxy-2-aminopyridine, as in 1, comprised a promising lead series because of their high ligand efficiency and excellent ADME profile. However, following metabolic oxidation, compounds in this series also demonstrated two significant safety risks: mutagenic potential and time-dependent drug-drug interaction (TDI). Metabolite identification studies revealed formation of a reactive metabolite. We hypothesized that minimizing or blocking the formation of such a metabolite would mitigate the safety liabilities. Our investigation demonstrated that structural modifications which either reduced the electron density of the 3-methoxy-2-aminopyridine ring or blocked the reactive site following metabolic oxidation were successful in reducing TDI and AMES mutagenicity.

The use of a tumor metastasis targeting peptide to deliver doxorubicin-containing liposomes to highly metastatic cancer.

Tumor metastasis is responsible for 90% of cancer-associated deaths and highly metastatic cancers are more prone to form metastasis foci and acquire the drug resistance. Here, a nanocarrier system (TMT-LS) has been constructed by modification of stealth liposomes with a metastatic cancer specific peptide, using the unmodified stealth liposomes (LS) as the control. The active targeted nanocarriers presented satisfactory particle size (about 100 nm) and drug release characteristics in vitro. Highly metastatic cancer cells (MDA-MB-435S and MDA-MB-231) and non-metastatic cancer cells (MCF-7) were applied as tumor cell models. The highly metastatic cancer cells were found to endocytose more TMT-LS in a faster way than TS, through a receptor-mediated pathway proved by specific receptor inhibition. Co-localization technique indicated the integrity of nanocarriers in cytoplasm. The significant targeting of TMT-LS to highly metastatic tumors was demonstrated in vivo and ex vivo in an orthotopic model as well as in a double tumor-bearing animal model with both metastatic and non-metastatic tumors in the same mouse. Importantly, the active targeted drug delivery system was found to penetrate deeper into tumor mass and have a longer retention within the malignant tissue. Further, TMT-LS greatly facilitated the efficacy of doxorubicin loaded in terms of inhibiting xenograft tumor growth and inducing cancer cell apoptosis, with only minor side effects. Together, the specific nanocarriers hold great potential in the development of nanomedicine for diagnosis and therapy of metastatic tumor.

Furanodiene, a natural product, inhibits breast cancer growth both in vitro and in vivo.

PURPOSE: Previous studies have reported that the Curcuma wenyujin Y.H. Chen et C. Ling extract, which has a high furanodiene content, showed anti-cancer effects in breast cancer cells in vitro. The present study was designed to evaluate the in vitro and in vivo anti-cancer activity of furanodiene. METHODS: The in vitro effects of furanodiene were examined on two human breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Assays of proliferation, LDH release, mitochondrial membrane potential (ΔΨm), cell cycle distribution, apoptosis and relevant signaling pathways were performed. The in vivo effect was determined with MCF7 tumor xenograft model in nude mice. RESULTS: Furanodiene significantly inhibited the proliferation and increased the LDH release in both cell lines in a dose-dependent manner. ΔΨm depolarization, chromatin condensation, and DNA fragmentation were also observed after furanodiene treatment. Furanodiene dose-dependently induced cell cycle arrest at the G0/G1 phase. The protein expressions of p-cyclin D1, total cyclin D1, p-CDK2, total CDK2, p-Rb, total Rb, Bcl-xL, and Akt were significantly inhibited by furanodiene, whereas the protein expressions of Bad and Bax, and the proteolytic cleavage of caspase-9, caspase-7, and poly-ADP-ribose polymerase (PARP) were dramatically increased. Furthermore, the z-VAD-fmk markedly reversed the furanodiene-induced cell cytotoxicity, the proteolytic cleavage of caspase-9, and DNA fragmentation but did not affect the proteolytic cleavage of PARP, whereas the Akt inhibitor VIII increased the furanodiene-induced cytotoxicity and PARP cleavage. In addition, furanodiene dose-dependently suppressed the tumor growth in vivo, achieving 32% and 54% inhibition rates after intraperitoneal injection of 15 mg/kg and 30 mg/kg, respectively. CONCLUSIONS: Taken together, we concluded that furanodiene suppresses breast cancer cell growth both in vitro and in vivo and could be a new lead compound for breast cancer chemotherapy.

[Design, synthesis and biological assay of novel tripeptidic tetrazoles as inhibitors of 20S proteasome].

Ubiquitin-proteasome pathway (UPP) is one of the ways utilized for selective degradation of many proteins in cells, and the 20S proteasome takes the functional machinery where hydrolysis of targeted proteins takes place. Based on existing peptide inhibitors, a series of novel tripeptidic tetrazoles have been designed, synthesized, and the structures have been confirmed with 1H NMR, MS and elemental analysis. Among them, three compounds (6b, 6d and 6h) showed inhibitory activities of ChT-L of 20S proteasome.