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.

[The synthesis of purine derivatives and its inhibitory activity on CD38 NADase].

CD38 is a multifunctional enzyme expressed in a variety of mammalian tissues, its catalytic activity was involved in a wide range of physiological processes. Based on the reported inhibitor of human CD38 NADase, 33 purine derivatives were designed and synthesized. The biological activity assay showed that compounds 20 and 38 exhibited almost the same extent of inhibitory activities on human CD38 NADase as the lead compound H2. The results also revealed that small substituents at C-6 of purine ring gave no obvious effect on inhibitory activity, but phenylpropionyl moiety at N-2 could affect the binding mode of the compound with CD38. This study provides a reliable basis for future rational design of inhibitors for CD38.

Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B.

AIM: Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca(2+)-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. METHODS: Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. RESULTS: Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 µmol/L. Br-IQ17B is selective over other subtypes such as α4ß2 and α3ß4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [(3)H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. CONCLUSION: We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.

CD38 Is Required for Neural Differentiation of Mouse Embryonic Stem Cells by Modulating Reactive Oxygen Species.

CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca(2+) mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production.

Treatment responses of procaterol and CD38 inhibitors in an ozone-induced airway hyperresponsiveness mice model.

Airway hyperresponsiveness (AHR) and airway inflammation are key pathophysiological features of many respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). To evaluate the treatment responses of procaterol and CD38 inhibitors in an ozone-induced AHR mice model, we hypothesized that procaterol and two synthetic CD38 inhibitors (Compounds T and H) might have therapeutic effects on the ozone-induced AHR mice model, and the nuclear factor-kappaB (NF-κB) pathway and the CD38 enzymatic activity might be involved in the mechanisms. With the exception of the Control group, ozone exposure was used to establish an AHR model. Male Kunming mice in the Procaterol and CD38 inhibitors groups were treated with an emulsifier of procaterol hydrochloride, Compound T or H. Results indicated that (1) no drug showed severe toxicity in this study; (2) ozone exposure induced airway inflammation and AHR; (3) intragastric treatment with procaterol and Compound T achieved potent therapeutic effects, but Compound H did not show any therapeutic effect; (4) the NF-κB pathway was involved in both the pathogenic mechanisms of ozone and therapeutic mechanisms of procaterol and Compound T; (5) however, the in vivo effect of Compound T was not caused by its inhibitory activity on CD38. Taken together, procaterol and Compound T are potentially good drugs to treat asthma and COPD complicated with ozone exposure.

Self-assembly cationic nanoparticles based on cholesterol-grafted bioreducible poly(amidoamine) for siRNA delivery.

In this study, a series of bioreducible poly(amidoamine)s grafting different percentages of cholesterol (rPAA-Ch14: 14%, rPAA-Ch29: 29%, rPAA-Ch57: 57% and rPAA-Ch87: 87%) was synthesized and used for siRNA delivery. These amphiphilic polymers were able to self-assemble into cationic nanoparticles in aqueous solution at low concentrations. The nanoparticle formation was evidenced via cryo-transmission electron microscope (Cryo-TEM) and dynamic light scattering analysis. The average hydrodynamic size of rPAA-Ch blank nanoparticles was about 80-160 nm with zeta potential of 50-60 mV. Also, the effects of different percentages of cholesterol grafted onto rPAA on physicochemical characteristics, in vitro cytotoxicity, cellular uptake, VEGF gene silencing efficacy and translocation mechanism of rPAA-Ch/siRNA complexes were investigated. The results showed that rPAA-Ch57 polymer was not only able to form stable nanocomplexes and possess high cell uptake, but also to exhibit the best in vitro VEGF gene silencing efficacy and the best in vivo tumor growth inhibition effect when it was formulated with VEGF-siRNA. Moreover, the observations of confocal laser scanning microscope (CLSM) and the study of cholesterol competitive inhibition demonstrated that endosomal/lysosomal escape and cytoplasmic dissociation of rPAA-Ch57/siRNA complexes were dependent on the "proton sponge effect" and disulfide cleavage, following internalization with cholesterol-related endocytosis pathway and subsequent transportion into endosomes/lysosomes. These findings indicated that the rPAA-Ch57 polymer should be a promising and potent carrier for siRNA delivery.

Self-assembly nanomicelles based on cationic mPEG-PLA-b-Polyarginine(R15) triblock copolymer for siRNA delivery.

Due to the absence of safe and effective carriers for in vivo delivery, the applications of small interference RNA (siRNA) in clinic for therapeutic purposes have been limited. In this study, a biodegradable amphiphilic tri-block copolymer (mPEG(2000)-PLA(3000)-b-R(15)) composed of monomethoxy poly(ethylene glycol), poly(d,l-lactide) and polyarginine was synthesized and further self-assembled to cationic polymeric nanomicelles for in vivo siRNA delivery, with an average diameter of 54.30 ± 3.48 nm and a zeta potential of approximately 34.8 ± 1.77 mV. The chemical structures of the copolymers were well characterized by (1)H NMR spectroscopy and FT-IR spectra. In vitro cytotoxicity and hemolysis assays demonstrated that the polymeric nanomicelles showed greater cell viability and haemocompatibility than those of polyethyleneimine (PEI) or R(15) peptide. In vitro experiments demonstrated that EGFR targeted siRNA formulated in micelleplexes exhibited approximately 65% inhibition of EGFR expression on MCF-7 cells in a sequence-specific manner, which was comparable to Lipofectamine™ 2000. The results of intravenous administration showed Micelleplex/EGFR-siRNA significantly inhibited tumor growth in nude mice xenografted MCF-7 tumors, with a remarkable inhibition of EGFR expression. Furthermore, no positive activation of the innate immune responses and no significant body weight loss was observed during treatment suggested that this polymeric micelle delivery system is non-toxic. In conclusion, the present nanomicelles based on cationic mPEG(2000)-PLA(3000)-b-R(15) copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA.

Enhanced anticancer activity of gemcitabine coupling with conjugated linoleic acid against human breast cancer in vitro and in vivo.

Gemcitabine (GEM) is a nucleoside analog agent against a wide variety of tumors. To overcome its limitation of rapid metabolism in vivo that results in short circulation time and poor antitumor efficacy, a novel prodrug (CLA-GEM conjugate) has been developed through the covalent coupling of conjugated linoleic acid (CLA) to N(4)-amino group of GEM. The chemical structure of CLA-GEM conjugate was identified by NMR, FTIR and other methods. From in vitro tests, it was demonstrated that the linkage with CLA increased the plasma stability of GEM as well as the antitumor activity against human breast tumor cells (MCF-7). Importantly, it also altered the transport pattern of GEM across cell membrane (MCF-7 and MDA-MB-231), evidenced by the little effect of nucleoside transporter inhibitors (NBMPR and dipyridamole) on the IC(50) values of CLA-GEM, instead of the great effect on that of unmodified GEM. In vivo pharmacokinetic study showed that the CLA-GEM conjugate had a longer plasma half-life and a higher bioavailability compared to that of unmodified GEM. Significant stronger antitumor activity was observed in the nude mice xenografted MCF-7 breast tumor after treated with CLA-GEM than that of unmodified GEM, while no significant body weight loss was found in all treatments. In conclusion, the novel CLA-GEM conjugate prepared in this study would be a promising prodrug of gemcitabine for future clinical use.

Synthesis, physicochemical and biological properties of oligonucleotides incorporated with amino-isonucleosides.

Antisense oligonucleotides (ASONs) and siRNAs have been applied extensively for the regulation of cellular and viral gene expression, and RNAi is currently one of the most promising new approaches for anti-tumor and anti-viral therapy. In order to improve bioactivity properties and physicochemical properties of siRNA, we synthesized a novel class of ASONs II-VII incorporated with amino-isonucleoside (isoA 1 and isoA 2 ) for investigation on basic physicochemical properties. Then we designed amino-isonucleoside (isoA 1 , isoA 2 and isoT 1 ) incorporated siRNA 2-7. Some meaningful results have been obtained from the physicochemical property experiments in ASONs. In RNAi potency experiments, we investigated RNAi potency of each strand of the siRNA. These amino-isonucleosides incorporated siRNAs showed promising bioactivity properties and had position specificity. Reduced off target effect from sense strand loading in siRNA application was observed.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Trifluoromethylated cyclic-ADP-ribose mimic: synthesis of 8-trifluoromethyl-N(1)-[(5''-O-phosphorylethoxy)methyl]-5'-O-phosphorylinosine-5',5''-cyclic pyrophosphate (8-CF(3)-cIDPRE) and its calcium release activity in T cells.

A convenient trifluoromethylation method was firstly applied to the synthesis of 8- CF(3)-purine nucleosides. On the basis of this method, new protection and deprotection strategies were developed for the successful synthesis of the trifluoromethylated cyclic-ADP-ribose mimic, 8-CF(3)-cIDPRE 1. Using intact, fura-2-loaded Jurkat T cells compound 1 and 2',3'-O-isopropylidene 8-CF(3)-cIDPRE 14 were characterized as membrane-permeant cADPR agonists. Contrary to the 8-substituted cADPR analogues that mainly act as antagonists of cADPR in cells, 8-substituted cIDPRE derivatives were shown to be Ca(2+) mobilizing agonists. Here we report that even compound 1, the 8-substituted cIDPRE with the strong electron withdrawing CF(3) group, behaves as an agonist in T cells. Interestingly, also the partially protected 2',3'-O-isopropylidene 8-CF(3)-cIDPRE activated Ca(2+) signaling indicating only a minor role for the hydroxyl groups of the southern ribose of cADPR for its biological activity. To our knowledge 8-CF(3)-cIDPRE 1 is the first reported fluoro substituted cADPR mimic and 8-CF(3)-cIDPRE 1 and compound 14 are promising molecular probes for elucidating the mode of action of cADPR.

Enhanced efficacy of functionalized epirubicin liposomes in treating brain glioma-bearing rats.

PURPOSE: The restriction of drug transporting across the blood-brain barrier (BBB) and the limit of drug penetrating into the tumor tissue remain the major obstacles for brain tumor chemotherapy. In the present study, we developed a functionalized liposomal nanoconstruct, epirubicin liposomes modified with tamoxifen (TAM) and transferrin (TF), for transporting drug across the BBB and afterwards targeting the brain glioma. METHODS: Evaluations were performed on the murine C6 glioma cells, the C6 glioma spheroids, the BBB model in vitro and the brain glioma-bearing rats. RESULTS: When compared with controls, epirubicin liposomes modified with TAM and TF showed the strongest inhibitory effect to C6 glioma cells or glioma spheroids in vitro, significant transport ability across the BBB model in vitro, an evident effect of targeting the brain tumor cells in vitro, and an extended median survival time in the brain glioma-bearing rats. CONCLUSION: Epirubicin liposomes modified with TAM and TF significantly improve the therapeutic efficacy of brain glioma in vitro and in animals, hence providing a new strategy for brain tumor chemotherapy.

Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals.

Chemotherapy for brain glioma has been of limited value due to the inability of transport of drug across the blood-brain barrier (BBB) and poor penetration of drug into the tumor. For overcoming these hurdles, the dual-targeting daunorubicin liposomes were developed by conjugating with p-aminophenyl-alpha-D-manno-pyranoside (MAN) and transferrin (TF) for transporting drug across the BBB and then targeting brain glioma. The dual-targeting effects were evaluated on the BBB model in vitro, C6 glioma cells in vitro, avascular C6 glioma tumor spheroids in vitro, and C6 glioma-bearing rats in vivo, respectively. After applying dual-targeting daunorubicin liposomes, the transport ratio across the BBB model was significantly increased up to 24.9%. The most significant uptake by C6 glioma was evidenced by flow cytometry and confocal microscope. The C6 glioma spheroid volume ratio was significantly lowered to 54.7%. The inhibitory rate to C6 glioma cells after crossing the BBB was significantly enhanced up to 64.0%. The median survival time of tumor bearing rats after administering dual-targeting daunorubicin liposomes (22 days) was significantly longer than that after giving free daunorubicin (17 days, P=0.001) or other controls. In conclusion, the dual-targeting daunorubicin liposomes are able to improve the therapeutic efficacy of brain glioma in vitro and in animals.

CD38/cADPR/Ca2+ pathway promotes cell proliferation and delays nerve growth factor-induced differentiation in PC12 cells.

Intracellular Ca(2+) mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca(2+) changes. Here we explored the role of one endogenous Ca(2+)-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca(2+) release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca(2+) increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.

N-(2,6-dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide as a novel tubulin ligand against human cancer.

PURPOSE: We have synthesized a new tubulin ligand N-(2,6-dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide (IG-105). This work investigates its anticancer effect and mechanism. EXPERIMENTAL DESIGN: Anticancer efficacy was evaluated at the molecular target, cancer cells and nude mice. The mechanism was explored at submolecular, molecular, and cellular levels. RESULTS: IG-105 showed a potent activity against human leukemia and solid tumors in breast, liver, prostate, lung, skin, colon, and pancreas with IC(50) values between 0.012 and 0.298 mumol/L. It was also active in drug-resistant tumor cells and not a P-glycoprotein substrate. It inhibited microtubule assembly followed by M-phase arrest, Bcl-2 inactivation, and then apoptosis through caspase pathways. The colchicine pocket on tubulin is the binding site of IG-105. Nude mice experiments showed that IG-105 monotherapy at 100 mg/kg i.p. (q2d) yielded 81% inhibition of Bel-7402 hepatoma growth and at 275 mg/kg i.p. (q2d) completely inhibited the tumor growth. MCF-7 breast cancer in nude mice showed a similar therapeutic response to IG-105. Acute toxicity of IG-105 was not found even at 1,000 mg/kg i.p. In combination with oxaliplatin or doxorubicin, IG-105 converted each of these subcurative compounds into a curative treatment with complete inhibition for tumor growth in the hepatoma-bearing nude mice. The combination was more active than either drug. In no experiment was toxicity increased by combination chemotherapy. CONCLUSIONS: IG-105 inhibits microtubule assembly by binding at colchicine pocket. It shows a potent anticancer activity in vitro and in vivo and has good safety in mice. We consider IG-105 merits further investigation.

A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide.

Peptide-oligonucleotide conjugates were synthesized using two strategies: a mimetic signal peptide-conjugated oligonucleotide was assembled stepwise on CPG by using 2,2-dimethyl-3-hydroxypropionic acid as a linker. To solve the precipitation problem in the coupling reaction caused by the electrostatic interaction of arginine-rich peptides and oligonucleotide, oligonucleotides were absorbed on an anion-exchange resin, and then the on-resin fragment was applied for the conjugation with arginine-rich peptide. The peptide-antisense oligonucleotides showed permeability to the cell membrane of HepG-2 cells.

Synthesis of antisense oligonucleotide-peptide conjugate targeting to GLUT-1 in HepG-2 and MCF-7 Cells.

A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxy-benzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.