Similar IC50 Values Research Articles

Short Proline-Rich Antimicrobial Peptides Inhibit Either the Bacterial 70S Ribosome or the Assembly of its Large 50S Subunit.

Short proline-rich antimicrobial peptides (PrAMPs) are a promising class of antibiotics that use novel mechanisms, thus offering the potential to overcome the health threat of multiresistant pathogens. The peptides bind to the bacterial 70S ribosome and can inhibit protein translation. We report that PrAMPs can be divided into two classes, with each class binding to a different site, and thus use different lethal mechanisms. Oncocin-type peptides inhibit protein translation in Escherichia coli by binding to the exit tunnel of the 70S ribosome with half maximal inhibitory concentrations (IC50 values) of around 2 to 6 μmol L(-1), whereas apidaecin-type peptides block the assembly of the large (50S) subunit of the ribosome, resulting in similar IC50 values. The revealed mechanisms should allow the design of new antibiotics to overcome current bacterial resistance mechanisms.

Abstract POSTER-THER-1402: Overcoming platinum resistance in ovarian cancer with niclosamide

Abstract Objective: Niclosamide, a salicyclamide derivative and FDA approved teniacide, exhibits potent effects against ovarian cancer in vitro by inhibiting the Wnt/β-catenin, STAT3, and mTOR pathways. Unfortunately, its low bioavailability as a chemotherapeutic agent in vivo necessitates investigation of analogs with improved pharmacokinetics and pharmacodynamics. The objective of this study was to investigate the role of targeting the Wnt/β-catenin and STAT3 pathways with niclosamide and niclosamide analogs (C-11 and C-32) in platinum-sensitive and -resistant ovarian cancer cell lines, primary ovarian cancer ascites cell populations, and tumor tissues. In addition, carboplatin and/or LLL12, a novel STAT3 inhibitor effective against ovarian cancer, were used in combination with niclosamide and its analogs to test for synergistic cytotoxicity. Methods: Two ovarian cancer cell lines A2780, SKOV3ip1 and their chemo-resistant derivatives, A2780.cp20 and SKOV3ip2.TRip2, were treated with niclosamide or C-11 or C-32 (0.1 to 5 μM) alone or in combination with carboplatin (5 to 150 μM) or LLL12 (0.1 nM - 10 µM). Tumorspheres were isolated from the ascites of ovarian cancer patients who underwent surgery. Tumor slices were prepared from freshly obtained ovarian cancer specimens. ATPLite assay was performed to measure cytotoxicity in cell lines, tumorspheres, and tumor slices. Western blot was used to assess expression of proteins implicated in the Wnt, mTOR, and STAT3 pathways. β-catenin signaling activity was measured using the TOPflash reporter assay. Results: All four cell lines were sensitive to niclosamide, C-11, and C-32 with similar IC50 values. Additive cytotoxicity was seen when niclosamide or its analogs were combined with carboplatin or LLL12 in the cell lines, tumorspheres, and tumor tissues. Ex vivo and in vitro samples from the same patient demonstrated similar cytotoxicity with niclosamide, C-11, and C-32 treatment. Synergism was also seen between carboplatin and LLL12 in vitro. The TOPflash assay showed that the combination of niclosamide (0.4 μM) and LLL12 (0.25 μM) decreased β-catenin activity more than either agent alone, suggesting that there is crosstalk between the STAT3 and Wnt/β-catenin pathways. Wnt/β-catenin, STAT3, and mTOR pathway target proteins (LRP6, pLRP6, Axin2, and Survivin) were downregulated with niclosamide and C-32 treatment, and to a greater degree with C-11. Conclusion: Combination treatment with niclosamide, or its analogs, with carboplatin or LLL12 induced greater cytotoxicity than either agent alone in ovarian cancer cell lines, primary tumorspheres, and tumor slices. The synergistic cytotoxicity seen in platinum-resistant cell lines with niclosamide, or its analogs, in combination with carboplatin suggest that targeting the Wnt/ β-catenin and STAT3 pathways may prove effective in overcoming chemoresistance in patients with platinum resistant ovarian cancer. Citation Format: Rebecca C. Arend, MD, Abhishek Gangrade, Christen L. Walters Haygood, MD, Chandrika Kurpad, Brandon J. Metge, Rajeev S. Samant, PhD, Pui-Kai Li, PhD, Yonghe Li, PhD, Deepak Bhasin, PhD, Charles Landen, MD, Ronald Alvarez, MD, J. Michael Straughn, MD, Donald J. Buchsbaum, PhD. Overcoming platinum resistance in ovarian cancer with niclosamide [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1402.

Abstract 1641: Tivantinib (ARQ 197) exhibits antitumor activity by directly interacting with tubulin and overcomes ABC transporter-mediated drug resistance

Abstract The receptor tyrosine kinase c-MET is recognized as a promising therapeutic target because c-MET-addicted cancers have been shown to be highly sensitive to c-MET kinase inhibitors. Tivantinib (ARQ197) was first reported as a highly selective c-MET inhibitor and is currently being investigated in a phase 3 clinical trial. However, as recently reported by us and other groups, tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules, we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Tivantinib could reduce tubulin polymerization independent of c-MET inhibition in cells and in mouse xenograft tumors. It is well known that microtubule destabilizing agents, such as vinblastine and colchicine, directly bind to tubulins at different binding sites. To determine if tivantinib directly binds to tubulin or not, we performed an in vitro competition assay using radiolabeled agents. Tivantinib competitively inhibited colchicine binding, but not vincristine or vinblastine binding, to purified tubulin, suggesting that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin, we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fit into the colchicine binding pocket of tubulin without steric hindrance. Currently, the microtubule inhibitors are widely used for the treatment of various cancers. However the development of acquired resistance is a major obstacle for the therapy. Overexpression of the drug efflux pump (ABC transporter) is one of the major mechanisms of drug resistance. We thus tested the cytotoxic activity of tivantinib against tubulin binding agent-resistant cells that we had established by incubating cells in sublethal concentrations of drug. Tivantinib showed similar IC50 values against parental and multidrug resistant cells. In contrast, other microtubule-targeting drugs, such as vincristine, paclitaxel, and colchicine, could not suppress the growth of resistant cells. Moreover, tivantinib equally suppressed growth and survival of ABCB1-, ABCC1-, or ABCG2- transfected cells. Our findings that tivantinib shows cytotoxic activity by disrupting tubulin polymerization, directly interacts with tubulin and overcomes ABC transporter-mediated drug resistance, suggest the possibility that tivantinib might be useful for treatment of wide variety of cancers. Citation Format: Aki Aoyama, Ryohei Katayama, Yasushi Okuno, Naoya Fujita. Tivantinib (ARQ 197) exhibits antitumor activity by directly interacting with tubulin and overcomes ABC transporter-mediated drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2015-1641

Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay

AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC50 values for AMPK activators AMP and A769662 and a similar IC50 value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z′ factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions.

Quinoa (Chenopodium quinoa Willd.) protein hydrolysates with in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant properties

The potential of quinoa to act as a source of dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant peptides was studied. A quinoa protein isolate (QPI) with a purity of 40.73 ± 0.90% was prepared. The QPI was hydrolysed at 50 °C for 3 h with two enzyme preparations: papain (P) and a microbial papain-like enzyme (PL) to yield quinoa protein hydrolysates (QPHs). The hydrolysates were evaluated for their DPP-IV inhibitory and oxygen radical absorbance capacity (ORAC) activities. Protein hydrolysis was observed in the QPI control, possibly due to the activity of quinoa endogenous proteinases. The QPI control had significantly higher DPP-IV half maximal inhibitory concentrations (IC50) and lower ORAC values than QPH-P and QPH-PL (P < 0.05). Both QPH-P and QPH-PL had similar DPP-IV IC50 and ORAC values. QPH-P had a DPP-IV IC50 value of 0.88 ± 0.05 mg mL−1 and an ORAC activity of 501.60 ± 77.34 μmol Trolox equivalent (T.E.) g−1. To our understanding, this is the first study demonstrating the in vitro DPP-IV inhibitory properties of quinoa protein hydrolysates. QPHs may have potential as functional ingredients with serum glucose lowering properties.

Development of novel monoclonal antibodies-based ultrasensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for aflatoxin B1 detection

Monoclonal antibodies (mAbs) that are specific to aflatoxin B1 (AFB1) were produced from hybridoma cell lines 3F6G11 and 9C7C11 by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells that were isolated from a BALB/c mouse that was immunized with AFB1-bovine serum albumin (BSA). Both 3F6G11 and 9C7C11 mAbs are the immunoglobulin G1 isotypes. Competitive direct enzyme-linked immunosorbent assays (cdELISA) were established to characterize these antibodies. In the 3F6G11 mAb based cdELISA, the concentrations causing 50% inhibition of binding of AFB1-horseradish peroxidase to the antibody by AFB1, AFB2, AFG1, and AFG2 were found to be 0.051, 0.050, 1.820, and 1.270 ng/ml, respectively. Using 9C7C11 mAbs, similar IC50 values for AFB1, AFB2, AFG1 and AFG2 were obtained as 0.045, 0.057, 2.530 and 2.120 ng/ml, respectively. A rapid and sensitive gold nanoparticle immunochromatographic strip (immunostrip) was also established for these antibodies. This strip has a detection limit of 1.0 ng/ml for AFB1 and the whole assay can be completed within 10 min. Extensive analysis of 20 samples by 3F6G11 mAb and 9C7C11 mAbs cdELISAs revealed that six samples were slightly contaminated by AFB1 at concentrations from 0.160 to 16.10 ng/g. Results of analyses of 20 samples with an immunostrip assay correlate well with those obtained using cdELISA. The proposed cdELISA and immunostrip methods are highly sensitive for the rapid screening of AFB1 in food samples.

Preparation of 7‐Methoxy Tacrine Dimer Analogs and Their In vitro/In silico Evaluation as Potential Cholinesterase Inhibitors

Novel types of symmetric bis‐7‐methoxytacrines connected by oligoethyleneoxy chains 3–5 and nonsymmetric monomeric 7‐methoxytacrines containing hydroxyl‐terminated oligoethyleneoxy chains 6–8 were prepared, and their in vitro/in silico effects on human recombinant AChE (hAChE) and human plasmatic butyrylcholinesterase (hBChE) were compared, with 7‐MEOTA (2) as the standard compound. The symmetric bis‐7‐MEOTA derivatives 3–5 showed hAChE inhibition similar to that of 2. On the other hand, their effects on hBChE revealed an increasing inhibition trend when the oligoethyleneoxy units between the two 7‐MEOTA moieties became longer. Accordingly, compounds 4 and 5 showed better selectivity towards hBChE. The most effective in the inhibition hAChE and hBChE was compound 8 with the longest oligoethyleneglycol chain, whereas compounds 6 and 7 resulted in similar IC50 values. A molecular modeling study using substrates 5 and 8 showed a possible binding conformation and protein–ligand interaction between the substrates and AChE/BChE.

Anticancer Agents: Does a Phosphonium Behave Like a Gold(I) Phosphine Complex? Let a "Smart" Probe Answer!

Gold phosphine complexes, such as auranofin, have been recognized for decades as antirheumatic agents. Clinical trials are now underway to validate their use in anticancer or anti-HIV treatments. However, their mechanisms of action remain unclear. A challenging question is whether the gold phosphine complex is a prodrug that is administered in an inactive precursor form or rather that the gold atom remains attached to the phosphine ligand during treatment. In this study, we present two novel gold complexes, which we compared to auranofin and to their phosphonium analogue. The chosen ligand is a phosphine-based smart probe, whose strong fluorescence depends on the presence of the gold atom. The in vitro biological action of the gold complexes and the phosphonium derivative were investigated, and a preliminary in vivo study in healthy zebrafish larvae allowed us to evaluate gold complex biodistribution and toxicity. The different analyses carried out showed that these gold complexes were stable and behaved differently from phosphonium and auranofin, both in vitro and in vivo. Two-photon microscopy experiments demonstrated that the cellular targets of these gold complexes are not the same as those of the phosphonium analogue. Moreover, despite similar IC50 values in some cancer cell lines, gold complexes displayed a low toxicity in vivo, in contrast to the phosphonium salt. They are therefore suitable for future in vivo investigations.

Investigation of the Potential of Hemp, Pea, Rice and Soy Protein Hydrolysates as a Source of Dipeptidyl Peptidase IV (DPP-IV) Inhibitory Peptides

Food protein hydrolysates contain peptide sequences with dipeptidyl peptidase IV (DPP-IV) inhibitory properties which may find use to improve serum glucose regulation in type 2 diabetics. Four plant protein isolates from hemp (H), pea (P), rice (R) and soy (S) were hydrolysed with three enzyme preparations: Corolase L10 (Cor), Promod 144MG (Prom) and Protamex (Prot). From the 12 hydrolysates generated after 4-h incubation, 7 yielded DPP-IV inhibitory activity <2.0 mg protein equivalent mL−1. Their IC50 values ranged from 0.73 ± 0.11 to 3.54 ± 0.24 mg dry weight (dw) hydrolysate mL−1 for the 4 h P_Prot and 4 h H_Cor hydrolysates, respectively. Simulated gastrointestinal digestion (SGID) of the intact proteins yielded DPP-IV IC50 values between 1.85 ± 0.34 and 4.50 ± 0.55 mg dw hydrolysate mL−1 for the R and H, respectively. The DPP-IV inhibitory potency of the 4 h hydrolysates subjected to SGID (DPP-IV IC50 ranging from 1.00 ± 0.42 to 3.83 ± 0.36 mg dw hydrolysate mL−1 for the SGID of the 4 h S_Prom and 4 h H_Prot, respectively) was generally higher than that of the corresponding intact protein subjected to SGID, with the exception of the SGID of R and that of the 4 h R_Prot hydrolysate which had similar IC50 values (P > 0.05). To our knowledge, this is the first study reporting that H and P protein hydrolysates inhibit DPP-IV in vitro. This study also demonstrates the potential benefit, in some instances, of hydrolysing plant protein substrates prior to oral ingestion with the view of releasing DPP-IV inhibitory peptides.

Efficacy of a novel, locally delivered TrkA inhibitor in preclinical models of OA and joint pain

Purpose: OA and associated joint pain is a major cause of disability, and there is a high unmet need for effective and safe analgesic therapies. Substantial pain alleviation has been reported for OA patients treated with intravenous (IV) infusions of antibodies which block nerve growth factor (NGF), however an increased drug-related risk for accelerated OA progression was observed in some patients. As an alternate strategy for targeting this pathway, we identified a novel small molecule compound, GZ389988, as an inhibitor of TrkA, the high-affinity receptor for NGF. GZ389988 was formulated for intraarticular (IA) delivery, and its effects on local knee pain were tested in rats using both a monosodium iodoacetate (MIA) induced model of OA, as well as an arthritis flare model mediated by streptococcal peptidoglycan polysaccharide (PGPS). Methods: Identification of TrkA inhibitor GZ389988: Drug candidate screening was conducted to select inhibitors of Trk family tyrosine kinases. Low solubility compounds were evaluated using a cell-based TrkA assay, and further screened against kinase and safety/liability panels. Acute cytotoxicity was assessed by neutral red assay for a variety of cell types, including primary human chondrocytes and synoviocytes (Articular Engineering). In vivo biocompatibility was tested by IA injection of GZ389988 into rat knee joints. Preclinical rat OA model: Unilateral knee OA was initiated by IA injection of MIA. One week later, diseased (ipsilateral) joints were treated with a single IA injection of GZ389988 or placebo/vehicle, and differences in hind-limb weight distribution were measured weekly for 4 weeks by incapacitance testing. In some cases, GZ389988 was administered to the contralateral knee instead, to test for the occurrence of systemic compound distribution affecting the ipsilateral joint. Preclinical rat arthritis flare model (Bolder BioPATH): Unilateral primary knee arthritis was induced by IA injection of PGPS. Two weeks later, animals received a single IA injection of GZ389988 or placebo/vehicle into the diseased (ipsilateral) joint. Disease reactivation flares were triggered by IV administration of PGPS at weeks 1 and 3 following drug (or placebo) treatment. Gait analysis was conducted by obtaining inked pawprint impressions from animals which were allowed to ambulate freely along a papered walkway. Gait scoring and quantification was performed using 4 distinct pawprint pairs per animal, collected daily for 4 days following each reactivation. Results: GZ389988 was identified as a potent inhibitor of TrkA (similar IC50 values observed for TrkA, TrkB, TrkC and cFMS/CSF1R), with a high level of selectivity against a wide panel of other kinases. In addition, GZ389988 has very low aqueous solubility, thus facilitating its formulation for local administration. No cytotoxicity was observed in cell-based assays using criteria of LD50/IC50 ratio >100. In vivo biocompatibility studies with GZ389988 showed neither a treatment effect on body weight or clinical appearance, nor any significant histopathologic changes in joint tissues. In the rat MIA model, a single ipsilateral dose of GZ389988 provided a significant reduction in local joint pain (weight-bearing imbalance) for 4 weeks. Treatment of the contralateral joint with GZ389988 had no effect on ipsilateral joint pain (Fig. 1), illustrating the absence of a substantial systemic effect following IA administration. GZ389988 was also efficacious in the PGPS arthritis model, demonstrating a significant improvement of gait analysis scores following painful disease reactivation flares (Fig. 2). Conclusions: Elevated levels of NGF are associated with tissue injury, and have been observed in a number of painful conditions including OA. In addition, TrkA is expressed by OA synovial fibroblasts and is upregulated in response to NGF. In OA pain trials, NGF-blocking Abs have demonstrated significant efficacy with high patient responder rates, however such studies have been subject to clinical hold by the FDA due to the occurrence of joint-related adverse events. We have targeted the NGF pain signaling pathway using a locally-effective inhibitor of TrkA. In addition to the efficacy data presented here, pharmacokinetic studies and safety pharmacology and preclinical toxicology data support the development of GZ389988 as a novel therapeutic approach for the treatment of OA and joint pain.Figure 2. Efficacy of GZ389988 in rat PGPS model of joint pain. Disease was inititated by a single IA injection of PGPS into the knee joint (ipsilateral) three weeks prior to the first reactivation. A single dose of GZ389988 was administered by IA injection into the knee one week prior to the first reactivation. Disease reactivation (painful flare) was induccd on study days 0 and 14 by IV administration of PGPS. Gait analysis was performed based on pawprint areas, and scored as follows: 1= mild limping/pain; 2=moderate limping/pain; 3=marked limping/pain; 4=severe hopping/pain. Data are presented as mean + SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

New heterocyclic hybrids of pyrazole and its bioisosteres: Design, synthesis and biological evaluation as dual acting antimalarial-antileishmanial agents

A new series of pyrazole derivatives were synthesized by hybridization with five-membered heterocyclic moieties such as thiazoles, thiazolidinones, 1,3,4-thiadiazoles and pyrazolines. The compounds were evaluated for their in vivo antimalarial activity against Plasmodium berghei infected mice and the most active derivatives were further examined for their in vitro antimalarial activity against chloroquine resistant (RKL9) strain of Plasmodium falciparum. Compounds 2c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b had more than 90% parasite suppression activity of that found with the antimalarial reference standard drug, chloroquine phosphate and had lower IC50 values than chloroquine. Compounds 4b and 9b were the most active derivatives, and their activities were 5-fold higher than chloroquine. All the newly synthesized compounds were evaluated for their in vitro antileishmanial activity against Leishmania aethiopica promastigotes and amastigote. The results showed that compounds 2c, 2d, 3d, 4b, 4c, 4d and 5a had lower or similar IC50 values than the reference standard drugs, amphotericin B and miltefosine. Compound 3d had the highest antileishmanial activity. Collectively, compounds 2c, 2d, 4b, 4c, 4d and 5a exhibited dual activity against malaria and leishmaniasis and were safe and well tolerated by the experimental animals orally up to 300 mg/kg and parenterally up to 100 mg/kg.

T-Type Ca2+ Channel Regulation by CO: A Mechanism for Control of Cell Proliferation.

T-type Ca(2+) channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe(2+) and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca(2+) channels.Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca(2+) channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC(50) values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca(2+) ](i) in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015).Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca(2+) channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca(2+) channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca(2+) channels.

Medicinal Mushroom Extracts Possess Differential Antioxidant Activity and Cytotoxicity to Cancer Cells

Many species of edible mushrooms are known to contain a wide array of compounds with high nutritional and medicinal values. However, these values vary widely among mushroom species because of the wide diversity of compounds with different solubilities to solvents used in extraction. We report here the comparison of antioxidant activity and cytotoxicity against cancer cells in extracts of Pleurotus ostreatus, P. sajor-caju, Agaricus campestris, and A. bisporus from 7 different solvents, including water, ethanol, ethyl acetate, acetone, chloroform, hexane, and petroleum ether. The extracts were analyzed for their antioxidant activities using the % DPPH (2,2-diphenyl-1-picrylhydrazylhydrate) scavenging activity method. Our results revealed that the water extracts exhibited the highest % DPPH scavenging activity in comparison to all other solvent extracts. The highest value was obtained from the water extract of P. sajor-caju (78.1%), and the lowest one was from the hexane extract of A. bisporus (0.8%). In general, extracts from nonpolar solvents exhibited much lower antioxidant activities than those from polar solvents. The cytotoxic effects of these extracts were evaluated using 2 cancer cell lines of larynx carcinoma (HEp-2) and breast carcinoma (MCF-7). When added into Hep-2 cells, the hexane extracts from P. ostreatus, P. sajor-caju, A. bisporus, and A. campestris yielded the highest IC50 values of 1.7 ± 1.56, 2.1 ± 2.82, 4.4 ± 1.71, and 2.2 ± 1.34 μg/mL, respectively, in comparison to all other solvent extracts. Similar IC50 values were obtained when the MCF-2 cancer cells were tested, suggesting that hexane is the preferred solvent to extract the anticancer compounds from these mushrooms. Our results also indicated that extracts from solvents with nonpolar or intermediate polarity were more potent than those with high polarity in their cytotoxicity against cancer cells, and extracts from different mushrooms by the same solvent possessed varied degrees of cytotoxicity.

アロマ精油成分による化学構造に依存した蛙坐骨神経の複合活動電位抑制

Aroma–oil compounds derived from plants have a variety of clinical effects including local anesthesia. We have previously reported that various aroma–oil compounds reduce the peak amplitudes of fast–conducting and Na+–channel blocker tetrodotoxin–sensitive compound action potentials (CAPs) recorded from the frog sciatic nerve in a manner dependent on their chemical structures. The present study further examined this structure–activity relationship by applying the air–gap method to the frog sciatic nerve. Cyclic alcohols ((+)–borneol, (–)–borneol, α–terpineol), chain alcohols ((–)–linalool, citronellol, geraniol) and esters (bornyl acetate, geranyl acetate) reduced CAP peak amplitudes with the half–maximal inhibitory concentration (IC50) values of 1.5 mM, 2.3 mM, 2.7 mM, 2.0 mM, 0.35 mM, 0.53 mM, 0.44 mM and 0.51 mM, respectively. This IC50 value for (–)–linalool was similar to that for (±)–linalool (1.7 mM), a value as reported previously. On the other hand, hydrocarbon (p–cymene) at a high concentration such as 2 mM reduced CAP amplitude by only 20%. The efficacy sequence of the aroma–oil compounds was esters ≧ alcohols ＞ hydrocarbons, and was thus consistent with one reported previously, i.e., phenols ≧ aldehydes ≧ esters ＞ alcohols ＞ ketones ＞ oxides ≫ hydrocarbons. There was a variation in IC50 value among the alcohols used; chain alcohols were more effective in inhibiting CAPs than cyclic ones. Similar IC50 values of (–)–linalool and (±)–linalool and also of (+)–borneol and (–)–borneol indicate no difference in efficacy between the steroisomers in inhibiting CAPs. There was no correlation between IC50 value for CAP inhibition by aroma–oil compound and its octanol–water partition coefficient value. The present study confirmed that aroma–oil compounds inhibit nerve conduction in a manner specific to their chemical structures.

Synthesis, characterization, and biological activity of novel metronidazole-piperazine amides

A new series of metronidazole derivatives containing piperazine rings were prepared via the reaction of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl chloride with selected substituted piperazines in the presence of a base. Structures of the new compounds were confirmed by NMR and MS spectral data and by elemental analyses. The antibacterial and anti-parasitic activities of these compounds were evaluated in vitro. Some of the newly synthesized compounds exhibited superior activity against Clostridium sporogenes bacteria compared to the standard drug metronidazole. On the other hand, other derivatives exhibited remarkable antigiardial activity and were found to be more active than metronidazole with IC 50 ranging from 1.8 to 6.7 µg/dm3. 1-Ethyl-4-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]piperazine also exhibited antigiardial activity with similar IC 50 value (7.6 µg/cm3) as compared to the reference drug metronidazole (IC 50 = 7.4 µg/cm3). Similarly, several of the new compounds exhibited significant antitrichomonal activity and found to be more active than metronidazole with IC 50 ranging from 6.7 to 8.65 µg/cm3 as compared to the reference drug metronidazole (8.9 µg/cm3).

Tivantinib (ARQ 197) exhibits antitumor activity by directly interacting with tubulin and overcomes ABC transporter-mediated drug resistance.

Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However, as recently reported by us and another group, tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules, we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report, tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin, we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin, we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore, tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast, other microtubule-targeting drugs, such as vincristine, paclitaxel, and colchicine, could not suppress the growth of cells overexpressing ABC transporters. Moreover, the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors.

Synthesis and biological activity of novel amidrazones incorporating 5-nitroimidazole, ciprofloxacin, and 7-chloro-4-piperazinylquinoline

Series of new amidrazones Ia–c, IIa–c, and IIIc, incorporating 5-nitroimidazole (a), ciprofloxacin (b), and 7-chloro-4-piperazinylquinoline (c) moieties, were synthesized by reacting hydrazonoyl chlorides I–III, derived from 4-fluoroaniline and 4-chloroaniline with the proper amine, in the presence of triethylamine. Structures of the prepared compounds were confirmed by 1H NMR, 13C NMR, and ESI-HRMS spectral data. The antitumor, antibacterial, and antiparasitic activities of the newly synthesized compounds were evaluated. Compounds Ic and IIc exhibited strong anticancer activity against all tested cancer cell lines. In addition, compounds Ia and IIa displayed stronger antimicrobial potency than metronidazole against microaerophilic bacteria. Compounds IIa, IIIc, and IIc, exhibited remarkable antigiardial activity and were found to be more active than metronidazole with IC50 of 5.6, 5.9, and 6.8 µg/mL, respectively. Compounds Ia, Ib, and Ic also exhibited antigiardial activity with similar IC50 values compared to the reference drug metronidazole (IC50 = 6.9 µg/mL). On the other hand, compounds Ia, Ib, and IIa exhibited significant antitrichomonal activity and found to be more active than metronidazole with IC50 of 3.79, 4.6, and 5.1 µg/mL, respectively, compared to the reference drug metronidazole (8.79 µg/mL).

Abstract DDT01-03: Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET

Abstract NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

Cytotoxic activity of 3,6-dihydroxyflavone in human cervical cancer cells and its therapeutic effect on c-Jun N-terminal kinase inhibition.

Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF) is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR) with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR) 4/CD14, p38 mitogen-activated protein kinase (MAPK), Jun-N terminal kinase (JNK), extracellular-signaling regulated kinase (ERK), and cyclooxygenase (COX)-2 pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM) value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM) in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M−1) and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.