Design and synthesis of novel pyrrolo[2,3-b]pyridine derivatives targeting V600EBRAF

Several pyrrolo[2,3-b]pyridine-based B-RAF inhibitors are well known and some of them are currently FDA approved as anticancer agents. Based on the structure of these FDA approved ^V600EB-RAF inhibitors, two series of pyrrolo[2,3-b]pyridine scaffold were designed and synthesized in attempt to develop new potent ^V600EB-RAF inhibitors. The 38 synthesized compounds were biologically evaluated for their ^V600EB-RAF inhibitory effect at single dose (10 μM). Compounds with high percent inhibition were tested to determine their IC₅₀ over ^V600EB-RAF. Compounds 34e and 35 showed the highest inhibitory effect with IC₅₀ values of 0.085 µM and 0.080 µM, respectively. Headed for excessive biological evaluation, the synthesized derivatives were tested over sixty diverse human cancer cell lines. Only compound 35 emerged as a potent cytotoxic agent against different panel of human cancer cell lines.     

Synthesis and in vitro evaluation of N-alkyl-7-methoxytacrine hydrochlorides as potential cholinesterase inhibitors in Alzheimer disease

All approved drugs for Alzheimer disease (AD) in clinical practice ameliorate the symptoms of the disease. Among them, acetylcholinesterase inhibitors (AChEIs) are used to increase the cholinergic activity. Among new AChEI, tacrine compounds were found to be more toxic compared to 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetrahydroacridine). In this Letter, series of 7-MEOTA analogues (N-alkyl-7-methoxytacrine) were synthesized. Their inhibitory ability was evaluated on recombinant human acetylcholinesterase (AChE) and plasmatic human butyrylcholinesterase (BChE). Three novel compounds showed promising results towards hAChE better to THA or 7-MEOTA. Three compounds resulted as potent inhibitors of hBChE. The SAR findings highlighted the C₆–C₇ N-alkyl chains for cholinesterase inhibition.     

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers

Two-pore channels (TPCs) are Ca²⁺-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca²⁺ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P₂-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

Selective publication of antidepressant trials and its influence on apparent efficacy: Updated comparisons and meta-analyses of newer versus older trials

BackgroundValid assessment of drug efficacy and safety requires an evidence base free of reporting bias. Using trial reports in Food and Drug Administration (FDA) drug approval packages as a gold standard, we previously found that the published literature inflated the apparent efficacy of antidepressant drugs. The objective of the current study was to determine whether this has improved with recently approved drugs.Methods and findingsUsing medical and statistical reviews in FDA drug approval packages, we identified 30 Phase II/III double-blind placebo-controlled acute monotherapy trials, involving 13,747 patients, of desvenlafaxine, vilazodone, levomilnacipran, and vortioxetine; we then identified corresponding published reports. We compared the data from this newer cohort of antidepressants (approved February 2008 to September 2013) with the previously published dataset on 74 trials of 12 older antidepressants (approved December 1987 to August 2002).Using logistic regression, we examined the effects of trial outcome and trial cohort (newer versus older) on transparent reporting (whether published and FDA conclusions agreed). Among newer antidepressants, transparent publication occurred more with positive (15/15 = 100%) than negative (7/15 = 47%) trials (OR 35.1, CI_95% 1.8 to 693). Controlling for trial outcome, transparent publication occurred more with newer than older trials (OR 6.6, CI_95% 1.6 to 26.4). Within negative trials, transparent reporting increased from 11% to 47%.We also conducted and contrasted FDA- and journal-based meta-analyses. For newer antidepressants, FDA-based effect size (ES_FDA) was 0.24 (CI_95% 0.18 to 0.30), while journal-based effect size (ES_Journals) was 0.29 (CI_95% 0.23 to 0.36). Thus, effect size inflation, presumably due to reporting bias, was 0.05, less than for older antidepressants (0.10).Limitations of this study include a small number of trials and drugs—belonging to a single class—and a focus on efficacy (versus safety).ConclusionsReporting bias persists but appears to have diminished for newer, compared to older, antidepressants. Continued efforts are needed to further improve transparency in the scientific literature.     

Alkylated/aminated nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates: Synthesis and anti-mycobacterial evaluation

The success in exploring anti-tubercular potency of nitroimidazole and quinoline, the core moieties of recently approved anti-tubercular drugs instigated us to synthesize a series of alkylated/aminated 2-methyl-5-nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates and to evaluate them for their activities against Mycobacterium tuberculosis as well as for their cytotoxicity towards the J774 murine macrophage cell line. Although the synthesized compounds did not surpass the activity of the standard drug Isoniazid, they have appreciable activities with minimal cytotoxicity. The synthesized nitroimidazole-7-chloroquinoline conjugate, 11c, having butyl chain as linker, proved to be the most potent among the series with an MIC₅₀ value of 2.2?μg/mL.     

CaCl2 Enhanced Somatic Embryogenesis in Manihot esculenta Crantz

Primary cassava somatic embryos were induced on a medium without CaCl₂, however, no or only a few secondary somatic embryos were formed from them. With 15 mM CaCl₂ in the medium for induction of cassava primary embryos, more secondary somatic embryos were produced from them, and they were much effective in maintaining their embryogenic capacity than the controls of embryos which were induced without CaCl₂.     

Morphological and cytogenetic characteristics of intergroup hybrids between closely related mating groups of the Closterium ehrenbergii species complex (Desmidiales, Chlorophyta)

Five F₁ hybrid strains were established from rare survivors in intergroup crosses between three closely related mating groups (A, B and H) of the Closterium ehrenbergii Meneghini ex Ralfs species complex. Cell sizes of these five strains studied under our standard culture conditions were compared to those of their parental stains and also to the total range of cell-size variation in each mating group. All five F₁ strains were larger in mean cell width than their parental strains. In cell length, three of them were larger than, one was the same as, and the other was intermediate between their parental strains. Their cell sizes were always larger than the range of their respective smaller parental mating group and three of them were larger than the range of their respective larger parental mating group.     

Analyses of Nivolumab Exposure and Clinical Safety Between 3-mg/kg Dosing and 240-mg Flat Dosing in Asian Patients with Advanced Renal Cell Carcinoma in the Real-World Clinical Setting

We aimed to identify the clinical characteristics related to increased nivolumab exposure in Japanese patients with renal cell carcinoma (RCC) in real-world clinical setting. Eleven patients were treated with the originally approved nivolumab dosing regimen of 3 mg/kg every 2 weeks (Q2W) (3-mg/kg group) and 8 patients with a flat dose of 240 mg Q2W (flat dosing group). Trough concentrations (C_min) until the fifth cycle were measured by sandwich enzyme-linked immunosorbent assay using anti-nivolumab monoclonal antibody established by the Autonomously Diversifying Library system. Mean C_min at four cycles of nivolumab were significantly higher in the flat dosing group than in the 3-mg/kg group. In an analysis of covariates related to nivolumab concentration, serum albumin (Alb) was significantly lower in the 3-mg/kg group than in the flat dose group. C_min correlated significantly with serum Alb at all cycles. In conclusion, serum Alb was a potential clinically relevant covariate for nivolumab pharmacokinetics in Japanese RCC patients. Further studies should verify whether serum Alb affects nivolumab efficacy and toxicity.     

Comparison of the Bacterial Communities in Anaerobic, Anoxic, and Oxic Chambers of a Pilot A2O Process Using Pyrosequencing Analysis

A₂O process is a sequential wastewater treatment process that uses anaerobic, anoxic, and oxic chambers for nitrogen and phosphorus removal. In this study, the bacterial communities among these chambers were compared, and the diversity of the bacteria involved in nitrogen and phosphorus removal was surveyed. A pilot-scale A₂O process (50 m³ day^?1) was operated for more than 6 months, and bacterial 16S rRNA gene diversity was analyzed using pyrosequencing. A total of 7,447 bacterial sequence reads were obtained from anaerobic (1,546), anoxic (2,158), and oxic (3,743) chambers. Even though there were differences in the atmospheric condition and functionality, no prominent differences could be found in the bacterial community of the three chambers of the pilot A₂O process. All sequence reads, which were taxonomically analyzed using the Eztaxon-e database, were assigned into 638 approved or tentative genera. Among them, about 72.2 % of the taxa were contained in the phyla Proteobacteria and Bacteroidetes. Phosphate-accumulating bacteria, Candidatus Accumulibacter phosphatis, and two other Accumulibacter were found to constitute 3.1 % of the identified genera. Ammonia-oxidizing bacteria, Nitrosomonas oligotropha, and four other phylotypes in the same family, Nitrosomonadaceae, constituted 0.2 and 0.9 %, respectively. Nitrite-oxidizing bacteria, Nitrospira defluvii, and other three phylotypes in the same family, Nitrospiraceae, constituted 2.5 and 0.1 %, respectively. In addition, Dokdonella and a phylotype of the phylum Chloroflexi, function in nitrogen and/or phosphate removal of which have not been reported in the A₂O process, constituted the first and third composition among genera at 4.3 and 3.8 %, respectively.     

Effect of soil moisture depletion on stem shrinkage and photosynthesis of tree seedlings

Effects of soil moisture depletion on stem contraction and photosynthesis of potted 3-year-old white ash (Fraxinus americana L.) and 4-year-old red pine (Pinus resinosa Ait.) seedlings were investigated in greenhouse experiments. White-ash seedlings transpired faster and consequently depleted soil moisture more rapidly than red-pine seedlings. Cumulative stem shrinkage occurred in both species as soils dried and appreciable stem expansion occurred only after soil was reirrigated. Following irrigation, seedlings rehydrated and their stems expanded to about their original diameters within 24 hours in white ash, and in up to 6 days in red pine seedlings. Photosynthesis was decreased in both species as soil dried, but it declined much faster in white ash than in red pine seedlings. Net emission of CO₂ by red pine seedlings occurred after 13 days of soil drying. In contrast, white-ash seedlings did not show net CO₂ release during severe drought. Several days after droughted white-ash and red-pine seedlings were reirrigated, photosynthesis recovered to pre-drought levels. Publication approved by Director of the Wisconsin Agricultural Experiment Station.     

Slow Receptor Dissociation Kinetics Differentiate Macitentan from Other Endothelin Receptor Antagonists in Pulmonary Arterial Smooth Muscle Cells

Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP₁) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K_b values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt_1/2) compared to bosentan and ambrisentan (ROt_1/2∶17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP₁ assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt_1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies.     

Synthesis,antimalarial activity and cytotoxic potential of new monocarbonyl analogues of curcumin

A series of novel monocarbonyl analogues of curcumin have been designed, synthesized and tested for their activity against Molt4, HeLa, PC3, DU145 and KB cancer cell lines. Six of the analogues showed potent cytotoxicity towards these cell lines with IC₅₀ values below 1 μM, which is better than doxorubicin, a US FDA approved drug. Several analogues were also found to be active against both CQ-resistant (W2 clone) and CQ-sensitive (D6) strains of Plasmodium falciparum in an in-vitro antimalarial screening. This level of activity warrants further investigation of the compounds for development as anticancer and antimalarial agents.     

Tryptophanol-derived oxazolopiperidone lactams: Identification of a hit compound as NMDA receptor antagonist

N-Methyl-d-aspartate receptors (NMDAR) exacerbated activation leads to neuron death through a phenomenon called excitotoxicity. These receptors are implicated in several neurological diseases (e.g., Alzheimer and Parkinson) and thus represent an important therapeutic target. We herein describe the study of enantiopure tryptophanol-derived oxazolopiperidone lactams as NMDA receptor antagonists. The most active hit exhibited an IC₅₀ of 63.4 μM in cultured rat cerebellar granule neurons thus being 1.5 fold more active than clinically approved NMDA antagonist amantadine (IC₅₀ = 92 μM).     

Comparison of Agar Dilution and Broth Microdilution Methods of Anaerobic Antimicrobial Susceptibility Testing using Several Veterinary Antibiotics against Clostridium perfringens Strains Originating from Porcine and Avian Sources

A broth microdilution and a reference agar dilution method was used to determine minimal inhibitory concentrations (MICs) of five veterinary antimicrobials when tested against 96 animal-derived and six American Type Culture Collection (ATCC) strains of Clostridium perfringens. These antimicrobials [bacitracin methylenedisalicylate (bacitracin-MD), tylosin, virginiamycin, erthromycin and tetracycline] are approved for use in animal feed at different levels for growth enhancement, control, and treatment of a variety of enteric diseases. For bacitracin-MD, MICs were higher using the broth microdilution method when compared to the agar dilution method. The two methods had the lowest agreement when using bacitracin-MD compared to the method agreements of other antimicrobials tested (only 34.3% of the C. perfringens tested within ±1 doubling dilution). Tylosin MICs were lower by the broth microdilution method but had better agreement between methods with 75.5% of the C. perfringens tested within ±1 doubling dilution. Good correlation between methods was found for virginiamycin, tetracycline, and erythromycin with 85.3, 76.5, 81.4% of the C. perfringens tested within ±1 doubling dilution, respectively. Differences in susceptibility to individual antimicrobials were found among the avian and porcine strains by both methods. For avian strains, bacitracin-MD, tylosin, and erthromycin MIC₉₀values had differences of at least four doubling dilutions between methods. There were biases toward higher bacitracin-MD and lower tylosin and erythromycin MIC₉₀values using the broth microdilution method. MIC₉₀values against porcine and ATCC strains were more comparable between methods for all five antimicrobials than those generated against avian strains but the biases were still present. Most animal-derived strains were inhibited by approved livestock feed levels of the antimicrobials. Caution should be used when evaluating the potential effectiveness of feed-based antimicrobials against C. perfringens when results are generated using an in vitro test that may not be in agreement with the reference agar dilution method.     

Edaravone directly reacts with singlet oxygen and protects cells from attack

AimsProtective effects of edaravone, an approved medicine for acute brain infarction in Japan, on cell death induced by singlet oxygen (¹O₂) were examined.Main methodThe ¹O₂ scavenging activity was examined by direct analysis of near-infrared luminescence in a cell-free system and by fluorospectrometry in the presence of cells. The protective effects of edaravone on ¹O₂-induced cell death were examined, using rat neuronal B50 cells. Cell death was evaluated by mitochondrial respiration (MTT assay), confocal microscopy and time-lapse imaging. The chemical reaction of edaravone with ¹O₂ was examined by production analysis using high performance liquid chromatography (HPLC).Key findingsWhen rose Bengal (RB) in D₂O was irradiated by a 514 nm laser beam, the signal of ¹O₂ was observed. Edaravone suppressed the ¹O₂ signal more potently than azide, a ¹O₂ scavenger. When B50 cells were irradiated by 525 nm green light in the RB solution, production of ¹O₂ and induction of cell death were observed. The fluorospectrometric study and the MTT assay revealed that 100–400 µM edaravone suppressed the ¹O₂ production and attenuated cell death in a concentration-dependent manner. Confocal microscopy and the time-lapse imaging revealed that edaravone prevented the impairment of membrane integrity and the progression of cell death induced by ¹O₂. The HPLC study revealed that edaravone chemically reacted with ¹O₂ and changed another compound.SignificanceSince ¹O₂ is possibly involved in post-ischemic neuronal damage, the clinically approved curative effects of edaravone on acute brain infarction might be attributed to its potent ¹O₂ scavenging activity.     

Potential of Chromium(III) Picolinate for Reproductive or Developmental Toxicity Following Exposure of Male CD-1 Mice Prior to Mating

Chromium(III) picolinate, [Cr(pic)₃], is a commonly used nutritional supplement in humans, which has also been approved for use in animals. Health concerns have arisen over the use of [Cr(pic)₃]. At high [Cr(pic)₃] doses, developmental toxicity tests in female mice have shown a higher litter incidence of split cervical arch in exposed fetuses, but this was not consistently reproducible. In the current study, male CD-1 mice were used to further assess the potential for reproductive or developmental toxicity. Four weeks prior to mating, the males were fed a diet providing 200 mg/kg/day [Cr(pic)₃] for comparison with untreated controls. Females were not treated. Each male was mated with two females, which were sacrificed on gestation day 17, and their litters were examined for adverse effects. Mating and fertility indices were not significantly altered by treatment. Male exposure to [Cr(pic)₃] also had no effect on prenatal mortality, fetal weight, or gross or skeletal morphology. These results suggest that paternal dietary exposure to chromium(III) picolinate has little potential for adverse reproductive effects, even at exposure levels considerably higher than expected human exposures from nutritional supplements (1 mg of Cr per day or less).     

Identification of old drugs as potential inhibitors of HIV-1 integrase – human LEDGF/p75 interaction via molecular docking

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, human protein Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. LEDGF/p75-HIV-1 IN interaction represents an attractive target for anti-HIV therapy. In this study, approved drugs were investigated for the finding of potential inhibitors on this target. Via molecular docking against the LEDGF/p75-binding pocket of HIV-1 IN, 26 old drugs were selected from the DrugBank and purchased for bioassays. Among them, eight, namely Atorvastatin, Bumetanide, Candesartan, Carbidopa, Diclofenac, Diflunisal, Eprosartan, and Sulindac, were identified as potential inhibitors of LEDGF/p75- HIV-1 IN interaction, whose IC₅₀ values ranged from 6.5?μM to 36.8?μM. In addition, Atorvastatin was previously reported to block HIV-1 replication and may have an important implication for the treatment of AIDS. Our results suggested a mechanism of action for the anti-HIV effects of Atorvastatin. This work provides a new example of inhibitors targeting protein-protein interaction and confirmed that old drugs were valuable sources for antiviral drug discovery.     

Expanding the structural diversity of diarylureas as multi-target tyrosine kinase inhibitors

Recently approved multi-target inhibitors of receptor tyrosine kinases (RTKs) have significantly improved the clinical treatment of cancers. A series of N,N′-diarylureas incorporated with aromatic heterocycle have been designed, synthesized and evaluated as novel multi-target RTK inhibitors. The preliminary biological evaluation indicated that several compounds exhibited comparable potency with Sorafenib. Among them, compound 6f was identified as the most potent multikinase inhibitor of EGFR, KDR and FGFR1 with IC₅₀ values of 14.83 nM, 21.57 nM, and 28.23 nM, respectively. These compounds expanded the structural diversity of diarylureas as RTK inhibitors. The results demonstrated that compound 6f could be served as novel lead compound for further development of multi-target RTK inhibitors.     

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC₅₀) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC₅₀, 12 nM) and L (IC₅₀, 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.