An efficient synthetic route for preparation of antimycobacterial wollamides and evaluation of their in vitro and in vivo efficacy

A convenient solid phase peptide synthetic (SPPS) route is reported for the preparation of antimycobacterial wollamides. The method is based on on-resin head-to-tail cyclization and is fast, efficient and amenable to automation. The in vitro antimycobacterial activities of the newly synthesized wollamides were evaluated against M. tuberculosis H37Rv (Mtb H37Rv). To assess their drug-likeness, in vitro pharmacokinetic (ADME) profiling was also performed. For wollamides with potent extracellular potency, intracellular activities and in vivo efficacy were determined. The results disclose the potent antimycobacterial (MIC_{Mtb H37Rv}?=?1.1?µM) and suitable drug-like properties of wollamide A (4b). Out of the synthesized wollamides, four compounds (4b–e) exhibited potent intracellular activities against Mtb H37Rv infected human macrophages (IC₅₀?=?0.2–1.3?µM). Results of in vivo blood exposure and efficacy assays for 4d and 4e are discussed.     

Natural products as sources of new fungicides (V): Design and synthesis of acetophenone derivatives against phytopathogenic fungi in vitro and in vivo

A series of acetophenone derivatives (10a–10i, 11, 12a–12g, 13a–13g, 14a–14d and 15a–15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure–activity relationship was summarized. Compounds 10a–10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC₅₀ values of 6.0–22.6?µg/mL, especially Cytospora sp. (IC₅₀?=?6.0?µg/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.     

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4′-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC₅₀) of 171 μg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC₅₀ of 260 μg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ∼50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita.     

Gastric intrinsic factor deficiency with combined GIF heterozygous mutations and FUT2 secretor variant

Several genome-wide association studies (GWAS) have identified a strong association between serum vitamin B12 and fucosyltransferase 2 (FUT2), a gene associated with susceptibility to Helicobacter pylori infection. Hazra et al. conducted a meta-analysis of three GWAS and found three additional loci in MUT, CUBN and TCN1. Other GWAS conducted in Italy and China confirmed the association for FUT2 gene. Alpha-2-fucosyltransferase (FUT2) catalyzes fucose addition to form H-type antigens in exocrine secretions. FUT2 non-secretor variant produces no secretion of H-type antigens and is associated with high-plasma vitamin B12 levels. This association was explained by the influence of FUT2 on H. pylori, which is a risk factor of gastritis, a main cause of vitamin B12 impaired absorption. However, we recently showed that H. pylori serology had no influence on FUT2 association with vitamin B12, in a large sample population, suggesting the involvement of an alternative mechanism. GIF is another gene associated with plasma levels of vitamin B12 and gastric intrinsic factor (GIF) is a fucosylated protein needed for B12 absorption. Inherited GIF deficiency produces B12 deficiency unrelated with gastritis. We report 2 families with heterozygous GIF mutation, 290T>C, M97T, with decreased binding affinity of GIF for vitamin B12 and one family with heterozygous GIF mutation 435_437delGAA, K145_N146delinsN and no B12 binding activity of mutated GIF. All cases with vitamin B12 deficit carried the FUT2 rs601338 secretor variant. Ulex europeus binding to GIF was influenced by FUT2 genotypes and GIF concentration was lower, in gastric juice from control subjects with the secretor genotype. GIF290C allele was reported in 5 European cases and no Africans among 1282 ambulatory subjects and was associated with low plasma vitamin B12 and anaemia in the single case bearing the FUT2 secretor variant. We concluded that FUT2 secretor variant worsens B12 status in cases with heterozygous GIF mutations by impairing GIF secretion, independently from H. pylori-related gastritis.     

Discovery of disubstituted xylylene derivatives as small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction

The Keap1–Nrf2–ARE system represents a crucial antioxidant defense mechanism that protects cells against reactive oxygen species. Targeting Keap1–Nrf2 protein–protein interaction (PPI) has become a promising drug target for several oxidative stress-related and inflammatory diseases including pulmonary fibrosis, chronic obstructive pulmonary disorder (COPD) and cancer chemoprevention. For the development of a potential therapeutic agent, drug-like properties and potency are important considerations. In this work, we focused on the modification of 4 as a lead through a molecular dissection strategy in an effort to improve its metabolic stability, leading to the discovery of a series of new disubstituted xylylene derivatives. The preliminary SAR of 9a indicated that compound 21a containing S-methylated acetate moieties exhibited comparable potency to the lead compound 4 in a fluorescent polarization assay but with improved metabolic stability in the presence of human liver microsomes.     

Sesquiterpene lactones from Vernonia scorpioides and their in vitro cytotoxicity

Fresh leaves of Vernonia scorpioides are widely used in Brazil to treat a variety of skin disorders. Previous in vivo studies with extracts of this species had also demonstrated a high antitumor potential. This paper reports isolation of four sesquiterpene lactones (hirsutinolides and glaucolides), together with diacetylpiptocarphol, 8-acetyl-13-etoxypiptocarphol, luteolin, apigenin, and ethyl caffeate from fresh leaves and flowers of Vernonia scorpioides. The hypothesis that hirsutinolide 3 is formed during extraction was verified theoretically using Density Functional Theory. The effects of isolated compounds on in vitro tumor cells were investigated, as well as their genotoxicity by means of an in vitro comet assay. The results indicate that glaucolide 2 and hirsutinolide 4 are toxic to HeLa cells. These compounds were genotoxic in vitro, a property that appears to be related to the presence of their epoxy groups, which has been a more reliable indication of toxicity than substitution on C-13 or the presence of α,β-unsaturated keto-groups. These results need to be replicated in vivo in order to ascertain their toxicity.     

Synthesis, in vitro and in vivo biological evaluation,COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives

The objective of this work was to obtain and evaluate anti-inflammatory in vitro, in vivo and in silico potential of novel indole-N-acylhydrazone derivatives. In total, 10 new compounds (3a–j) were synthesized in satisfactory yields, through a condensation reaction in a single synthesis step. In the lymphoproliferation assay, using mice splenocytes, 3a and 3b showed inhibition of lymphocyte proliferation of 62.7% (±3.5) and 50.7% (±2), respectively, while dexamethasone presented an inhibition of 74.6% (±2.4). Moreover, compound 3b induced higher Th2 cytokines production in mice splenocytes cultures. The results for COX inhibition assays showed that compound 3b is a selective COX-2 inhibitor, but with less potency when compared to celecoxib, and compound 3a not presented selectivity towards COX-2. The molecular docking results suggest compounds 3a and 3b interact with the active site of COX-2 in similar conformations, but not with the active site of COX-1, and this may be the main reason to the COX-2 selectivity of compound 3b. In vivo carrageenan-induced paw edema assays were adopted for the confirmation of the anti-inflammatory activity. Compound 3b showed better results in suppressing edema at all tested concentrations and was able to induce an edema inhibition of 100% after 5?h of carrageenan injection at the 30?mg?kg^?1 dosage, corroborating with the COX inhibition and lymphoproliferation results. I addition to our experimental results, in silico analysis suggest that compounds 3a and 3b present a well-balanced profile between pharmacodynamics and pharmacokinetics. Thus, our preliminary results revealed the potentiality of a new COX-2 selective derivative in the modulation of the inflammatory process.     

Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro

Barrett N. J., Smyth J. D. and Ong S. J. 1982. Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro. International Journal for Parasitology12: 315–322. Tetrathyridia of Mesocestoides corti, from the body cavity of mice, maintained in the laboratory by intraperitoneal infection, were used for in vitro culture. In an initial experiment, after 50 days asexual multiplication in vitro one tetrathyridium spontaneously segmented and developed into a sexually mature adult. Further experiments were carried out in an attempt to determine the conditions favouring segmentation and sexual differentiation. A combination of 5 or 10 ml liquid medium S1OE.H (basically composed of CMRL 1066 and foetal calf serum with supplements) changed every 3 days, in a Leighton tube (19 × 105 mm), rotated at 38°C and gassed with 10 or 20% CO₂, containing between 100 and 200 tetrathyridia, has proved to be most suitable so far. Numerous adult worms with normal male and female genitalia have been obtained in this system. However, segmentation is sporadic, rather than consistent and only a few shelled eggs with hooked oncospheres have so far been obtained, suggesting that impregnation and fertilization in vitro is not fully comparable with that in vivo.     

Cultivation in vitro of Schistosomatium douthitti (trematoda: Schistosomatidae)

Cultivation in vitro of Schistosomatium douthitti (Trematoda : Schistosomatidae). International Journal for Parasitology12: 541–545. We grew S. douthitti in vitro from the cercaria to pairing ovigerous adults, using methods and media previously reported for cultivation of Schistosoma mansoni. Growth of S. douthitti in vitro was much more rapid than that of S. mansoni, but the cultures achieved a similar stage of maximal development. Cultured S. douthitti worms were smaller than those from animals and eggs were inviable. We determined that females of this species, known to produce eggs in unisexual animal infections, will also do so in culture in the absence of males.     

In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues

Resveratrol is a natural polyphenol found mainly on red grapes and in red wine, pointed as an important anti-inflammatory/immunomodulatory molecule. However, its bioavailability problems have limited its use encouraging the search for new alternatives agents. Thus, in this study, we synthetize 12 resveratrol analogues (6 imines, 1 thioimine and 5 hydrazones) and investigated its cytotoxicity, antioxidant activity and in vitro anti-inflammatory/immunomodulatory properties. The most promising compounds were also evaluated in vivo. The results showed that imines presented less cytotoxicity, were more effective than resveratrol on DPPH scavenger and exhibited an anti-inflammatory profile. Among them, the imines with a radical in the para position, on the ring B, not engaged in an intramolecular hydrogen-interaction, showed more prominent anti-inflammatory activity modulating, in vivo, the edema formation, the inflammatory infiltration and cytokine levels. An immunomodulatory activity also was observed in these molecules. Thus, our results suggest that imines with these characteristics presents potential to control inflammatory disorders.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Discovery of tetrahydroindazoles as a novel class of potent and in vivo efficacious gamma secretase modulators

The identification and optimization of a novel series of centrally efficacious gamma secretase modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described. Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyridines were identified as structurally distinct novel series of GSMs. Representative compound BI-1408 ((R)-42) was demonstrated to be centrally efficacious in rats at a 30?mg/kg oral dose.     

Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells

Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could play a role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene expression is known to modify inflammation, a mechanistic role in viral susceptibility and clearance has yet to be elucidated. Therefore, we utilized differentiated human nasal epithelial cells (NEC) and an enzymatic virus-like particle entry assay, to examine the role Nrf2-dependent gene expression has on viral entry and replication. Herein, lentiviral vectors that express Nrf2-specific short hairpin (sh)-RNA effectively decreased both Nrf2 mRNA and Nrf2 protein expression in transduced human NEC from healthy volunteers. Nrf2 knockdown correlated with a significant increase in influenza virus entry and replication. Conversely, supplementation with the potent Nrf2 activators sulforaphane (SFN) and epigallocatechin gallate (EGCG) significantly decreased viral entry and replication. The suppressive effects of EGCG on viral replication were abolished in cells with knocked-down Nrf2 expression, suggesting a causal relationship between the EGCG-induced activation of Nrf2 and the ability to protect against viral infection. Interestingly, the induction of Nrf2 via nutritional supplements SFN and EGCG increased antiviral mediators/responses: RIG-I, IFN-β, and MxA at baseline in the absence of infection. Our data indicate that there is an inverse relationship between the levels of Nrf2 expression and the viral entry/replication. We also demonstrate that supplementation with Nrf2-activating antioxidants inhibits viral replication in human NEC, which may prove to be an attractive therapeutic intervention. Taken together, these data indicate potential mechanisms by which Nrf2-dependent gene expression regulates susceptibility to influenza in human epithelial cells.     

Ornithine decarboxylase in Phycomyces: In vitro and in vivo properties

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5′-phosphate are 90 and 0.37 μm, respectively. Putrescine is a potent competitive inhibitor with a K_i of 75 μm. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.     

Discovery of novel,potent, and orally bioavailable pyrido[2,3-d][1]benzazepin-6-one antagonists for parathyroid hormone receptor 1

Structural modification of a 1,4-benzodiazepin-2-one-based PTHR1 antagonist 5, a novel type of PTHR1 antagonist previously synthesized in our laboratories, yielded compound 10, which had better chemical stability than compound 5. Successive optimization of the lead 10 improved aqueous solubility, metabolic stability, and animal pharmacokinetics, culminating in the identification of DS37571084 (12). Our study paves the way for the discovery of novel and orally bioavailable PTHR1 antagonists.     

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-x_L in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the R_S versus S_S sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-x_L.     

Dinuclear copper(II) complexes containing 6-(benzylamino)purines as bridging ligands: Synthesis, characterization, and in vitro and in vivo antioxidant activities

A series of dinuclear copper(II) complexes involving 6-(benzylamino)purine derivatives, (HL_n), as bridging ligands were synthesized, characterized and tested for both their in vitro and in vivo antioxidant activities. Based on results of elemental analyses, temperature dependence of magnetic susceptibility measurements, UV-vis, FTIR, EPR, NMR and MALDI-TOF mass spectroscopy, conductivity measurements and thermal analyses, the complexes with general compositions of [Cu₂(μ-HL_n)₄Cl₂]Cl₂ · 2H₂O (1-4) and [Cu₂(μ-HL_n)₂(μ-Cl)₂Cl₂] (5-7) were prepared {where n = 1-4; HL₁ = 6-[(2-methoxybenzyl)amino]purine, HL₂ = 6-[(4-methoxybenzyl)amino]purine, HL₃ = 6-[(2,3-dimethoxybenzyl)amino]purine and HL₄ = 6-[(3,4-dimethoxybenzyl)amino]purine}. In the case of complexes 2, 3, 5 and 7, the antioxidant activities were studied by both in vitro {superoxide dismutase-mimic (SOD-mimic) activity} and in vivo {cytoprotective effect against the alloxan-induced diabetes (antidiabetic activity)} methods. The obtained IC₅₀ value of the SOD-mimic activity for the complex 5 (IC₅₀ = 0.253 μM) was shown to be even better than that of the native bovine Cu,Zn-SOD enzyme (IC₅₀ = 0.480 μM), used as a standard. As for the antidiabetic activity, the pretreatment of mice with complexes 3 and 7 led to the complete elimination of cytotoxic attack of alloxan and its free radical metabolites, used as a diabetogenic agent. The cytoprotective effect of these compounds was proved by the preservation of the initial blood glucose levels of the pretreated animals, as against the untreated control group.     

Synthesis, characterization and in vitro anti-cancer activity of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters

N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2-7) were prepared by coupling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGlyGly(OEt) and GlyGlyGlyGly(OEt) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The compounds were characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The anti-proliferative effects of the ortho derivatives 2 and 5 were measured in vitro against H1299 lung cancer cells and both gave IC₅₀ values greater than 50 μM. Therefore, extending the length of the peptide chain had a negative effect on activity, relative to N-(ferrocenyl)benzoyl amino acid and dipeptide derivatives.     

In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

E3 ubiquitin (Ub) ligases play diverse roles in cellular regulation in eukaryotes. Three homologous AtRmas (AtRma1, AtRma2, and AtRma3) were recently identified as ER-localized Arabidopsis homologs of human RING membrane-anchor E3 Ub ligase. Here, auxin binding protein 1 (ABP1), one of the auxin receptors in Arabidopsis, was identified as a potential substrate of AtRma2 through a yeast two-hybrid assay. An in vitro pull-down assay confirmed the interaction of full-length AtRma2 with ABP1. AtRma2 was transiently expressed in tobacco (Nicotiana benthamiana) plants through an Agrobacterium-mediated infiltration method and bound ABP1 in vivo. In vitro ubiquitination assays revealed that bacterially-expressed AtRma2 ubiquitinated ABP1. ABP1 was poly-ubiquitinated in tobacco cells and its stability was significantly increased in the presence of MG132, a 26S proteasome inhibitor. This suggests that ABP1 is controlled by the Ub/26S proteasome system. Therefore, AtRma2 is likely involved in the cellular regulation of ABP1 expression levels.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.