Novel Cerium(IV) Coordination Compounds of Monensin and Salinomycin.

The largely uncharted complexation chemistry of the veterinary polyether ionophores, monensic and salinomycinic acids (HL) with metal ions of type M4+ and the known antiproliferative potential of antibiotics has provoked our interest in exploring the coordination processes between MonH/SalH and ions of Ce4+. (1) Methods: Novel monensinate and salinomycinate cerium(IV)-based complexes were synthesized and structurally characterized by elemental analysis, a plethora of physicochemical methods, density functional theory, molecular dynamics, and biological assays. (2) Results: The formation of coordination species of a general composition [CeL2(OH)2] and [CeL(NO3)2(OH)], depending on reaction conditions, was proven both experimentally and theoretically. The metal(IV) complexes [CeL(NO3)2(OH)] possess promising cytotoxic activity against the human tumor uterine cervix (HeLa) cell line, being highly selective (non-tumor embryo Lep-3 vs. HeLa) compared to cisplatin, oxaliplatin, and epirubicin.

Synthesis and evaluation of antibacterial and trypanocidal activity of derivatives of monensin A.

The synthesis and biological evaluation of eleven derivatives of the natural polyether ionophore monensin A (MON), modified at the C-26 position, is presented. Eight urethane and three ester derivatives were tested for their antimicrobial activity against different strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. In addition, their antiparasitic activity was also evaluated with bloodstream forms of Trypanosoma brucei. The majority of the modified ionophores were active against a variety of Gram-positive bacterial strains, including methicillin-resistant S. epidermidis, and showed better antibacterial activity than the unmodified MON. The phenyl urethane derivative of MON exhibited the most promising antibacterial activity of all tested compounds, with minimal inhibitory concentration values of 0.25-0.50 µg/ml. In contrast, none of the MON derivatives displayed higher antitrypanosomal activity than the unmodified ionophore.

Polyether Cyclization Cascade Alterations in Response to Monensin Polyketide Synthase Mutations.

The targeted manipulation of polyketide synthases has in recent years led to numerous new-to-nature polyketides. For type I polyketide synthases the response of post-polyketide synthases (PKS) processing enzymes onto the most frequently polyketide backbone manipulations is so far insufficiently studied. In particular, complex processes such as the polyether cyclisation in the biosynthesis of ionophores such as monensin pose interesting objects of research. We present here a study of the substrate promiscuity of the polyether cyclisation cascade enzymes in monensin biosynthesis in the conversion of redox derivatives of the nascent polyketide chain. LC-HRMS/MS2 -based studies revealed a remarkable flexibility of the post-PKS enzymes. They acted on derivatized polyketide backbones based on the three possible polyketide redox states within two different modules and gave rise to an altered polyether structure. One of these monensin derivatives was isolated and characterized by 2D-NMR spectroscopy, crystallography, and bioactivity studies.

Expanding the antibacterial selectivity of polyether ionophore antibiotics through diversity-focused semisynthesis.

Polyether ionophores are complex natural products capable of transporting cations across biological membranes. Many polyether ionophores possess potent antimicrobial activity and a few selected compounds have the ability to target aggressive cancer cells. Nevertheless, ionophore function is believed to be associated with idiosyncratic cellular toxicity and, consequently, human clinical development has not been pursued. Here, we demonstrate that structurally novel polyether ionophores can be efficiently constructed by recycling components of highly abundant polyethers to afford analogues with enhanced antibacterial selectivity compared to a panel of natural polyether ionophores. We used classic degradation reactions of the natural polyethers lasalocid and monensin and combined the resulting fragments with building blocks provided by total synthesis, including halogen-functionalized tetronic acids as cation-binding groups. Our results suggest that structural optimization of polyether ionophores is possible and that this area represents a potential opportunity for future methodological innovation.

Calcium salts of long-chain fatty acids from linseed oil decrease methane production by altering the rumen microbiome in vitro.

Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH4) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH4 production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH4 production, and rumen microbiome in vitro. We compared five treatments: three CSFA concentrations-0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis-15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P < 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P < 0.05), DM digestibility-adjusted CH4 production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P < 0.05). The genera Ruminobacter, Succinivibrio, Succiniclasticum, Streptococcus, Selenomonas.1, and Megasphaera, which are related to propionate production, were increased (P < 0.05), while Methanobrevibacter and protozoa counts, which are associated with CH4 production, were decreased in FAH, compared with CON (P < 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH4 production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH4 production, CSFA may be a promising candidate for reduction of CH4 emission from ruminants.

Synthesis and Anticancer Activity of Dimeric Polyether Ionophores.

Polyether ionophores represent a group of natural lipid-soluble biomolecules with a broad spectrum of bioactivity, ranging from antibacterial to anticancer activity. Three seem to be particularly interesting in this context, namely lasalocid acid, monensin, and salinomycin, as they are able to selectively target cancer cells of various origin including cancer stem cells. Due to their potent biological activity and abundant availability, some research groups around the world have successfully followed semi-synthetic approaches to generate original derivatives of ionophores. However, a definitely less explored avenue is the synthesis and functional evaluation of their multivalent structures. Thus, in this paper, we describe the synthetic access to a series of original homo- and heterodimers of polyether ionophores, in which (i) two salinomycin molecules are joined through triazole linkers, or (ii) salinomycin is combined with lasalocid acid, monensin, or betulinic acid partners to form 'mixed' dimeric structures. Of note, all 11 products were tested in vitro for their antiproliferative activity against a panel of six cancer cell lines including the doxorubicin resistant colon adenocarcinoma LoVo/DX cell line; five dimers (14-15, 17-18 and 22) were identified to be more potent than the reference agents (i.e., both parent compound(s) and commonly used cytostatic drugs) in selective targeting of various types of cancer. Dimers 16 and 21 were also found to effectively overcome the resistance of the LoVo/DX cancer cell line.

Sorption and abiotic transformation of monensin by iron and manganese oxides.

Monensin, an ionophore antibiotic, is commonly administered as a feed additive to cattle and poultry. A large percentage of the administered dose is excreted in animal waste, which is often applied to agricultural fields as fertilizer. The objective of this work is to gain insight into the fate of monensin in soil by investigating the interactions between monensin and common soil minerals, including sorption and transformation to unmonitored partial oxidation products. Batch sorption experiments across varying conditions (i.e., pH, ionic strength) and desorption experiments (i.e., methanol, PO43-, methyl tert-butyl ether) were used to determine the extent to which a selection of common redox-active soil minerals [birnessite (δ-MnO2), goethite (α-FeOOH), hematite (α-Fe2O3)] can bind and transform monensin. Monensin was bound by hematite (pH < 7.5, up to 7.5 mmol kg-1), goethite (pH < 7.5, up to 3.4 mmol kg-1), and birnessite (pH < 7, up to 0.1 mmol kg-1). Combined sorption and transformation were the greatest for hematite and the lowest for birnessite. Sorption to hematite was more reversible than to goethite. Each desorption from goethite recovered <10% of sorbed monensin, whereas desorption from hematite recovered up to 69% of sorbed monensin, dependent on the solution. The potential for iron and manganese (hydr)oxides to abiotically transform monensin through reductive dissolution to partial oxidation products was evaluated by mass spectral analysis following sorption experiments. Additionally, the dominant sorption mechanism was inferred through ATR-FTIR spectroscopy, via examination of the carboxylate peak separation differences, on goethite and hematite to be bridging bidentate.

Prediction of Transformation Products of Monensin by Electrochemistry Compared to Microsomal Assay and Hydrolysis.

The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.

Antiproliferative activity of ester derivatives of monensin A at the C-1 and C-26 positions.

Monensin A (MON) is a polyether ionophore antibiotic, which shows a wide spectrum of biological activity, including anticancer activity. A series of structurally diverse monensin esters including its C-1 esters (1-9), C-26-O-acetylated derivatives (10-15), and lactone (16) was synthesized and for the first time evaluated for their antiproliferative activity against four human cancer cell lines with different drug-sensitivity level. All of the MON derivatives exhibited in vitro antiproliferative activity against cancer cells at micromolar concentrations. The majority of the compounds was able to overcome the drug resistance of LoVo/DX and MES-SA/DX5 cell lines. The most active compounds proved to be MON C-26-O-acetylated derivatives (10-15) which exhibited very good resistance index and high selectivity index.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

Chemotherapeutic Potential of Monensin as an Anti-microbial Agent.

Monensin is a lipid-soluble naturally occurring bioactive ionophore produced by Streptomyces spp. Its antimicrobial activity is mediated by its ability to exchange Na+ and K+ ions across the cell membrane thereby disrupting ionic gradients and altering cellular physiology. It is approved by Food and Drug Administration as a veterinary antibiotic to treat coccidiosis. Besides veterinary applications, monensin exhibits a broad spectrum activity against opportunistic pathogens of humans such as bacteria, virus, fungi and parasites in both drug sensitive and resistant strains. This ionophore can selectively kill pathogens with negligible toxic effect on mammalian cells. In this review, we discuss the therapeutic potential of monensin as a new broad-spectrum anti-microbial agent that warrants further studies for clinical use.

One-pot synthesis and antiproliferative activity of novel double-modified derivatives of the polyether ionophore monensin A.

Monensin A (MON) is a polyether ionophore antibiotic, which shows a wide spectrum of biological activity. New MON derivatives such as double-modified ester-carbonates and double-modified amide-carbonates were obtained by a new and efficient one-pot synthesis with triphosgene as the activating reagent and the respective alcohol or amine. All new derivatives were tested for their antiproliferative activity against two drug-sensitive (MES-SA, LoVo) and two drug-resistant (MES-SA/DX5, LoVo/DX) cancer cell lines, and were also studied for their antimicrobial activity against different Staphylococcus aureus and Staphylococcus epidermidis bacterial strains. For the first time, the activity of MON and its derivatives against MES-SA and MES-SA/DX5 were evaluated.

In vitro activity of salinomycin and monensin derivatives against Trypanosoma brucei.

BACKGROUND: African trypanosomes are the causative agents of sleeping sickness in humans and nagana disease in livestock animals. As the few drugs available for treatment of the diseases have limited efficacy and produce adverse reactions, new and better tolerated therapies are required. Polyether ionophores have been shown to display anti-cancer, anti-microbial and anti-parasitic activity. In this study, derivatives of the polyether ionophores, salinomycin and monensin were tested for their in vitro activity against bloodstream forms of Trypanosoma brucei and human HL-60 cells. RESULTS: Most polyether ionophore derivatives were less trypanocidal than their corresponding parent compounds. However, two salinomycin derivatives (salinomycin n-butyl amide and salinomycin 2,2,2-trifluoroethyl ester) were identified that showed increased anti-trypanosomal activity with 50 % growth inhibition values in the mid nanomolar range and minimum inhibitory concentrations of below 1 µM similar to suramin, a drug used in the treatment of sleeping sickness. In contrast, human HL-60 cells were considerably less sensitive towards all polyether ionophore derivatives. The cytotoxic to trypanocidal activity ratio (selectivity) of the two promising compounds was greater than 250. CONCLUSIONS: The data indicate that polyether ionophore derivatives are interesting lead compounds for rational anti-trypanosomal drug development.

Estimation of environmentally relevant chemical properties of veterinary ionophore antibiotics.

Monensin (MON) and salinomycin (SAL), known as polyether ionophore antibiotics (IPAs), are extensively used in livestock industry and can enter the environment via animal manure and agricultural runoff. Although some studies have investigated the environmental fate and transformation of IPAs, the lack of information on IPAs' aqueous-phase chemical properties is a major hindrance for further in-depth research. This study was able to experimentally determine the acidity constants (pKa), metal-complex dissociation constants (Kdiss), and intrinsic aqueous solubility of MON species, and some of these properties of SAL. The pKa value of MON was found to be 4.5, close to other aliphatic carboxylic acids and the predicted value by the computer program ChemAxon. The metal-complex dissociation constants of MON were estimated to be 0.058 and 0.573 with sodium ion (Na(+)) and potassium ion (K(+)), respectively. The Kdiss value of SAL with sodium ion was found to be 1.31. Compared to the previous values determined in organic solvents, the Kdiss of MON in aqueous phase are several orders of magnitude higher but maintain the same relative selectivity toward metal ions (Na(+) versus K(+)). The determined pKa and Kdiss values were also used to assess the aqueous solubility limits of different IPA species under different pH and metal ion concentrations. Results from this study provide more accurate information for the properties of IPAs. The obtained constants can be applied to predict the speciation of IPAs in various aquatic systems and help shed light on the environmental fate of IPAs.

Comparative assessment of the effects of salinomycin and monensin on the biodistribution of lead and some essential metal ions in mice, subjected to subacute lead intoxication.

In this study, we present a comparative assessment of the effects of two polyether ionophorous antibiotics (monensin and salinomycin) on the concentrations of lead (Pb), cooper (Cu), zinc (Zn) and iron (Fe) in the kidneys, spleen, liver and brain of Pb-intoxicated animals. Our data demonstrated that the intoxication of ICR male mice with Pb salt resulted in a significant accumulation of Pb in all studied organs of the mice compared to the untreated control animals. The biodistribution of the toxic metal was in the order kidneys>spleen>liver>brain. The treatment of the Pb-intoxicated animals with tetraethylammonium salts of monensic and salinomycinic acids significantly decreased the concentration of the toxic metal ion compared to the toxic control. The effect varied in the interval 38% (for kidneys) to 52% (for brain) compared to the toxic control group (Pb). The tetraethylammonium salt of salinomycinic acid was more effective in reducing the Pb concentration in the brain of the Pb-treated mice compared to monensin. Pb-intoxication did not affect significantly the Zn endogenous concentration compared to the normal values. The treatment of ICR male mice with Pb-salt decreased the Cu concentration in the spleen and increased the Cu concentration in the liver compared to the untreated control animals. The detoxification of the Pb-intoxicated mice with tetraethylammonium salts of salinomycinic and monensic acids restored the Cu concentration in the spleen, but did not affect the Cu levels in the liver. The Pb-intoxication of the ICR mice resulted in a significant decrease of the Fe-concentration in the spleen and liver compared to the untreated control animals. The administration of the tetraethylammonium salts of salinomycinic and monensic acids to the Pb-treated animals restored the levels of Fe in both organs.

Anti-proliferative activity of Monensin and its tertiary amide derivatives.

New tertiary amide derivatives of polyether ionophore Monensin A (MON) were synthesised and their anti-proliferative activity against cancer cell lines was studied. Very high activity (IC50=0.09 µM) and selectivity (SI=232) of MON against human biphenotypic myelomonocytic leukemia cell line (MV4-11) was demonstrated. The MON derivatives obtained exhibit interesting anti-proliferative activity, high selectivity index and also are able to break the drug-resistance of cancer cell line.

Synthesis and Antiproliferative Activity of Silybin Conjugates with Salinomycin and Monensin.

Aiming at development of multitarget drugs for the anticancer treatment, new silybin (SIL) conjugates with salinomycin (SAL) and monensin (MON) were synthesized, in mild esterification conditions, and their antiproliferative activity was studied. The conjugates obtained exhibit anticancer activity against HepG2, LoVo and LoVo/DX cancer cell lines. Moreover, MON-SIL conjugate exhibits higher anticancer potential and better selectivity than the corresponding SAL-SIL conjugate.

Antiproliferative Activity of Polyether Antibiotic--Cinchona Alkaloid Conjugates Obtained via Click Chemistry.

A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained by the Cu(I)-catalysed 1,3-dipolar Huisgen cycloaddition (click chemistry) of respective N-propargyl amides of salinomycin or monensin with four different Cinchona alkaloid derived azides. In vitro antiproliferative activity of these conjugates evaluated against three cancer cell lines (LoVo, LoVo/DX, HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00 µm) and appeared to be less toxic and more selective against normal cells than two standard anticancer drugs.

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester.

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczynski et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108-2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.

Acidocalcisome is required for autophagy in Trypanosoma brucei.

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca(2+) and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.