In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.

Antivitamins B12 -Some Inaugural Milestones.

The recently delineated structure- and reactivity-based concept of antivitamins B12 has begun to bear fruit by the generation, and study, of a range of such B12 -dummies, either vitamin B12 -derived, or transition metal analogues that also represent potential antivitamins B12 or specific B12 -antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B12 -chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B12 have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B12 -deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B12 to induce functional B12 -deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.

Drug repurposing to overcome resistance to various therapies for colorectal cancer.

Emergence of novel treatment modalities provides effective therapeutic options, apart from conventional cytotoxic chemotherapy, to fight against colorectal cancer. Unfortunately, drug resistance remains a huge challenge in clinics, leading to invariable occurrence of disease progression after treatment initiation. While novel drug development is unfavorable in terms of time frame and costs, drug repurposing is one of the promising strategies to combat resistance. This approach refers to the application of clinically available drugs to treat a different disease. With the well-established safety profile and optimal dosing of these approved drugs, their combination with current cancer therapy is suggested to provide an economical, safe and efficacious approach to overcome drug resistance and prolong patient survival. Here, we review both preclinical and clinical efficacy, as well as cellular mechanisms, of some extensively studied repurposed drugs, including non-steroidal anti-inflammatory drugs, statins, metformin, chloroquine, disulfiram, niclosamide, zoledronic acid and angiotensin receptor blockers. The three major treatment modalities in the management of colorectal cancer, namely classical cytotoxic chemotherapy, molecular targeted therapy and immunotherapy, are covered in this review.

Weak Interaction of the Antimetabolite Drug Methotrexate with a Cavitand Derivative.

Formation of inclusion complexes involving a cavitand derivative (as host) and an antimetabolite drug, methotrexate (as guest) was investigated by photoluminescence measurements in dimethyl sulfoxide solvent. Molecular modeling performed in gas phase reflects that, due to the structural reasons, the cavitand can include the methotrexate in two forms: either by its opened structure with free androsta-4-en-3-one-17α-ethinyl arms or by the closed form when all the androsta-4-en-3-one-17α-ethinyl arms play role in the complex formation. Experiments reflect enthalpy driven complex formation in higher temperature range while at lower temperature the complexes are stabilized by the entropy gain.

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Flow field-flow fractionation and multi-angle light scattering as a powerful tool for the characterization and stability evaluation of drug-loaded metal-organic framework nanoparticles.

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. Graphical abstract ᅟ.

Inhibition of mTOR/eIF4E by anti-viral drug ribavirin effectively enhances the effects of paclitaxel in oral tongue squamous cell carcinoma.

Upregulation of eIF4E is associated with poor clinical outcome in many human cancers and represents a potential therapeutic target. However, the function of eIF4E remains unknown in oral tongue squamous cell carcinoma (OTSCC). In this work, we show that ribavirin, an anti-viral drug, effectively augments sensitivity of OTSCC cells to paclitaxel via inhibiting mTOR/eIF4E signaling pathway. Ribavirin dose-dependently inhibits proliferation and induces apoptosis in SCC-9 and CAL27 cells. Combination of ribavirin and paclitaxel are more effective in inhibiting proliferation and inducing apoptosis in OTSCC cells. Importantly, the in vivo efficacy of ribavirin and its synergism with paclitaxel is confirmed by two independent OTSCC xenograft mouse models. Mechanistically, ribavirin significantly decreases mTOR/eIF4E signaling pathway in OTSCC cells via suppressing phosphorylation of Akt, mTOR, 4EBP1 and eIF4E. Overexpression of the phosphor-mimetic form of eIF4E (eIF4E S209D) but not the nonphosphorylatable form (eIF4E S209A) reverses the effects of ribavirin, confirming that eIF4E inhibition is the mechanism of action of ribavirin in OTSCC cells. In addition, eIF4E depletion significantly enhances the anti-proliferative and pro-apoptotic effects of paclitaxel, demonstrating the critical role of eIF4E in OTSCC cell response to paclitaxel. Our work is the first to demonstrate the efficacy of ribavirin as a single agent and synergism as combination with paclitaxel in OTSCC in vitro and in vivo. Our findings also demonstrate the therapeutic value of inhibiting eIF4E in OTSCC treatment.

Effect of nucleoside analogue antimetabolites on the structure of PEO-PPO-PEO micelles investigated by SANS.

The effect of three nucleoside analogue antimetabolites (5-fluorouracil, floxuridine, and gemcitabine) on the structure of Pluronic L62 copolymer micelles was investigated using small-angle neutron scattering. These antimetabolites used for cancer chemotherapy have analogous molecular structures but different molecular sizes and aqueous solubilities. It was found that the addition of the three antimetabolites slightly reduced the micellar size and aggregation number, and the micellar anisotropy. The added antimetabolites also changed the internal molecular distribution of the micelles as measured by the scattering length densities, resulting in enhanced hydration of the hydrophobic core region of the micelle. The strength of the effect was found to correlate with the molecular properties of the model drugs, i.e. a larger molecular size and a higher aqueous solubility lead to enhanced hydration of the micellar core.

EXPERIMENTAL ASSESSMENT OF THE SPECIFIC EFFICIENCY OF NEW METABOLIC DRUG RUNIKHOL ON MODELS OF NON-ALCOHOLIC FATTY LIVER DISEASE.

We present results of preclinical evaluation of the specific efficiency of new metabolic corrector runikhol on models of non-alcoholic fatty liver disease in laboratory rats (228 albino males of gray Wistar rats weighing 220 - 240 g). The drug based on succinic acid positively influences the key types of metabolism impaired under conditions of development of the metabolic syndrome. Results of research testify to high efficiency and good prospects of using runikhol in the treatment of metabolic syndrome accompanied by organ disorders.

Antivitamins B12--A Structure- and Reactivity-Based Concept.

B12 -antimetabolites are compounds that counteract the physiological effects of vitamin B12 and related natural cobalamins. Presented here is a structure- and reactivity-based concept of the specific 'antivitamins B12 ': it refers to analogues of vitamin B12 that display high structural similarity to the vitamin and are 'locked chemically' to prevent their metabolic conversion into the crucial organometallic B12 -cofactors. Application of antivitamins B12 to healthy laboratory animals is, thus, expected to induce symptoms of B12 -deficiency. Antivitamins B12 may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B12 -deficiency, and also in recognizing physiological roles of B12 that probably still remain to be discovered.

[Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

Effects of carbon monoxide on quality, nutrients and antioxidant activity of post-harvest jujube.

BACKGROUND: To effectively extend the shelf life of fresh jujube, carbon monoxide (CO), as a small molecular gas, was applied to fumigate fresh jujube. The quality and antioxidant activity of jujubes fumigated with carbon monoxide at concentrations of 0 (control), 5, 10, 20 and 40 µmol L⁻¹ for 1 h under ambient temperature were investigated. RESULTS: The jujube fumigated with 10 µmol L⁻¹ CO showed the best preserving effect amongst all samples. At 30 days, the decay incidence of jujube fumigated with 10 µmol L⁻¹ CO is only two-thirds of that of control sample; its red index and weight loss rate were 22.8% and 19.4% lower, and its firmness, soluble solids content (SSC) and acidity were 18.7%, 5.4% and 12.2% higher than that of control samples, respectively. Its vitamin C and total flavonoid contents were also the highest. However, no significant difference in total polyphenol content was found. The jujubes treated with 10 µmol L⁻¹ CO exhibited the highest antioxidant activity in terms of reducing power and 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl radical scavenging activity. However, the jujubes fumigated with 40 µmol L⁻¹ CO showed inferior characteristics compared with the control sample. CONCLUSION: Fumigating jujubes with proper concentration of CO probably is a potential novel method for post-harvest jujube preservation in the future.

Variability in tannin content, chemistry and activity in a diverse group of tannin containing sorghum cultivars.

BACKGROUND: Tannins are large polyphenolic polymers and are known to bind proteins, limiting their digestibility, but are also excellent antioxidants. Numerous studies investigating the functional properties of sorghum tannin have been conducted by comparing grain samples from different sorghum lines without considering the other intrinsic characteristics of the grain. The purpose of this study was to remove the confounding intrinsic factors present in the endosperm so the effect of the tannins could be evaluated utilizing a unique decortication/reconstitution procedure. RESULTS: The tannin content of the 14 cultivars tested ranged from 2.3 to 67.2 catechin equivalents. The bran fractions were studied for their impact on protein binding and antioxidant capacity. Protein digestibility by pepsin ranged from 8% to 58% at the highest tannin level addition. Protein binding ranged from 3.11 to 16.33 g blue bovine serum albumin kg⁻¹ bran. Antioxidant capacity ranged from 81.33 to 1122.54 µmol Trolox equivalents g⁻¹ bran. High-performance size-exclusion chromatography detailed molecular size distributions of the tannin polymers and relationship to tannin functionality. CONCLUSION: The tannin content and composition play a significant role in determining tannin functionality. These differences will allow for selections of high-tannin sorghums with consideration of the biological activities of the tannins.

Diarylheptanoid glycosides from Tacca plantaginea and their effects on NF-κB activation and PPAR transcriptional activity.

In the screening search for NF-κB inhibitory and PPAR transactivational agents from medicinal plants, a methanol extract of the whole plant of Tacca plantaginea and its aqueous fraction showed the significant activities. Bioassay-guided fractionation combined with repeated chromatographic separation of the aqueous fraction of the methanol extract of T. plantaginea resulted in the isolation of two new diarylheptanoid glycosides, plantagineosides A (1) and B (2), an unusual new cyclic diarylheptanoid glycoside, plantagineoside C (3), and three known compounds (4-6). Their structures were determined by extensive spectroscopic and chemical methods. Compounds 3-6 significantly inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC(50) values ranging from 0.9 to 9.4 µM. Compounds 1-6 significantly activated the transcriptional activity of PPARs in a dose-dependent manner, with EC(50) values ranging from 0.30 to 10.4 µM. In addition, the transactivational effects of compounds 1-6 were evaluated on three individual PPAR subtypes, including PPARα, γ, and ß(δ). Compounds 1-6 significantly enhanced the transcriptional activity of PPARß(δ), with EC(50) values in a range of 11.0-30.1 µM. These data provide the rationale for using T. plantaginea and its components for the prevention and treatment of inflammatory and metabolic diseases.

Optimization of process for reduction of antinutritional factors in edible cereal brans.

Reduction of various antinutritional factors in cereal brans by different treatments (microwave heating, dry heating and wet heating) were studied. There was significant difference (p ≤ 0.05) in reduction of antinutritional factors of treated cereal brans except for dry heating at low temperature. Microwave heating at 2450 MHz for 2.5 min resulted in 53.85%, 57.21%, 65.00% and 100% reduction in phytic acid, polyphenols, oxalates and saponins, respectively. Wet heating resulted in maximum reduction in trypsin inhibitor activity (83.07%) at 110 °C for 25 min. Processing treatment resulted in increase in bulk density and slight darkening of the brans. The most effective method of detoxifying most of the toxicants was microwave heating for 2.5 min, and therefore it could be exploited for making treated brans an ideal source for potential food application.

Tridentate ionic hydrogen-bonding interactions of the 5-fluorocytosine cationic dimer and other 5-fluorocytosine analogues characterized by IRMPD spectroscopy and electronic structure calculations.

Ionic hydrogen-bonding interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, along with the cationic (proton-bound) dimer have been characterized by infrared multiple-photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD action spectra, in combination with calculated spectra and relative energetics, indicate that it is most probable that predominantly a single isomer exists in each experiment. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest-energy isomer convincingly matches the experimental spectrum. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) involving a tridentate ionic hydrogen-bonding interaction. The three sites of intermolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significant calculated binding energy of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, suggests that a tridentate-proton-bound dimer was observed predominantly in the experiments. Comparison of the calculated relative Gibbs free energies (298 K) for this species and several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV.

Drug release and its relationship with kinetic and thermodynamic parameters of drug sorption onto starch acetate fibers.

Drug release and its relationship with kinetic and thermodynamic parameters of drug sorption onto starch acetate (SA) fibers have been studied using Diclofenac, 5-Fluorouracil (5-Fu), and Metformin as model drugs. The sorption method is more flexible and can avoid limitations or problems which occur with molten or dissolution methods. To understand drug release of sorption loading, kinetic and apparent thermodynamic parameters, such as diffusion coefficient, activation energy for diffusion, affinity, and sorption enthalpy and entropy, have been investigated. The quantitative relationship between drug release and drug-loading concentration, affinity, and activation energy for diffusion has been established to predict the initial burst and subsequent release of the drugs. Up to 12% of Diclofenac, based on the weight of SA, can be loaded onto fibers using the sorption method. Drugs with higher activation energy for diffusion, lower diffusion coefficients, and higher affinity for SA fiber, such as Diclofenac, are more suitable for sorption loading. It has also been found that elevated temperatures will achieve higher loading capacity and a more constant release rate.

Biocompatible and self-recoverable succinoglycan dialdehyde-crosslinked alginate hydrogels for pH-controlled drug delivery.

We fabricated polysaccharide-based hydrogels, which are biocompatible, self-recoverable and pH-sensitive. Succinoglycan dialdehyde (SGDA) was first synthesized from bacterial succinoglycan directly isolated from Sinorhizobium meliloti and then hydrazine-functionalized alginate (HZ-Alg) was prepared to form SGDA-crosslinked alginate hydrogels (SGDA/HZ-Alg) without any catalyst. Due to structural characteristics of SGDA, SGDA/HZ-Alg were effectively obtained in a short time even at low concentrations (0.94-1.57 wt%) where they exhibited self-recoverable and tunable rheological properties corresponding to efficiency of recovery from 93.2%-97.9%. Moreover, SGDA/HZ-Alg showed the pH-responsive degradation as well as pH-controlled release behavior for 5-fluorouracil. 5-Fluorouracil was released approximately 98 % at pH 2.0 within 12 h, but not completely released even after 24 h at pH 7.4. The WST-8 assay results also demonstrated that SGDA/HZ-Alg did not show any cytotoxicity against HEK-293 cells. Since the suggested hydrogels are biocompatible, rheologically self-recoverable and tunable, and pH-controllable, they would be potential biomaterials for the hydrogel-based drug delivery systems.

Acacetin, a flavone with diverse therapeutic potential in cancer, inflammation, infections and other metabolic disorders.

BACKGROUND: Acacetin is a di-hydroxy and mono-methoxy flavone present in various plants, including black locust, Damiana, Silver birch. Literature information revealed that acacetin exhibits an array of pharmacological potential including chemopreventive and cytotoxic properties in cancer cell lines, prevents ischemia/reperfusion/myocardial infarction-induced cardiac injury, lipopolysaccharide (LPS), 1-methyl-4-phenyl pyridinium ion (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced neuroinflammation, LPS and sepsis-induced lung injury, rheumatoid and collagen-induced arthritis, inhibit the microbial growth, obesity, viral-mediated infections as well as hepatic protection. PURPOSE: This review highlights the therapeutic potential of acacetin, with updated and comprehensive information on the biological sources, chemistry, and pharmacological properties along with the possible mechanism of action, safety aspects, and future research opportunities. STUDY DESIGN: The information was retrieved from various search engines, including Pubmed, SciFinder, Science direct, Inxight:drugs, Google scholar, and Meta cyc. RESULT: The first section of this review focuses on the detailed biological source of acacetin, chromatographic techniques used for isolation, chemical characteristics, the method for the synthesis of acacetin, and the available natural and synthetic derivatives. Subsequently, the pharmacological activities, including anti-cancer, anti-inflammatory, anti-viral, anti-microbial, anti-obesity, have been discussed. The pharmacokinetics data and toxicity profile of acacetin are also discussed. CONCLUSION: Acacetin is a potent molecule reported for its strong anti-inflammatory and anti-cancer activity, however further scientific evidence is essential to validate its potency in disease models associated with inflammation and cancer. There is limited information available for toxicity profiling of acacetin; therefore, further studies would aid in establishing this natural flavone as a potent candidate for research studies at clinical setup.

Design of polysaccharidic nano-in-micro soft agglomerates as primary oral drug delivery vehicle for colon-specific targeting.

Microencapsulation of polysaccharidic nanoparticles is met with nanoscale and biological performance changes. This study designs soft agglomerates as nanoparticle vehicle without nanoparticles undergoing physical processes that alter their geometry. The nanoparticles were made of high molecular weight chitosan/pectin with covalent 5-fluorouracil/folate. Nanoparticle aggregation vehicle was prepared from low molecular weight chitosan. The nanoparticles and aggregation vehicle were blended in specific weight ratios to produce soft agglomerates. Nanoparticles alone are unable to agglomerate. Adding aggregation vehicle (< 2 µm) promoted soft agglomeration with nanoparticles deposited onto its surfaces with minimal binary coalescence. The large and rough-surfaced aggregation vehicle promoted nanoparticles deposition and agglomeration. A rounder vehicle allowed assembly of nanoparticles-on-aggregation vehicle into agglomerates through interspersing smaller between larger populations. Soft agglomeration reduced early drug release, and was responsive to intracapsular sodium alginate coat to further sustain drug release. The soft agglomerates can serve as a primary oral colon-specific vehicle.