Level Of Growth Inhibition Research Articles

Synthesis and Preclinical Evaluation of Small-Molecule Prostate-Specific Membrane Antigen-Targeted Abiraterone Conjugate.

Prostate cancer is the second most common type of cancer among men. The main method of its treatment is androgen deprivation therapy, which has a wide range of side effects. One of the solutions to this challenge is the targeted delivery of drugs to prostate cancer cells. In this study, we performed the synthesis of a novel small-molecule PSMA-targeted conjugate based on abiraterone. Cytotoxicity, the induction of intracellular reactive oxygen species, and P450-cytochrome species inhibition were investigated for this conjugate PSMA-abiraterone. The conjugate demonstrated a preferential effect on prostate tumor cells, remaining inactive at up to 100 µM in human fibroblast cells. In addition, it revealed preferential efficacy, specifically on PSMA-expressing lines with a 65% tumor growth inhibition level on 22Rv1 (PSMA+) xenografts after 14-fold oral administration of PSMA-Abi at a single dose of 500 mg/kg (7.0 g/kg total dose) was observed. This compound showed significantly reduced acute toxicity with comparable efficacy compared to AbiAc.

Allelopathic Activity of Annona reticulata L. Leaf Extracts and Identification of Three Allelopathic Compounds for the Development of Natural Herbicides

Using plant-based allelopathic compounds might be a potent substitute to help mitigate the effects of synthetic herbicides. Annona reticulata L. is often planted for its fruit in residential gardens. This plant is well-documented for its diverse ethnomedicinal uses. However, there is no information in the literature on the allelopathic potential of A. reticulata leaves. Therefore, the allelopathic potential and relevant allelopathic compounds of A. reticulata leaves were investigated in this study. The bioassays were carried out using a completely randomized experimental layout (CRD), and the resulting data were analyzed using one-way ANOVA at p ≤ 0.05. Aqueous methanol extracts of A. reticulata leaves significantly inhibited the growth of three dicots and three monocots (Lepidium sativum L., Medicago sativa L., Lactuca sativa L., Echinochloa crus-galli (L.) P. Beauv., Lolium multiflorum Lam., and Phleum pratense L., respectively). The level of growth inhibition was proportional to the A. reticulata extract concentration. Three compounds were purified through different chromatographic steps, and their structures were determined using spectroscopy and identified as loliolide, 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one, and 3,4-dihydroxyphenylethanol. The 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one had the greatest effect on suppressing cress root growth, while loliolide had the greatest effect on suppressing timothy shoot growth. The values for 50% seedling growth suppression showed that the compound with the maximum inhibitory activity was loliolide, followed by 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one and 3,4-dihydroxyphenylethanol. Therefore, this result suggests that the three compounds might be responsible for the allelopathic effects of A. reticulata leaf extracts, and these compounds have the potential to be used to develop effective bioherbicides.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

Abstract 1008: Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells

Abstract Assessment of chemosensitivity and resistance varies with cancer cell of origin, drug and culture conditions. However, it is also not uncommon to find multiple different IC50 values for the same chemotherapeutic treatments acting on the same tumor cell lines. The aim of this research was to investigate this discrepancy in phenotypes and elucidate relevant variables that influence them. We assessed chemosensivity as a function of tumor cell density by plating TOV-21G ovarian, HT29 colorectal, and U2OS osteosarcoma cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin, 5-fluorouracil (5-FU), and etoposide, respectively. CellTiter Glo assays were performed at 72 hours of treatment to determine IC50. Cisplatin IC50 for TOV-21G cells plated at 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, and 500 cells/well were 12.00 μM, 5.47 μM, 3.16 μM, 2.45 μM, 2.54 μM, 2.83 μM, and 2.66 μM, respectively. Etoposide IC50 for U2OS cells plated at the same seeding densities were 6.50 μM, 5.19 μM, 3.23 μM, 2.98 μM, 2.49 μM, 2.13 μM, and 1.88 μM, respectively. IC50 values for HT29 cells treated with 5-FU at 2,000, 1,000, and 500 cells/well were 35.89 μM, 19.74 μM, and 13.42 μM, respectively. TOV-21G, U2OS, and HT29 cells all showed differences in IC50 values depending on cell plating density. TOV-21G and U2OS cells both showed a gradual decrease and plateau in IC50 as the cancer cell density decreased from 50,000 cells/well to 500 cells/well. HT29 cells displayed a sharper IC50 decrease as the cell density decreased from 2,000 cells/well to 500 cells/well. Our experiments demonstrate that IC50 values are not static at 72 hours and that tumor cell density is an important variable in influencing observed tumor resistance to traditionally effective drugs. As the density of cancer cells increases, the cells develop more resistance to the associated chemotherapy and require higher quantities of drug to achieve the same level of growth inhibition. We are currently exploring underlying molecular mechanisms that may influence these changes in IC50 including cell survival signaling proteins, cell-cell contact or growth factors that may influence cell density-related chemoresistance. Citation Format: Thomas Brown, Ujwal Punyamurtula, Jill Strandberg, Wafik S. El-Deiry. Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1008.

Vesicle transport related protein Synaptotagmin-1 mediates paraquat transport to antagonize paraquat toxicity

In this study, A549/PQ cells with moderate resistance to paraquat (PQ) were obtained by treating A549 cells with PQ, their growth rate was slowed down, the accumulation concentration of PQ and the levels of growth inhibition, injury and early apoptosis induced by PQ were significantly lower than those of parental A549 cells. Microarray screening and RT-qPCR detection found that Synaptotagmin-1 (SYT1) expression in drug-resistant cells was significantly increased, and PQ further enhanced its expression. After inhibiting SYT1 expression in A549/PQ cells, cell viability, intracellular PQ concentration and the expression of Bcl-2, SNAP25 and RAB26 were significantly reduced, while the mortality, early apoptosis rate and Bax expression were significantly increased. In vivo experiments also further showed that PQ promoted the expression of SYT1, SNAP25 and RAB26 in PQ-poisoned mice; when inhibiting SYT1 expression, PQ concentration in lung tissues was significantly increased, and the levels of lung injury and apoptosis were also significantly enhanced, while the expression of SNAP25 and RAB26 was significantly reduced. This indicates that PQ poisoning leads to compensatory up-regulation of vesicle transport related proteins such as SYT1 in vivo, thereby promoting PQ transmembrane transport, and then reducing the pulmonary accumulation of PQ and PQ-caused lung injury.

Anti-Proliferative and Anti-Telomerase Effects of Blackberry Juice and Berry- Derived Polyphenols on HepG2 Liver Cancer Cells and Normal Human Blood Mononuclear Cells.

Previous studies have provided strong evidence for the anticancer activity of berry fruits. In this study, we investigated the effects of blackberry juice and three berry- polyphenolic compounds on cell proliferation and telomerase activity in human hepatoma HepG2 and normal peripheral blood mononuclear cells (PBMCs). The cell viability and telomerase activity were measured by MTT and TRAP assay, respectively. Berry effects on the expression of genes were determined by quantitative RT-PCR assay. Blackberry, gallic acid, and resveratrol inhibited proliferation of both HepG2 and PBMC cells in a dosedependent manner. Resveratrol was more effective than gallic acid for reducing the viability of HepG2 cells, but both showed the same level of growth inhibition in PBMC cells. Berry, resveratrol, and gallic acid significantly inhibited telomerase activity in HepG2 cells. The antiproliferative effect of berry was associated with apoptotic DNA fragmentation. Gallic acid was more effective for reducing telomerase activity than resveratrol, but anthocyanin moderately increased telomerase activity in cancer cells. Telomerase activity was induced by all three polyphenols in PBMCs. Overall, Krumanin chloride was more effective to induce telomerase than gallic acid and resveratrol in PBMC cells. There was no significant difference in hTERT, hTR, and Dnmts expressions between berry treated and the control untreated HepG2 cells. But, a significant downregulation of HDAC1 and HDAC2 and upregulation of SIRT1 were observed in berry-treated cells. These data indicate that the berry anticancer effect is associated with antitelomerase activity and changes in HDACs expression. The data also suggest that berry antitelomerase activity is mainly related to its gallic acid and resveratrol, but not anthocyanin content.

Evaluation of Anti-Colorectal Cancer Activity in Methanolic Extract of Coleus forskohlii Root

In a multistage process that involves moving from a precancerous lesion to a malignant tumour state, cancer develops when normal cells undergo the change into tumour cells. Colon cancer, also known as colorectal cancer (CRC), is the third most common and second-deadliest malignant tumour in the world. The current study's objective was to assess the anticancer potential of a methanolic extract of Coleus forskohlii root on the cancer cell line HT-29 using the MTT assay. A variety of assay methods were used, including morphological investigations, cell growth inhibition, and cell viability. At various concentrations (12.5, 25, 50, 100, and 200 µg/ml), the Coleus forskohlii extract was tested against the HT-29 cell line for its ability to inhibit cell proliferation. This study revealed that when concentrations rise the cell growth inhibition levels rise. The conventional medicine Doxorubicin had a growth inhibition of 72.70% at a dose of 5 µg/ml while the lowest growth inhibition was determined to be 9.55% at 12.5 µg/ml and the highest growth inhibition was 71.16% at 200 µg/ml. IC50 was greater than 123.90 µg/ml. According to the study's findings, a human colon adenocarcinoma cell line could be resistant to the anti-colorectal cancer effects of Coleus forskohlii root methanolic extract (HT-29).

The Effect of Andrographis paniculata Nees on Oxidative Stress and Parasitemia Levels of Plasmodium berghei Infected Rats

Background: During malaria infection, oxidative stress arises due to the high metabolic rate of the multiplying parasite within the erythrocyte. Malondialdehyde (MDA), a product of lipid peroxidation and glutathione (GSH) has been suggested as a biomarker of oxidative stress. The Ethyl acetate (EA) fraction from the ethanol extract of Andrographis paniculata was shown to inhibit Plasmodium berghei in vivo. However, the antimalarial mechanism of the EA fraction, specifically on oxidative stress has not been investigated previously. Therefore, this study aimed to investigate the effects of the EA fraction on parasitemia levels, GSH and MDA levels of P. berghei infected rats. Methods: Female Wistar rats infected with P. berghei were divided into three groups. Group one received no treatment (negative control), group two was treated with 1.4 mg/200 g body weight of chloroquine diphosphate as positive control, and group three was treated with the EA fraction at a dose equal to andrographolide 3.5 mg/200 g body weight. The treatments lasted for four days (day 0 to day 3) and parasitemia was observed from day 0 to day 4. Rats were sacrificed and blood taken intracardially on day 4 after parasitemia observation. GSH was measured using an ELISA reader at a wavelength of 415 nm. MDA was observed via spectrophotometry at a wavelength of 532 nm. Results: The EA fraction at a dose equal to andrographolide 3.5 mg/200 g body weight was able to inhibit parasite growth by 81.97±9.14%. The GSH levels of the negative control, positive control and EA fraction treated group were 139.30±75.93 μMol/mL, 81.06±53.26 μMol/mL and 105.71±76.00 μMol/mL, respectively. Furthermore, the MDA level of negative control, positive control and EA fraction treated group were 11.18±0.70 nMol mL, 8.81±1.26 nMol/mL and 9.40±0.74 nMol/mL, respectively. No significant differences were detected between treatment groups regarding their GSH levels. Additionally, there was a significant difference in MDA levels between the negative control and positive control groups; as well as a significant difference between the negative control and the EA treated group. However, no significant difference in MDA levels between the EA fraction treated group and positive control group. Interestingly, a correlation was found between parasite growth inhibition and MDA levels among groups (p&lt;0.05). Conclusion: The EA fraction of A. paniculata significantly decreased MDA levels which correlated significantly with parasitemia levels of P. berghei infected rats. The antimalarial activity of the EA fraction may have been correlated with oxidative stress mechanisms and this correlation could be explained in part by the decreased production of MDA.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.

Sequential Application of Calcium Phosphate and ε-Polylysine Show Antibacterial and Dentin Tubule Occluding Effects In Vitro.

In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial effects of ε-polylysine were evaluated with broth dilution assay against P. gingivalis. CPP solution from MCPM, DCPD, and TTCP was prepared. Four concentrations of ε-polylysine(ε-PL) solutions (0.125%, 0.25%, 0.5%, 1%) were prepared. Dentin discs were prepared from recently extracted human third molars. Dentin discs were incubated with P. gingivalis (ATCC 33277) bacterial suspension (ca. 105 bacteria) containing Brain Heart Infusion medium supplemented with 0.1 g/mL Vitamin K, 0.5 mg/mL hemin, 0.4 g/mL L-cysteine in anaerobic jars (37 °C) for 7 days to allow for biofilm formation. P. g–infected dentin specimens were randomly divided into four groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL. On each dentin specimen, CPP solution was applied followed by polylysine solution with microbrush and immersed in artificial saliva. Precipitate formation, antibacterial effects, and occlusion of dentinal tubules were characterized in vitro over up to 72 h using scanning electron microscopy. ε-PL showed 34.97% to 61.19% growth inhibition levels against P. gingivalis (P. g) after 24 h of incubation. On P. g-infected dentin specimens, DCPD + 0.25% ε-PL, and DCPD + 0.5% ε-PL groups showed complete bacterial inhibition and 78.6% and 98.1% dentin tubule occlusion, respectively (p < 0.001). The longitudinal analysis on fractured dentin samples in DCPD and TTCP groups revealed deeply penetrated hydroxyapatite-like crystal formations in dentinal tubules after 72 h of incubation in artificial saliva.

Silver Nanoparticles Stabilized with Phosphorus-Containing Heterocyclic Surfactants: Synthesis, Physico-Chemical Properties, and Biological Activity Determination.

Phosphorus-containing heterocyclic cationic surfactants alkyldimethylphenylphospholium bromides with the alkyl chain length 14 to 18 carbon atoms were used for the stabilization of silver nanodispersions. Zeta potential of silver nanodispersions ranges from +35 to +70 mV, which indicates the formation of stable silver nanoparticles (AgNPs). Long-chain heptadecyl and octadecyl homologs of the surfactants series provided the most intensive stabilizing effect to AgNPs, resulting in high positive zeta potential values and smaller diameter of AgNPs in the range 50–60 nm. A comparison with non-heterocyclic alkyltrimethylphosphonium surfactants of the same alkyl chain length showed better stability and more positive zeta potential values for silver nanodispersions stabilized with heterocyclic phospholium surfactants. Investigations of biological activity of phospholium-capped AgNPs are represented by the studies of antimicrobial activity and cytotoxicity. While cytotoxicity results revealed an increased level of HepG2 cell growth inhibition as compared with the cytotoxicity level of silver-free surfactant solutions, no enhanced antimicrobial action of phospholium-capped AgNPs against microbial pathogens was observed. The comparison of cytotoxicity of AgNPs stabilized with various non-heterocyclic ammonium and phosphonium surfactants shows that AgNPs capped with heterocyclic alkyldimethylphenylphospholium and non-heterocyclic triphenyl-substituted phosphonium surfactants have the highest cytotoxicity among silver nanodispersions stabilized by the series of ammonium and phosphonium surfactants.

Abstract 1706: Hydrogel encapsulated TLR9 agonists show sustained tumor growth inhibition and prolong survival of CT26 tumor-bearing mice

Abstract CpG-oligodeoxynucleotides (CpG ODNs) are known ligands of innate immune receptor, toll-like receptor 9 (TLR9). Activation of TLR9 by its ligands leads to stimulation of an immune cascade represented by secretion of Th1-type cytokine and chemokine milieu, including IFN-α and activation of immune cell surface markers. TLR9 agonists have a number of therapeutic applications. Currently, several TLR9 agonists are in late stage clinical trials in combination with checkpoint inhibitors (CPI) in cancer patients. In these trials, TLR9 agonists are commonly administered once a week for several weeks, suggesting that they may be degraded in the local tissues or dissipated from the site of action rapidly requiring once a week dosing. To improve the stability, delivery, and durability of TLR9 agonist activity with reduced frequency of administration, we encapsulated TLR9 agonists in injectable hydrogels containing poloxamer. TLR9 agonist/hydrogel formulations can be used to slow release of TLR9 agonist following peritumoral/intratumoral or by other routes of administration. In here, we have studied TLR9 agonists with and without encapsulation in hydrogels for tumor growth inhibition and toxicity in syngeneic tumor models in mice. In CT26 colon cancer model, peritumoral and intratumoral administration of TLR9 agonist in five doses in 12 days without hydrogel encapsulation showed potent tumor growth inhibition and increased survival of tumor bearing mice compared with mice injected with vehicle. Peritumoral administration of encapsulated TLR9 agonist in two different types of hydrogels, one that releases the TLR9 agonist at slower rate or the other that releases little faster at the same total dose in single or two doses, respectively, showed similar levels of tumor growth inhibition up to 11 days as that of the TLR9 agonist dosed five times without hydrogel formulation. Both hydrogel formulations prolonged survival of tumor bearing mice to a similar extent. Moreover, the mice administered with hydrogel encapsulated TLR9 agonist showed lower body weight loss compared with unformulated TLR9 agonist. Benefit of hydrogel encapsulation in tumor bearing mice has been confirmed with a second TLR9 agonist as well. Taken together, the current results demonstrate that hydrogel encapsulated TLR9 agonists are released slowly over a time, resulting in sustained tumor growth inhibition with minimal effect on body weight. The encapsulation of TLR9 agonist in hydrogels may permit less frequent dosing in patients. The hydrogel encapsulation can also be expanded to other therapeutic modalities of oligonucleotides such as antisense, siRNA and gene editing technologies. Citation Format: Ekambar R. Kandimalla, Jun Jiang, Lokendrakumar C. Bengani, Xinyu Huang, Jason Zhang. Hydrogel encapsulated TLR9 agonists show sustained tumor growth inhibition and prolong survival of CT26 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1706.

Comparative Evaluation of Two Commercial Propolis Extracts as Plant Defense Activator and Antimicrobial Agent Against Pseudomonas syringae pv. tomato (Pst) strain DC3000

Propolis is a natural bee product that protects the beehives from internal and external factors. It is a natural-complex compound with a wide potential use as an antimicrobial, anti-fungal, and anti-inflammatory effect. The role of propolis as a plant protection agent and an alternative to pesticides is an unexplored area. Here, two different commercially available propolis extracts were evaluated for antibacterial effects on Pseudomonas syringae pv. tomato (Pst) strain DC3000. Also, the activities of defense response genes WRKY70 and CaBP22 in Arabidopsis thaliana under propolis application were compared. According to the results, each propolis extract and dose had a different effect on gene expressions as well as antibacterial activity. One of the commercial brands had a significant effect at all doses while another brand’s propolis extract had its activity at only 1% concentration. Propolis reduced bacterial growth up to 93% with a 2% concentration. For the first time, propolis is also evaluated for its capacity as a plant defense activator agent and it induced WRKY70 and CaBP22 gene expression. The differences in gene expression and bacterial growth inhibition levels suggest the importance of the origins of propolis, such as plant species and regions it collected. While preliminary in nature, these results suggest a significant potential of propolis in plant protection in commercial and organic agriculture.

Eupomatenes A – E: Neolignans isolated from the leaves of Australian rainforest plant Eupomatia laurina

A detailed phytochemical investigation of the leaves of the Australian rainforest tree Eupomatia laurina, led to the discovery of five new neolignans, eupomatenes A – E and eight known compounds, eupomatenoid-2, trans-(2′S)-2-[1′-(4-methoxyphenyl)prop-2′-yl]anethol, chlorogenic acid, chlorogenic acid-methyl ester, tyrosol-1-O-β-xylopyranosyl-1(1 → 6)-O-β-glucopyranoside, leucoside, kaempferol-3-O-neohesperidoside, and pachypodol. The structures of all the compounds were determined by detailed spectroscopic analysis. All compounds were also evaluated for their anti-inflammatory properties by assessing their inhibitory effects on nitric oxide (NO) production and TNF- α release in RAW 264.7 macrophages. Whilst slight anti-inflammatory activity (in terms of inhibition of NO production) was observed with eupomatenes A – E, this was also associated with high levels of cell growth inhibition.

Exploring the Role of the NO-Detoxifying Enzyme HmpA in the Evolution of Domesticated Alfalfa Rhizobia.

We have previously shown the extensive loss of genes during the domestication of alfalfa rhizobia and the high nitrous oxide emission associated with the extreme genomic instability of commercial inoculants. In the present note, we describe the molecular mechanism involved in the evolution of alfalfa rhizobia. Genomic analysis showed that most of the gene losses in inoculants are due to large genomic deletions rather than to small deletions or point mutations, a fact consistent with recurrent DNA double-strand breaks (DSBs) at numerous locations throughout the microbial genome. Genetic analysis showed that the loss of the NO-detoxifying enzyme HmpA in inoculants results in growth inhibition and high DSB levels under nitrosative stress, and large genomic deletions in planta but not in the soil. Therefore, besides its known function in the effective establishment of the symbiosis, HmpA can play a critical role in the preservation of the genomic integrity of alfalfa rhizobia under host-derived nitrosative stress.

Polyunsaturated Fatty Acids (PUFA) Increases the Sensitivity of Saccharomyces cerevisiae to Ethanol by Impairing the Function of the Mitochondrial Unselective Channel (MUC)

Yeast cells uncouple its respiration by opening a mitochondrial unselective channel (MUC) that dissipates the membrane potential and accelerates oxygen consumption in order to avoid a deleterious buildup of ROS. Yeast incorporates polyunsaturated fatty acids (PUFA) into membranes when available in its environment, increasing the sensitivity of membranes to oxidative damage. One factor causing oxidative stress in yeast is ethanol accumulation during fermentation. The MUC is constituted by the voltage-dependent anionic channel (VDAC), the adenine nucleotide translocator and the cyclophilin D, being the VDAC a sensor for Ca2+ levels that promotes MUC closing under high Ca2+ concentrations. Moreover, MUC function is dependent on the phospholipid cardiolipin. In this work, we propose that yeast become more susceptible to ethanol stress when grow in the presence of PUFA by the impairment of MUC function due to enhanced lipid peroxidation. Yeast cells were grown in liquid YPD medium with (+C18:3) or without (-C18:3) 1mM linolenic acid and 10% ethanol. Ethanol decreased yeast growth by 20% and 90% in –C18:3 and +C18:3 cells, respectively. Ethanol also promoted lipid peroxidation only in +C18:3 cells. The addition of amiodarone, which increases cytosolic Ca2+ levels and promotes the closure of MUC, decreased 80% the growth in –C18:3 cells, while the +C18:3 cells showed the same levels of growth inhibition than without amiodarone. In – C18:3 cells respiring in uncoupled state (U), amiodarone decreased respiration by promoting MUC closing and coupling. Conversely, respiration was slightly inhibited by amiodarone in the +C18:3 cells. The role of MUC in the deleterious effects of ethanol in +C18:3 cells was confirmed in yeast mutants depleted in cardiolipin and VDAC, as these cells became insensitive to the effects of amiodarone. These results suggest that increased sensitivity to ethanol elicited by C18:3 may be due to impaired MUC function due to enhanced lipid peroxidation.

Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Plasmodium Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner 1 and its active enantiomer 1-S exhibited low-double-digit nanomolar activity against resistant Plasmodium falciparum (Pf) laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of Pf and P. vivax. The slow killing profile and the relative high propensity to develop resistance in vitro (minimum inoculum resistance of 8 × 105 parasites at a selection pressure of 3 × IC50) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of Pf malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target Plasmodium PRS.

Effect of 5-hydroxypyrimidine derivatives on tumor growth and cytokine concentration in blood serum of female CBA mice with cervical cancer (RSHM-5)

The effects of intraperitoneal administration of SNK-411 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine) in a dose of 25 mg/kg (the total dose of 350 mg/kg) and SNK-578 (hydrochloride of 2-isobutyl-4,6-dimethyl-5- hydroxypyrimidine) in a dose of 10 mg/kg (the total dose of 140 mg/kg) on tumor growth and concentration of cytokines in the blood serum were studied in female CBA mice. Substances were administrated from the 2nd to 15th days of tumor development. Tumor growth inhibition (TGI) and serum cytokine level were studied on the 7th day after the end of compounds administration (21st day of tumor growth). In intact control group (n=10) median tumor mass was 1255 mg. TGI in the group of animals treated with SNK-411 was 47%; in the group of mice treated with SNK-578 TGI was 87%, tumor mass demonstrated 7.4-fold reduction. Serum concentrations of cytokines (IL-6, IL-10, IL-17A and IFN-γ) in tumor-bearing group of mice were higher versus the intact control group by 229%, 40%, 60% and 81%, respectively. Highly active SNK-578 decreased concentrations of prooncogenic IL-10, IL-17A and proinflammatory IL-6, by 61%, 70% and 29% as compared to tumor-bearing control group. SNK-411 decreased concentrations of prooncogenic IL-10 and IL-17A by 48% and 60%, respectively, and did not affect concentration of IL-6. Taking into consideration that IL-6 participates in autoimmune reactions, we can assume that the immune control is one of the crucial mechanisms of antitumor effect of SNK-578. All results are statistically significant.

New Paracyclophanylthiazoles with Anti-Leukemia Activity: Design, Synthesis, Molecular Docking, and Mechanistic Studies.

A new series of methyl 2-(2-(4′-[2.2]paracyclophanyl)-hydrazinylidene)-3-substituted-4-oxothiazolidin-5-ylidene)acetates 3a–f were synthesized from the reaction of paracyclophanyl-acylthiosemicarbazides 2a–f with dimethyl acetylenedicarboxylate. Based upon nuclear magnetic resonance (NMR), infrared (IR), and mass spectra (HRMS), the structure of the obtained products was elucidated. X-ray structure analysis was also used as unambiguous tool to elucidate the structure of the products. The target compounds 3a–f were screened against 60 cancer cell lines. They displayed anticancer activity against a leukemia subpanel, namely, RPMI-8226 and SR cell lines. The activity of compound 3a was found as the most cytotoxic potency against 60 cancer cell lines. Consequently, it was selected for further five doses analysis according to National Cancer Institute (NCI) protocol. The cytotoxic effect showed selectivity ratios ranging between 0.63 and 1.28 and between 0.58 and 5.89 at the GI50 and total growth inhibition (TGI) levels, respectively. Accordingly, compound 3a underwent further mechanistic study against the most sensitive leukemia RPMI-8226 and SR cell lines. It showed antiproliferation with IC50 = 1.61 ± 0.04 and 1.11 ± 0.03 µM against RPMI-8226 and SR cell lines, respectively. It also revealed a remarkable tubulin inhibitory activity, compared to colchicine with IC50 = 4.97 µM/mL. Caspase-3, BAX, and Bcl-2 assays for 3a using annexin V-FITC staining revealed significant pro-apoptotic activity. Furthermore, multidrug-resistant leukemia SR cells were used to show better resistance indices (1.285 ng/mL, 1.15-fold) than the reference. Docking studies with β-tubulin indicate that most of the tested compounds illustrated good binding at the colchicine binding site of the enzyme, especially for compound 3a, which made several interactions better than that of the reference colchicine.