Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

Construction of Anthocyanin Biosynthesis System Using Chalcone as a Substrate in Lactococcus lactis NZ9000.

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.

Bioreduction of 4'-Hydroxychalcone in Deep Eutectic Solvents: Optimization and Efficacy with Various Yeast Strains.

4'-dihydrochalcones are secondary metabolites isolated from many medicinal plants and from the resin known as 'dragon's blood'. Due to their biological potential, our research objective was to determine the possibilities of using biocatalysis processes carried out in deep eutectic solvents (DESs) to obtain 4'-dihydrochalcones as a model compound. The processes were carried out in a culture of the yeast Yarrowia lipolytica KCh 71 and also in cultures of strains of the genera Rhodotorula and Debaryomyces. Based on the experiments carried out, an optimum process temperature of 35 °C was chosen, and the most suitable DES contained glycerol as a hydrogen bond donor (HBD). For a medium with 30% water content (DES 11), the conversion observed after 24 h exceeded 70%, while increasing the amount of water to 50% resulted in a similar level of conversion after just 1 h. A fivefold increase in the amount of added substrate resulted in a reduction in conversion, which reached 30.3%. Of the other yeast strains tested, Rhodotorula marina KCh 77 and Rhodotorula rubra KCh 4 also proved to be good biocatalysts for the bioreduction process. For these strains, the conversion reached 95.4% and 95.1%, respectively. These findings highlight the potential of yeast as a biocatalyst for the selective reduction of α,ß-unsaturated ketones and the possibility of using a DESs as a reaction medium in this process.

Exogenous Melatonin Enhances Dihydrochalcone Accumulation in Lithocarpus litseifolius Leaves via Regulating Hormonal Crosstalk and Transcriptional Profiling.

Dihydrochalcones (DHCs) constitute a specific class of flavonoids widely known for their various health-related advantages. Melatonin (MLT) has received attention worldwide as a master regulator in plants, but its roles in DHC accumulation remain unclear. Herein, the elicitation impacts of MLT on DHC biosynthesis were examined in Lithocarpus litseifolius, a valuable medicinal plant famous for its sweet flavor and anti-diabetes effect. Compared to the control, the foliar application of MLT significantly increased total flavonoid and DHC (phlorizin, trilobatin, and phloretin) levels in L. litseifolius leaves, especially when 100 µM MLT was utilized for 14 days. Moreover, antioxidant enzyme activities were boosted after MLT treatments, resulting in a decrease in the levels of intracellular reactive oxygen species. Remarkably, MLT triggered the biosynthesis of numerous phytohormones linked to secondary metabolism (salicylic acid, methyl jasmonic acid (MeJA), and ethylene), while reducing free JA contents in L. litseifolius. Additionally, the flavonoid biosynthetic enzyme activities were enhanced by the MLT in leaves. Multiple differentially expressed genes (DEGs) in RNA-seq might play a crucial role in MLT-elicited pathways, particularly those associated with the antioxidant system (SOD, CAT, and POD), transcription factor regulation (MYBs and bHLHs), and DHC metabolism (4CL, C4H, UGT71K1, and UGT88A1). As a result, MLT enhanced DHC accumulation in L. litseifolius leaves, primarily by modulating the antioxidant activity and co-regulating the physiological, hormonal, and transcriptional pathways of DHC metabolism.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Anti-inflammatory effects of new human histamine H3 receptor ligands with flavonoid structure on BV-2 neuroinflammation.

OBJECTIVE: Microglia play an important role in the neuroinflammation developed in response to various pathologies. In this study, we examined the anti-inflammatory effect of the new human histamine H3 receptor (H3R) ligands with flavonoid structure in murine microglial BV-2 cells. MATERIAL AND METHODS: The affinity of flavonoids (E243 -flavone and IIIa-IIIc-chalcones) for human H3R was evaluated in the radioligand binding assay. The cytotoxicity on BV-2 cell viability was investigated with the MTS assay. Preliminary evaluation of anti-inflammatory properties was screened by the Griess assay in an in vitro neuroinflammation model of LPS-treated BV-2 cells. The expression and secretion of pro-inflammatory cytokines were evaluated by real-time qPCR and ELISA, respectively. The expression of microglial cell markers were determined by immunocytochemistry. RESULTS: Chalcone derivatives showed high affinity at human H3R with Ki values < 25 nM. At the highest nontoxic concentration (6.25 µM) compound IIIc was the most active in reducing the level of nitrite in Griess assay. Additionally, IIIc treatment attenuated inflammatory process in murine microglia cells by down-regulating pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) at both the level of mRNA and protein level. Our immunocytochemistry studies revealed expression of microglial markers (Iba1, CD68, CD206) in BV-2 cell line. CONCLUSIONS: These results emphasize the importance of further research to accurately identify the anti-inflammatory mechanism of action of chalcones.

Phenolic chalcones as agents against Trichomonas vaginalis.

Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world's most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4'-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Genetic mapping of sexually dimorphic volatile and non-volatile floral secondary chemistry of a dioecious willow.

Secondary chemistry often differs between sexes in dioecious plant species, a pattern attributed to its possible role in the evolution and/or maintenance of dioecy. We used GC-MS to measure floral volatiles emitted from, and LC-MS to quantitate non-volatile secondary compounds contained in, female and male Salix purpurea willow catkins from an F2 family. Using the abundance of these chemicals, we then performed quantitative trait locus (QTL) mapping to locate them on the genome, identified biosynthetic candidate genes in the QTL intervals, and examined expression patterns of candidate genes using RNA-seq. Male flowers emitted more total terpenoids than females, but females produced more benzenoids. Male tissue contained greater amounts of phenolic glycosides, but females had more chalcones and flavonoids. A flavonoid pigment and a spermidine derivative were found only in males. Male catkins were almost twice the mass of females. Forty-two QTL were mapped for 25 chemical traits and catkin mass across 16 of the 19 S. purpurea chromosomes. Several candidate genes were identified, including a chalcone isomerase associated with seven compounds. A better understanding of the genetic basis of the sexually dimorphic chemistry of a dioecious species may shed light on how chemically mediated ecological interactions may have helped in the evolution and maintenance of dioecy.

New 1,2,3-triazole linked ciprofloxacin-chalcones induce DNA damage by inhibiting human topoisomerase I& II and tubulin polymerization.

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC50s ranged from 2.53-8.67 µM, 8.67-62.47 µM, and 4.19-24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC50 values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC50 values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC50 = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.

Protective Effects of the Chalcone-Based Derivative AN07 on Inflammation-Associated Myotube Atrophy Induced by Lipopolysaccharide.

Inflammation is a major cause of skeletal muscle atrophy in various diseases. 2-Hydroxy-4'-methoxychalcone (AN07) is a chalcone-based peroxisome-proliferator-activated receptor gamma (PPARγ) agonist with various effects, such as antiatherosclerosis, anti-inflammation, antioxidative stress, and neuroprotection. In this study, we examined the effects of AN07 on protein homeostasis pathway and mitochondrial function in inflammation-associated myotube atrophy induced by lipopolysaccharides (LPS). We found that AN07 significantly attenuated NF-κB activation, inflammatory factors (TNF-α, IL-1ß, COX-2, and PGE2), Nox4 expression, and reactive oxygen species levels in LPS-treated C2C12 myotubes. Moreover, AN07 increased SOD2 expression and improved mitochondrial function, including mitochondrial membrane potential and mitochondrial oxygen consumption rate. We also demonstrated that AN07 attenuated LPS-induced reduction of myotube diameter, MyHC expression, and IGF-1/IGF-1R/p-Akt-mediated protein synthesis signaling. Additionally, AN07 downregulated LPS-induced autophagy-lysosomal protein degradation molecules (LC3-II/LC3-I and degraded p62) and ubiquitin-proteasome protein degradation molecules (n-FoxO1a/MuRF1/atrogin-1). However, the regulatory effects of AN07 on protein synthesis and degradation signaling were inhibited by the IGF-1R inhibitor AG1024 and the PI3K inhibitor wortmannin. In addition, the PPARγ antagonist GW9662 attenuated the effects of AN07 against LPS-induced inflammation, oxidation, and protein catabolism. In conclusion, our findings suggest that AN07 possesses protective effects on inflammation-induced myotube atrophy and mitochondrial dysfunction.

Antioxidant and Anti-Melanogenic Activities of Heat-Treated Licorice (Wongam, Glycyrrhiza glabra × G. uralensis) Extract.

Melanin is a brown or black pigment that protects skin from ultraviolet radiation and reactive oxygen species (ROS). However, overproduction of melanin is associated with lentigines, melasma, freckles and skin cancer. Licorice has shown antioxidant, anti-tumor, anti-platelet, anti-inflammatory and immunomodulatory activities and is used as a natural treatment for skin whitening. We aimed to confirm the potential of Wongam, a new cultivar of licorice developed by the Rural Development Administration (RDA), as a whitening agent in cosmetics. In addition, we verified the effect of heat treatment on the bioactivity of licorice by comparing antioxidant and anti-melanogenic activities of licorice extract before and after heating (130 °C). The heat-treated licorice extract (WH-130) showed higher radical-scavenging activities in the ABTS+ (2,2'-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays. In addition, WH-130 inhibited melanogenesis more effectively due to downregulation of tyrosinase in B16F10 melanoma cells than non-heated licorice extract. Moreover, heat treatment increased total phenolic content. In particular, isoliquiritigenin, an antioxidant and anti-melanogenic compound of licorice, was produced by heat treatment. In conclusion, WH-130, with increased levels of bioactive phenolics such as isoliquiritigenin, has potential for development into a novel skin whitening material with applications in cosmetics.

Biosynthesis of the Dihydrochalcone Sweetener Trilobatin Requires Phloretin Glycosyltransferase2.

Epidemics of obesity and type 2 diabetes drive strong consumer interest in plant-based low-calorie sweeteners. Trilobatin is a sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain trilobatin's bitter positional isomer phloridzin. Variation in trilobatin content was mapped to the Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples. Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed protein purification and differential gene expression analysis in samples high in trilobatin but low in phloridzin. Markers developed for PGT2 cosegregated strictly with the Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4'-o-glycosylation of phloretin to trilobatin as well as 3-hydroxyphloretin to sieboldin. Transient expression of double bond reductase, chalcone synthase, and PGT2 genes reconstituted the apple pathway for trilobatin production in Nicotiana benthamiana Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing trilobatin, and for high amounts of this natural low-calorie sweetener to be produced via biopharming and metabolic engineering in yeast.

Microbial Transformation of Broussochalcones A and B by Aspergillus niger.

Broussochalcones A (BCA, 1) and B (BCB, 2) are major bioactive constituents isolated from Broussonetia papyrifera, a polyphenol-rich plant belonging to the family Moraceae. Due to their low yields from natural sources, BCA (1) and BCB (2) were prepared synthetically by employing Claisen-Schmidt condensation, and these were used as substrates for microbial transformation to obtain novel derivatives. Microbial transformation of BCA (1) and BCB (2) with the endophytic fungus Aspergillus niger KCCM 60332 yielded 10 previously undescribed chalcones (1a-1e and 2a-2e). Their structures were established based on the spectroscopic methods. The cytotoxicity of BCA (1), BCB (2), and their metabolites (1a-1e and 2a-2e) was determined by human cancer cell lines A375P, A549, HT-29, MCF-7, and HepG2, with 1e shown to be most cytotoxic.

Combined 3D-QSAR and docking analysis for the design and synthesis of chalcones as potent and selective monoamine oxidase B inhibitors.

Monoamine oxidases (MAOs) are important targets in medicinal chemistry, as their inhibition may change the levels of different neurotransmitters in the brain, and also the production of oxidative stress species. New chemical entities able to interact selectively with one of the MAO isoforms are being extensively studied, and chalcones proved to be promising molecules. In the current work, we focused our attention on the understanding of theoretical models that may predict the MAO-B activity and selectivity of new chalcones. 3D-QSAR models, in particular CoMFA and CoMSIA, and docking simulations analysis have been carried out, and their successful implementation was corroborated by studying twenty-three synthetized chalcones (151-173) based on the generated information. All the synthetized molecules proved to inhibit MAO-B, being ten out of them MAO-B potent and selective inhibitors, with IC50 against this isoform in the nanomolar range, being (E)-3-(4-hydroxyphenyl)-1-(2,2-dimethylchroman-6-yl)prop-2-en-1-one (152) the best MAO-B inhibitor (IC50 of 170 nM). Docking simulations on both MAO-A and MAO-B binding pockets, using compound 152, were carried out. Calculated affinity energy for the MAO-A was +2.3 Kcal/mol, and for the MAO-B was -10.3 Kcal/mol, justifying the MAO-B high selectivity of these compounds. Both theoretical and experimental structure-activity relationship studies were performed, and substitution patterns were established to increase MAO-B selectivity and inhibitory efficacy. Therefore, we proved that both 3D-QSAR models and molecular docking approaches enhance the probability of finding new potent and selective MAO-B inhibitors, avoiding time-consuming and costly synthesis and biological evaluations.

Design and synthesis of methoxyphenyl- and coumarin-based chalcone derivatives as anti-inflammatory agents by inhibition of NO production and down-regulation of NF-κB in LPS-induced RAW264.7 macrophage cells.

Exaggerated inflammatory responses may cause serious and debilitating diseases such as acute lung injury and rheumatoid arthritis. Two series of chalcone derivatives were prepared as anti-inflammatory agents. Methoxylated phenyl-based chalcones 2a-l and coumarin-based chalcones 3a-f were synthesized and compared for their inhibition of COX-2 enzyme and nitric oxide production suppression. Methoxylated phenyl-based chalcones showed better inhibition to COX-2 enzyme and nitric oxide suppression than the coumarin-based chalcones. Among the 18 synthesized chalcone derivatives, compound 2f exhibited the highest anti-inflammatory activity by inhibition of nitric oxide concentration in LPS-induced RAW264.7 macrophages (IC50 = 11.2 µM). The tested compound 2f showed suppression of iNOS and COX-2 enzymes. Moreover, compound 2f decreases in the expression of NF-κB and phosphorylated IκB in LPS-stimulated macrophages. Finally, docking studies suggested the inhibition of IKKß as a mechanism of action and highlighted the importance of 2f hydrophobic interactions.

Design, synthesis and drug resistance reversal potential of novel curcumin mimics Van D: Synergy potential of curcumin mimics.

Being crucial part of plant-based novel discovery of drug from natural resources, a study was done to explore the antibacterial potential of curcumin mimics in combination with antibiotics against multidrug resistant isolates of Pseudomonas aeruginosa. The best candidate Van D, a curcumin mimics reduced the MIC of tetracycline (TET) up to 16 folds against multidrug resistant clinical isolates. VanD further inhibited the efflux pumps as evident by ethidium bromide efflux and by in-silico docking studies. In another experiment, it was also found that Van D inhibits biofilm synthesis. This derivative kills the KG-P2, an isolate of P. aeruginosa in a time dependent manner, the post-antibiotic effect (PAE) of tetracycline was extended as well as mutant prevention concentration (MPC) of TET was also decreased. In Swiss albino mice, Van D reduced the proinflammatory cytokines concentration. In acute oral toxicity study, this derivative was well tolerated and found to be safe up to 1000 mg/kg dose. To the best of our knowledge, this is the first report on curcumin mimics as synergistic agent via inhibition of efflux pump.

Novel urea linked ciprofloxacin-chalcone hybrids having antiproliferative topoisomerases I/II inhibitory activities and caspases-mediated apoptosis.

A novel series of urea-linked ciprofloxacin (CP)-chalcone hybrids 3a-j were synthesized and screened by NCI-60 cancer cell lines as potential cytotoxic agents. Interestingly, compounds 3c and 3j showed remarkable antiproliferative activities against both colon HCT-116 and leukemia SR cancer cells compared to camptothecin, topotecan and staurosporine with IC50 = 2.53, 2.01, 17.36, 12.23 and 3.1 µM for HCT-116 cells, respectively and IC50 = 0.73, 0.64, 3.32, 13.72 and 1.17 µM for leukemia SR cells, respectively. Also, compounds 3c and 3j exhibited inhibitory activities against Topoisomerase (Topo) I with % inhibition = 51.19% and 56.72%, respectively, compared to camptothecin (% inhibition = 60.05%) and Topo IIß with % inhibition = 60.81% and 60.06%, respectively, compared to topotecan (% inhibition = 71.09%). Furthermore, compound 3j arrested the cell cycle of leukemia SR cells at G2/M phase. It induced apoptosis both intrinsically and extrinsically via activation of proteolytic caspases cascade (caspases-3, -8, and -9), release of cytochrome C from mitochondria, upregulation of proapoptotic Bax and down-regulation of Bcl-2 protein level. Thus, the new ciprofloxacin derivative 3j could be considered as a potential lead for further optimization of antitumor agent against leukemia and colorectal carcinoma.

Novel piperazine-chalcone hybrids and related pyrazoline analogues targeting VEGFR-2 kinase; design, synthesis, molecular docking studies, and anticancer evaluation.

New piperazine-chalcone hybrids and related pyrazoline derivatives have been designed and synthesised as potential vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. The National Cancer Institute (NCI) has selected six compounds to evaluate their antiproliferative activity in vitro against 60 human cancer cells lines. Preliminary screening of the examined compounds indicated promising anticancer activity against number of cell lines. The enzyme inhibitory activity against VEGFR-2 was evaluated and IC50 of the tested compounds ranged from 0.57 µM to 1.48 µM. The most potent derivatives Vd and Ve were subjected to further investigations. A cell cycle analysis showed that both compounds mainly arrest HCT-116 cell cycle in the G2/M phase. Annexin V-FITC apoptosis assay showed that Vd and Ve induced an approximately 18.7-fold and 21.2-fold total increase in apoptosis compared to the control. Additionally, molecular docking study was performed against VEGFR (PDB ID: 4ASD) using MOE 2015.10 software and Sorafenib as a reference ligand.