Synthesis, Biological Evaluation and Molecular Modelling of 2'-Hydroxychalcones as Acetylcholinesterase Inhibitors.

A series of 2'-hydroxy- and 2'-hydroxy-4',6'-dimethoxychalcones was synthesised and evaluated as inhibitors of human acetylcholinesterase (AChE). The majority of the compounds were found to show some activity, with the most active compounds having IC50 values of 40-85 µM. Higher activities were generally observed for compounds with methoxy substituents in the A ring and halogen substituents in the B ring. Kinetic studies on the most active compounds showed that they act as mixed-type inhibitors, in agreement with the results of molecular modelling studies, which suggested that they interact with residues in the peripheral anionic site and the gorge region of AChE.

Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model.

BACKGROUND: The limited antibiotic options for effective control of methicillin-resistant Staphylococcus aureus infections has led to calls for new therapeutic approaches to combat this human pathogen. An alternative approach to control MRSA is through the use of anti-infective agents that selectively disrupt virulence-mediated pathways without affecting microbial cell viability or by modulating the host natural immune defenses to combat the pathogen. METHODS: We established a C. elegans - S. aureus liquid-based assay to screen for potential anti-infectives against S. aureus. The assay was utilized to screen 37 natural extracts and 29 synthetic compounds for the ability to extend the lifespan of infected nematodes. Disc diffusion and MIC microdilution tests were used to evaluate the anti-microbial properties of these natural extracts and synthetic compounds whilst in vivo bacterial CFU within the C. elegans gut were also enumerated. RESULTS: We screened a total of 37 natural extracts and 29 synthetic compounds for anti-infective properties. The screen successfully revealed 14 natural extracts from six plants (Nypa fruticans, Swietenia macrophylla, Curcuma longa, Eurycoma longifolia, Orthosiphon stamineus and Silybum eburneum) and one marine sample (Faunus ater) that improved the survival of S. aureus-infected worms by at least 2.8-fold as well as 14 synthetic compounds that prolonged the survival of S. aureus-infected nematodes by 4-fold or greater. An anti-microbial screen of all positive hits demonstrated that 8/28 hits had no effect on S. aureus growth. Of these 8 candidates, 5 of them also protected the worms from MRSA infection. We also noted that worms exposed to N. fruticans root and O. stamineus leaf extracts showed reduced intestinal colonization by live S. aureus. This suggests that these extracts could possibly activate host immunity to eliminate the bacteria or interfere with factor/s that prevents pathogen accumulation. CONCLUSION: We have successfully demonstrated the utility of this liquid-based screen to identify anti-infective substances that prolong S. aureus-infected host survival without affecting bacterial cell viability.

Boesenbergia rotunda: From Ethnomedicine to Drug Discovery.

Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.

Computational-aided design: minimal peptide sequence to block dengue virus transmission into cells.

Dengue virus (DV) infection is one of the main public health concerns, affecting approximately 390 million people worldwide, as reported by the World Health Organization. Yet, there is no antiviral treatment for DV infection. Therefore, the development of potent and nontoxic anti-DV, as a complement for the existing treatment strategies, is urgently needed. Herein, we investigate a series of small peptides inhibitors of DV antiviral activity targeting the entry process as the promising strategy to block DV infection. The peptides were designed based on our previously reported peptide sequence, DN58opt (TWWCFYFCRRHHPFWFFYRHN), to identify minimal effective inhibitory sequence through molecular docking and dynamics studies. The in silico designed peptides were synthesized using conventional Fmoc solid-phase peptide synthesis chemistry, purified by RP-HPLC and characterized using LCMS. Later, they were screened for their antiviral activity. One of the peptides, AC 001, was able to reduce about 40% of DV plaque formation. This observation correlates well with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis - AC 001 showed the most favorable binding affinity through 60 ns simulations. Pairwise residue decomposition analysis has revealed four key residues that contributed to the binding of these peptides into the DV2 E protein pocket. This work identifies the minimal peptide sequence required to inhibit DV replication and explains the behavior observed on an atomic level using computational study.Communicated by Ramaswamy H. Sarma.

Challenges and Complications of Poly(lactic-co-glycolic acid)-Based Long-Acting Drug Product Development.

Poly(lactic-co-glycolic acid) (PLGA) is one of the preferred polymeric inactive ingredients for long-acting parenteral drug products that are constituted of complex formulations. Despite over 30 years of use, there are still many challenges faced by researchers in formulation-related aspects pertaining to drug loading and release. Until now, PLGA-based complex generic drug products have not been successfully developed. The complexity in developing these generic drug products is not just due to their complex formulation, but also to the manufacturing process of the listed reference drugs that involve PLGA. The composition and product attributes of commercial PLGA formulations vary with the drugs and their intended applications. The lack of standard compendial methods for in vitro release studies hinders generic pharmaceutical companies in their efforts to develop PLGA-based complex generic drug products. In this review, we discuss the challenges faced in developing PLGA-based long-acting injectable/implantable (LAI) drug products; hurdles that are associated with drug loading and release that are dictated by the physicochemical properties of PLGA and product manufacturing processes. Approaches to overcome these challenges and hurdles are highlighted specifically with respect to drug encapsulation and release.

3-(2-Amino-phenyl-sulfanyl)-1,3-diphenyl-propan-1-one.

In the title compound, C(21)H(19)NOS, the three aromatic rings are not coplanar, the dihedral angles between them being 84.75 (7), 88.01 (8) and 8.36 (16)°. In the crystal, two types of C-H⋯ π inter-actions, one of which is weak, and N-H⋯π inter-actions link the mol-ecules into layers parallel to the ab plane.

Halocarbon emissions by selected tropical seaweeds exposed to different temperatures.

Four tropical seaweeds, Gracilaria manilaensis Yamamoto & Trono, Ulva reticulata Forsskål, Kappaphycus alvarezii (Doty) L.M.Liao and Turbinaria conoides (J.Agardh) Kützing, collected from various habitats throughout Malaysia, were subjected to temperatures of 40, 35, 30, 25 and 20 °C in the laboratory. An exposure range of 21-38 °C is reported for Malaysian waters. The effect of the temperature exposures on the halocarbon emissions of the seaweeds were determined 4 and 28 h after treatment. The emission rates for a suite of six halocarbons commonly emitted by seaweeds, bromoform (CHBr3), dibromomethane (CH2Br2), diiodomethane (CH2I2), iodomethane (CH3I), dibromochloromethane (CHBr2Cl) and dichlorobromomethane (CHBrCl2), were measured using a cryogenic purge-and-trap sample preparation system coupled to a gas chromatography-mass spectrometry. The emission rate of CHBr3 was the highest of the six halocarbons for all the seaweeds under all the temperatures tested, followed by CH2Br2, and CH2I2. The emission rates were affected by temperature change and exposure duration, but overall responses were unique to each seaweed species. Larger decreases in the emissions of CHBr3, CH2Br2, CH2I2 and CHBr2Cl were found for K. alvarezii and T. conoides after 4 h at 40 °C. In both cases there was a >90% (p < 0.05) reduction in the Fv/Fm value suggesting that photosynthetic actitivity was severely compromised. After a 28 h exposure period, strong negative correlations (r = -0.69 to -0.95; p < 0.01) were observed between temperature and the emission of CHBr3, CH2Br2 and CH2I2 for U. reticulata, K. alvarezii and T. conoides. This suggests a potential decrease in the halocarbon emissions from these tropical seaweeds, especially where the temperature increase is a prolonged event. Strong correlations were also seen between seaweed chlorophyll and carotenoid pigment contents and the emission rates for CHBr3, CH2Br2 and CH2I2 (r = 0.48 to 0.96 and -0.49 to -0.96; p < 0.05). These results suggest that the regulation of halocarbon production versus reactive oxygen species production in seaweeds is an area worthy of further exploration.

Effect of irradiance on the emission of short-lived halocarbons from three common tropical marine microalgae.

Marine algae have been reported as important sources of biogenic volatile halocarbons that are emitted into the atmosphere. These compounds are linked to destruction of the ozone layer, thus contributing to climate change. There may be mutual interactions between the halocarbon emission and the environment. In this study, the effect of irradiance on the emission of halocarbons from selected microalgae was investigated. Using controlled laboratory experiments, three tropical marine microalgae cultures, Synechococcus sp. UMACC 371 (cyanophyte), Parachlorella sp. UMACC 245 (chlorophyte) and Amphora sp. UMACC 370 (diatom) were exposed to irradiance of 0, 40 and 120 µmol photons m-2s-1. Stress in the microalgal cultures was indicated by the photosynthetic performance (Fv/Fm, maximum quantum yield). An increase in halocarbon emissions was observed at 120 µmol photons m-2s-1, together with a decrease in Fv/Fm. This was most evident in the release of CH3I by Amphora sp. Synechococcus sp. was observed to be the most affected by irradiance as shown by the increase in emissions of most halocarbons except for CHBr3 and CHBr2Cl. High positive correlation between Fv/Fm and halocarbon emission rates was observed in Synechococcus sp. for CH2Br2. No clear trends in correlation could be observed for the other halocarbons in the other two microalgal species. This suggests that other mechanisms like mitochondria respiration may contribute to halocarbon production, in addition to photosynthetic performance.

Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling.

Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 µM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 µM and 16 µM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.

In vitro functional evaluation of isolaureline, dicentrine and glaucine enantiomers at 5-HT2 and α1 receptors.

Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT2 and α1 adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5-HT2 and adrenergic α1 receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT2 and α1 receptors with (R)-isolaureline showing the greatest potency (pKb  = 8.14 at the 5-HT2C receptor). Both (R)- and (S)-glaucine also antagonized α1 receptors, but they behaved very differently to the other compounds at 5-HT2 receptors: (S)-glaucine acted as a partial agonist at all three 5-HT2 receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT2A receptor.

Suppression of Staphylococcus aureus biofilm formation and virulence by a benzimidazole derivative, UM-C162.

Staphylococcus aureus is a major cause of nosocomial infections and secretes a diverse spectrum of virulence determinants as well as forms biofilm. The emergence of antibiotic-resistant S. aureus highlights the need for alternative forms of therapeutics other than conventional antibiotics. One route to meet this need is screening small molecule derivatives for potential anti-infective activity. Using a previously optimized C. elegans - S. aureus small molecule screen, we identified a benzimidazole derivative, UM-C162, which rescued nematodes from a S. aureus infection. UM-C162 prevented the formation of biofilm in a dose-dependent manner without interfering with bacterial viability. To examine the effect of UM-C162 on the expression of S. aureus virulence genes, a genome-wide transcriptome analysis was performed on UM-C162-treated pathogen. Our data indicated that the genes associated with biofilm formation, particularly those involved in bacterial attachment, were suppressed in UM-C162-treated bacteria. Additionally, a set of genes encoding vital S. aureus virulence factors were also down-regulated in the presence of UM-C162. Further biochemical analysis validated that UM-C162-mediated disruption of S. aureus hemolysins, proteases and clumping factors production. Collectively, our findings propose that UM-C162 is a promising compound that can be further developed as an anti-virulence agent to control S. aureus infections.

Correction: Synthesis and evaluation of nuciferine and roemerine enantiomers as 5-HT2 and α1 receptor antagonists.

[This corrects the article DOI: 10.1039/C7MD00629B.].

Synthesis and evaluation of nuciferine and roemerine enantiomers as 5-HT2 and α1 receptor antagonists.

In this study, the (S)-enantiomers of the aporphine alkaloids, nuciferine and roemerine, were prepared via a synthetic route involving catalytic asymmetric hydrogenation and both stereoisomers were evaluated in vitro for functional activity at human 5-HT2 and adrenergic α1 receptor subtypes using a transforming growth factor-α shedding assay. Both enantiomers of each of the compounds were found to act as antagonists at 5-HT2 and α1 receptors. (R)-roemerine was the most potent compound at 5-HT2A and 5-HT2C receptors (pKb = 7.8-7.9) with good selectivity compared to (S)-roemerine at these two receptors and compared to its activity at 5-HT2B, α1A, α1B and α1D receptors.

Halocarbon emissions by selected tropical seaweeds: species-specific and compound-specific responses under changing pH.

Five tropical seaweeds, Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, Padina australis Hauck, Sargassum binderi Sonder ex J. Agardh (syn. S. aquifolium (Turner) C. Agardh), Sargassum siliquosum J. Agardh and Turbinaria conoides (J. Agardh) Kützing, were incubated in seawater of pH 8.0, 7.8 (ambient), 7.6, 7.4 and 7.2, to study the effects of changing seawater pH on halocarbon emissions. Eight halocarbon species known to be emitted by seaweeds were investigated: bromoform (CHBr3), dibro-momethane (CH2Br2), iodomethane (CH3I), diiodomethane (CH2I2), bromoiodomethane (CH2BrI), bromochlorometh-ane (CH2BrCl), bromodichloromethane (CHBrCl2), and dibro-mochloromethane (CHBr2Cl). These very short-lived halocarbon gases are believed to contribute to stratospheric halogen concentrations if released in the tropics. It was observed that the seaweeds emit all eight halocarbons assayed, with the exception of K. alvarezii and S. binderi for CH2I2 and CH3I respectively, which were not measurable at the achievable limit of detection. The effect of pH on halocarbon emission by the seaweeds was shown to be species-specific and compound specific. The highest percentage changes in emissions for the halocarbons of interest were observed at the lower pH levels of 7.2 and 7.4 especially in Padina australis and Sargassum spp., showing that lower seawater pH causes elevated emissions of some halocarbon compounds. In general the seaweed least affected by pH change in terms of types of halocarbon emission, was P. australis. The commercially farmed seaweed K. alvarezii was very sensitive to pH change as shown by the high increases in most of the compounds in all pH levels relative to ambient. In terms of percentage decrease in maximum quantum yield of photosynthesis (Fv∕Fm) prior to and after incubation, there were no significant correlations with the various pH levels tested for all seaweeds. The correlation between percentage decrease in the maximum quantum yield of photosynthesis (Fv∕Fm) and halocarbon emission rates, was significant only for CH2BrCl emission by P. australis (r = 0.47; p ≤ 0.04), implying that photosynthesis may not be closely linked to halocarbon emissions by the seaweeds studied. Bromine was the largest contributor to the total mass of halogen emitted for all the seaweeds at all pH. The highest total amount of bromine emitted by K. alvarezii (an average of 98% of total mass of halogens) and the increase in the total amount of chlorine with decreasing seawater pH fuels concern for the expanding seaweed farming activities in the ASEAN region.

AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei--Crystal structure, mode of action, and biological activity.

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and ß-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.

Chalcones with electron-withdrawing and electron-donating substituents: anticancer activity against TRAIL resistant cancer cells, structure-activity relationship analysis and regulation of apoptotic proteins.

In the present study, a series of 46 chalcones were synthesised and evaluated for antiproliferative activities against the human TRAIL-resistant breast (MCF-7, MDA-MB-231), cervical (HeLa), ovarian (Caov-3), lung (A549), liver (HepG2), colorectal (HT-29), nasopharyngeal (CNE-1), erythromyeloblastoid (K-562) and T-lymphoblastoid (CEM-SS) cancer cells. The chalcone 38 containing an amino (-NH2) group on ring A was the most potent and selective against cancer cells. The effects of the chalcone 38 on regulation of 43 apoptosis-related markers in HT-29 cells were determined. The results showed that 20 apoptotic markers (Bad, Bax, Bcl-2, Bcl-w, Bid, Bim, CD40, Fas, HSP27, IGF-1, IGFBP-4, IGFBP-5, Livin, p21, Survivin, sTNF-R2, TRAIL-R2, XIAP, caspase-3 and caspase-8) were either up regulated or down regulated.

Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 µM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 µM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.