Biocatalytic Generation of o-Quinone Imines in the Synthesis of 1,4-Benzoxazines and Its Comparative Green Chemistry Metrics.

1,4-Benzoxazines are important motifs in many pharmaceuticals and can be formed by a reaction sequence involving the oxidation of o-aminophenols to their corresponding quinone imine followed by an in situ inverse electron demand Diels-Alder (IEDDA) cycloaddition with a suitable dienophile. Reported herein is the development of a reaction sequence that employs horseradish peroxidase to catalyze the oxidation of the aminophenols prior to the IEDDA as a more sustainable alternative to the use of conventional stoichiometric oxidants. The synthesis of 10 example benzoxazines is demonstrated in this "one-pot, two-step" procedure with yields between 42% and 92%. The green chemistry metrics, including the E-factor and generalized reaction mass efficiency, for this biocatalytic reaction were compared against the conventional chemical approach. It was found that the reported biocatalytic route was approximately twice as green by these measures.

Conjugated ß-Cyclodextrin Enhances the Affinity of Folic Acid towards FRα: Molecular Dynamics Study.

Drug targeting is a progressive area of research with folate receptor alpha (FRα) receiving significant attention as a biological marker in cancer drug delivery. The binding affinity of folic acid (FA) to the FRα active site provides a basis for recognition of FRα. In this study, FA was conjugated to beta-cyclodextrin (ßCD) and subjected to in silico analysis (molecular docking and molecular dynamics (MD) simulation (100 ns)) to investigate the affinity and stability for the conjugated system compared to unconjugated and apo systems (ligand free). Docking studies revealed that the conjugated FA bound into the active site of FRα with a docking score (free binding energy < -15 kcal/mol), with a similar binding pose to that of unconjugated FA. Subsequent analyses from molecular dynamics (MD) simulations, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) demonstrated that FA and FA-ßCDs created more dynamically stable systems with FRα than the apo-FRα system. All systems reached equilibrium with stable RMSD values ranging from 1.9-2.4 Å and the average residual fluctuation values of the FRα backbone atoms for all residues (except for terminal residues ARG8, THR9, THR214, and LEU215) were less than 2.1 Å with a consistent Rg value of around 16.8 Å throughout the MD simulation time (0-100 ns). The conjugation with ßCD improved the stability and decreased the mobility of all the residues (except residues 149-151) compared to FA-FRα and apo-FRα systems. Further analysis of H-bonds, binding free energy (MM-PBSA), and per residue decomposition energy revealed that besides APS81, residues HIS20, TRP102, HIS135, TRP138, TRP140, and TRP171 were shown to have more favourable energy contributions in the holo systems than in the apo-FRα system, and these residues might have a direct role in increasing the stability of holo systems.

Molecular Dynamics Simulations in Designing DARPins as Phosphorylation-Specific Protein Binders of ERK2.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play key roles in promoting cell survival and proliferation through the phosphorylation of various substrates. Remarkable antitumour activity is found in many inhibitors that act upstream of the ERK pathway. However, drug-resistant tumour cells invariably emerge after their use due to the reactivation of ERK1/2 signalling. ERK1/2 inhibitors have shown clinical efficacy as a therapeutic strategy for the treatment of tumours with mitogen-activated protein kinase (MAPK) upstream target mutations. These inhibitors may be used as a possible strategy to overcome acquired resistance to MAPK inhibitors. Here, we report a class of repeat proteins-designed ankyrin repeat protein (DARPin) macromolecules targeting ERK2 as inhibitors. The structural basis of ERK2-DARPin interactions based on molecular dynamics (MD) simulations was studied. The information was then used to predict stabilizing mutations employing a web-based algorithm, MAESTRO. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations. Two mutations, Ala → Asp and Ser → Leu, were found to perform better than the original sequence (DARPin E40) based on the associated energy and key residues involved in protein-protein interaction. MD simulations and analysis of the data obtained on these mutations supported our predictions.

Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda.

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.

Analogues of 2'-hydroxychalcone with modified C4-substituents as the inhibitors against human acetylcholinesterase.

A series of C4-substituted tertiary nitrogen-bearing 2'-hydroxychalcones were designed and synthesised based on a previous mixed type acetylcholinesterase inhibitor. Majority of the 2'-hydroxychalcone analogues displayed a better inhibition against acetylcholinesterase (AChE) than butyrylcholinesterase (BuChE). Among them, compound 4c was identified as the most potent AChE inhibitor (IC50: 3.3 µM) and showed the highest selectivity for AChE over BuChE (ratio >30:1). Molecular docking studies suggested that compound 4c interacts with both the peripheral anionic site (PAS) and catalytic anionic site (CAS) regions of AChE. ADMET analysis confirmed the therapeutic potential of compound 4c based on its blood-brain barrier penetrating. Overall, the results suggest that this 2'-hydroxychalcone deserves further investigation into the therapeutic lead for Alzheimer's disease (AD).

A review on chitosan and its development as pulmonary particulate anti-infective and anti-cancer drug carriers.

Chitosan, as a biodegradable and biocompatible polymer, is characterized by anti-microbial and anti-cancer properties. It lately has received a widespread interest for use as the pulmonary particulate backbone materials of drug carrier for the treatment of infectious disease and cancer. The success of chitosan as pulmonary particulate drug carrier is a critical interplay of their mucoadhesive, permeation enhancement and site/cell-specific attributes. In the case of nanocarriers, various microencapsulation and micro-nano blending systems have been devised to equip them with an appropriate aerodynamic character to enable efficient pulmonary aerosolization and inhalation. The late COVID-19 infection is met with acute respiratory distress syndrome and cancer. Chitosan and its derivatives are found useful in combating HCoV and cancer as a function of their molecular weight, substituent type and its degree of substitution. The interest in chitosan is expected to rise in the next decade from the perspectives of drug delivery in combination with its therapeutic performance.

Recent advances in celluloses and their hybrids for stimuli-responsive drug delivery.

The limitations of existing drug delivery systems (DDS) such as non-specific bio-distribution and poor selectivity have led to the exploration of a variety of carrier platforms to facilitate highly desirable and efficient drug delivery. Stimuli-responsive DDS are one of the most versatile and innovative approach to steer the compounds to the intended sites by exploiting their responsiveness to a range of various triggers. Preparation of stimuli-responsive DDS using celluloses and their derivatives offer a remarkable advantage over conventional polymer materials. In this review, we highlight on state-of-art progress in developing cellulose/cellulose hybrid stimuli-responsive DDS, which covers the preparation techniques, physicochemical properties, basic principles and, mechanisms of stimuli effect on drug release from various types of cellulose based carriers, through recent innovative investigations. Attention has been paid to endogenous stimuli (pH, temperature, redox gradient and ionic-strength) responsive DDS and exogenous stimuli (light, magnetic field and electric field) responsive DDS, where the cellulose-based materials have been extensively employed. Furthermore, the current challenges and future prospects of these DDS are also discussed at the end.

Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda.

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.

Dynamics and binding interactions of peptide inhibitors of dengue virus entry.

In this study, we investigate the binding interactions of two synthetic antiviral peptides (DET2 and DET4) on type II dengue virus (DENV2) envelope protein domain III. These two antiviral peptides are designed based on the domain III of the DENV2 envelope protein, which has shown significant inhibition activity in previous studies and can be potentially modified further to be active against all dengue strains. Molecular docking was performed using AutoDock Vina and the best-ranked peptide-domain III complex was further explored using molecular dynamics simulations. Molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) was used to calculate the relative binding free energies and to locate the key residues of peptide-protein interactions. The predicted binding affinity correlated well with the previous experimental studies. DET4 outperformed DET2 and is oriented within the binding site through favorable vdW and electrostatic interactions. Pairwise residue decomposition analysis has revealed several key residues that contribute to the binding of these peptides. Residues in DET2 interact relatively lesser with the domain III compared to DET4. Dynamic cross-correlation analysis showed that both the DET2 and DET4 trigger different dynamic patterns on the domain III. Correlated motions were seen between the residue pairs of DET4 and the binding site while binding of DET2 results in anti-correlated motion on the binding site. This work showcases the use of computational study in elucidating and explaining the experiment observation on an atomic level.

Production of green biofuel by using a goat manure supported Ni-Al hydrotalcite catalysed deoxygenation process.

The high oxygen content in natural biomass resources, such as vegetable oil or biomass-pyrolysed bio oil, is the main constraint in their implementation as a full-scale biofuel for the automotive industry. In the present study, renewable fuel with petrodiesel-like properties was produced via catalytic deoxygenation of oleic acid in the absence of hydrogen (H2). The deoxygenation pathway of oleic acid to bio-hydrocarbon involves decarboxylation/decarbonylation of the oxygen content from the fatty acid structure in the form of carbon dioxide (CO2)/carbon monoxide (CO), with the presence of a goat manure supported Ni-Al hydrotalcite (Gm/Ni-Al) catalyst. Goat manure is an abundant bio-waste, containing a high mineral content, urea as well as cellulosic fiber of plants, which is potentially converted into activated carbon. Synthesis of Gm/Ni-Al was carried out by incorporation of pre-activated goat manure (GmA) during co-precipitation of Ni-Al catalyst with 1 : 3, 1 : 1 and 3 : 1 ratios. The physico-chemical properties of the catalysts were characterized by X-ray diffractometry (XRD), Brunauer-Emmet-Teller (BET) surface area, field emission surface electron microscopy (FESEM) and temperature program desorption ammonia (TPD-NH3) analysers. The catalytic deoxygenation reaction was performed in a batch reactor and the product obtained was characterized by using gas chromatography-mass spectroscopy (GCMS) for compound composition identification as well as gas chromatography-flame ionisation detector (GC-FID) for yield and selectivity determination. The optimization and evaluation were executed using response surface methodology (RSM) in conjunction with central composite design (CCD) with 5-level-3-factors. From the RSM reaction model, it was found that the Gm/Ni-Al 1 : 1 catalysed deoxygenation reaction gives the optimum product yield of 97.9% of hydrocarbon in the range of C8-C20, with diesel selectivity (C17: heptadecane and heptadecene compounds) of 63.7% at the optimal reaction conditions of: (1) reaction temperature: 327.14 °C, (2) reaction time: 1 h, and (3) catalyst amount: 5 wt%.

Development of a NS2B/NS3 protease inhibition assay using AlphaScreen® beads for screening of anti-dengue activities.

BACKGROUND: Dengue infection is an endemic infectious disease and it can lead to dengue fever, dengue hemorrhagic fever, and/or dengue shock syndromes. Dengue NS2B/NS3 protease complex is essential for viral replication and is a primary target for anti-dengue drug development. In this study, a NS2B/NS3 protease inhibition assay was developed using AlphaScreen® beads and was used to screen compounds for their protease inhibition activities. METHODS: The assay system utilized a known NS2B/NS3 peptide substrate, a recombinant of NS2B/NS3 protease with proprietary StrepTactin® donor and nickel chelate acceptor beads in 384-well format. RESULTS: The optimized assay to screen for NS2B/NS3 protease inhibitors was demonstrated to be potentially useful with reasonable z' factor, coefficient variance and signal to background ratio. However, screening of synthesized thioguanine derivatives using the optimized AlphaScreen® assay revealed weak NS2B/NS3 inhibition activities. CONCLUSION: The AlphaScreen® assay to screen for NS2B/NS3 protease inhibitors is potentially applicable for high throughput screening.

Lung cancer: active therapeutic targeting and inhalational nanoproduct design.

INTRODUCTION: Pulmonary drug delivery is organ-specific and benefits local drug action for lung cancer. The use of nanotechnology and targeting ligand enables cellular-specific drug action. Combination approaches increase therapeutic efficacy and reduce adverse effects of cancer chemotherapeutics that have narrow therapeutic index window and high cytotoxicity levels. The current progress of inhaled cancer chemotherapeutics has not been examined with respect to targeting strategy and clinical application potential. AREAS COVERED: This review examines the state of the art in passive (processing and formulation) and active (targeting ligand and receptor binding) technologies in association with the use of nanocarrier to combat lung cancer. It highlights routes to equip nanocarrier with targeting ligands as a function of the chemistry of participating biomolecules and challenges in inhalational nanoproduct development and clinical applications. Both research and review articles were examined using the Scopus, Elsevier, Web of Science, Chemical Abstracts, Medline, CASREACT, CHEMCATS, and CHEMLIST database with the majority of information retrieved between those of 2000-2018. EXPERT COMMENTARY: The therapeutic efficacy of targeting ligand-decorated nanocarriers needs to be demonstrated in vivo in the form of finished inhalational products. Their inhalation efficiency and medical responses require further examination. Clinical application of inhaled nanocancer chemotherapeutics is premature.

Phosphodiesterase-5 inhibitors and their analogues as adulterants of herbal and food products: analysis of the Malaysian market, 2014-16.

Adulteration of herbal health supplements with phosphodiesterase-5 (PDE-5) inhibitors and their analogues is becoming a worldwide problem. The aim of this study was to investigate herbal and food products sold in the Malaysian market for the presence of these adulterants. Sixty-two products that claim to enhance men's sexual health were sampled between April 2014 and April 2016. These products included unregistered products seized by the Pharmacy Enforcement Division of the Ministry of Health (n = 39), products sent to the National Pharmaceutical Regulatory Agency for pre-registration testing (n = 9) and products investigated under the post-registration market surveillance programme (n = 14). The products were tested against an in-house spectral library consisting of 61 PDE-5 inhibitors and analogues using a validated liquid chromatography-mass spectrometry ion-trap-time-of-flight (LC-MS IT-TOF) method. Thirty-two (82%) of the unregistered products and two (14%) of the registered products were found to be adulterated with at least one PDE-5 inhibitor or analogue, while none of the pre-registration products contained adulterants. A total of 16 different adulterants were detected and 36% of the adulterated products contained a mixture of two or more adulterants. This study has demonstrated that the adulteration of unregistered herbal products in the Malaysian market is an alarming issue that needs to be urgently addressed by the relevant authorities.

Conformational and energy evaluations of novel peptides binding to dengue virus envelope protein.

Effective novel peptide inhibitors which targeted the domain III of the dengue envelope (E) protein by blocking dengue virus (DENV) entry into target cells, were identified. The binding affinities of these peptides towards E-protein were evaluated by using a combination of docking and explicit solvent molecular dynamics (MD) simulation methods. The interactions of these complexes were further investigated by using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) and Molecular Mechanics Generalized Born Surface Area (MMGBSA) methods. Free energy calculations of the peptides interacting with the E-protein demonstrated that van der Waals (vdW) and electrostatic interactions were the main driving forces stabilizing the complexes. Interestingly, calculated binding free energies showed good agreement with the experimental dissociation constant (Kd) values. Our results also demonstrated that specific residues might play a crucial role in the effective binding interactions. Thus, this study has demonstrated that a combination of docking and molecular dynamics simulations can accelerate the identification process of peptides as potential inhibitors of dengue virus entry into host cells.

Anti-inflammatory trends of new benzimidazole derivatives.

AIM: In present study, the anti-inflammatory activities of a new series of benzimidazole derivatives were studied, investigating their inhibition of secretory phospholipase A2, lipoxygenase, COXs and lipopolysaccharide-induced secretion of TNF-α and IL-6 in mouse RAW264.7 macrophages. RESULTS: Synthesized compounds effectively inhibited proinflammatory enzymes and cytokines. CONCLUSION: A strong inhibition of secretory phospholipases A2 was exhibited by benzimidazole derivatives with trifluoromethyl and methoxy substitutions at position 4 of attached phenyl, whereas compound 8 containing pyridine ring substituted with amino group showed very potent 5-lipoxygenase inhibition. Molecular docking experiments were carried out to elucidate the molecular basis of the observed inhibitory activities.

Benzimidazole derivatives as potential dual inhibitors for PARP-1 and DHODH.

Poly (ADP-ribose) polymerases (PARPs) play diverse roles in various cellular processes that involve DNA repair and programmed cell death. Amongst these polymerases is PARP-1 which is the key DNA damage-sensing enzyme that acts as an initiator for the DNA repair mechanism. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the pyrimidine biosynthetic pathway which is an important target for anti-hyperproliferative and anti-inflammatory drug design. Since these enzymes share a common role in the DNA replication and repair mechanisms, it may be beneficial to target both PARP-1 and DHODH in attempts to design new anti-cancer agents. Benzimidazole derivatives have shown a wide variety of pharmacological activities including PARP and DHODH inhibition. We hereby report the design, synthesis and bioactivities of a series of benzimidazole derivatives as inhibitors of both the PARP-1 and DHODH enzymes.

Distribution of Flavonoids and Cyclohexenyl Chalcone Derivatives in Conventional Propagated and In Vitro-Derived Field-Grown Boesenbergia rotunda (L.) Mansf.

The distribution patterns of flavonoids and cyclohexenyl chalcone derivatives in conventional propagated (CP) and in vitro-derived (CPA) field-grown plants of an important medicinal ginger, Boesenbergia rotunda, are described. A total of eight compounds were extracted from six organs (rootlet, rhizome, shoot base, maroon stem, stalk, and leaf) of the CP and CPA plants. Five major chromatographic peaks, namely, alpinetin, pinocembrin, pinostrobin, 4-hydroxypanduratin A, and panduratin A, were consistently observed by high performance liquid chromatography. Nonaerial organs had higher levels of flavonoids than the aerial ones for all types of samples. Among the compounds detected, pinostrobin and 4-hydroxypanduratin A were the most abundant flavonoid and cyclohexenyl chalcone derivative, respectively. The distribution and abundance of the bioactive compounds suggested that the shoot base could be more potentially useful for medicinal application than other organs of the plant and may be the site of storage or occurrence of biosynthetic enzymatic activities.

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 µM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources.

Flavonoids with M1 muscarinic acetylcholine receptor binding activity.

Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer's disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki=40-110 µM), comparable to that of acetylcholine (Ki=59 µM). Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.