Hispidin oligomers are styrylpyrone pigments isolated from the medicinal fungi Inonotus xeranticus and Phellinus linteus. They exhibit diverse biological activities and strong free radical scavenging activity. To rationalize the antioxidant activity of a series of four hispidin oligomers and determine the favored mechanism involved in free radical scavenging, DFT calculations were carried out at the B3P86/6-31+G (d, p) level of theory in gas and solvent. The results showed that bond dissociation enthalpies of OH groups of hispidin oligomers (ArOH) and spin density delocalization of related radicals (ArO•) are the appropriate parameters to clarify the differences between the observed antioxidant activities for the four oligomers. The effect of the number of hydroxyl groups and presence of a catechol moiety conjugated to a double bond on the antioxidant activity were determined. Thermodynamic and kinetic studies showed that the PC-ET mechanism is the main mechanism involved in free radical scavenging. The spin density distribution over phenoxyl radicals allows a better understanding of the hispidin oligomers formation.
Extensive chromatographic separations performed on the basic (pH=8-10) chloroform soluble fraction of Aconitum heterophyllum resulted in the isolation of three new diterpenoid alkaloids, 6β-Methoxy, 9β-dihydroxylheteratisine (1), 1α,11,13β-trihydroxylhetisine (2), 6,15β-dihydroxylhetisine (3), and the known compounds iso-atisine (4), heteratisine (5), hetisinone (6), 19-epi-isoatisine (7), and atidine (8). Structures of the isolated compounds were established by means of mass and NMR spectroscopy as well as single crystal X-ray crystallography. Compounds 1-8 were screened for their antioxidant and enzyme inhibition activities followed by in silico studies to find out the possible inhibitory mechanism of the tested compounds. This work is the first report demonstrating significant antioxidant and anticholinesterase potentials of diterpenoid alkaloids isolated from a natural source.
Phenolic Schiff bases are known for their diverse biological activities and ability to scavenge free radicals. To elucidate (1) the structure-antioxidant activity relationship of a series of thirty synthetic derivatives of 2-methoxybezohydrazide phenolic Schiff bases and (2) to determine the major mechanism involved in free radical scavenging, we used density functional theory calculations (B3P86/6-31+(d,p)) within polarizable continuum model. The results showed the importance of the bond dissociation enthalpies (BDEs) related to the first and second (BDEd) hydrogen atom transfer (intrinsic parameters) for rationalizing the antioxidant activity. In addition to the number of OH groups, the presence of a bromine substituent plays an interesting role in modulating the antioxidant activity. Theoretical thermodynamic and kinetic studies demonstrated that the free radical scavenging by these Schiff bases mainly proceeds through proton-coupled electron transfer rather than sequential proton loss electron transfer, the latter mechanism being only feasible at relatively high pH.
The progress of novel sorbents and their function in preconcentration techniques for determination of trace elements is a topic of great importance. This review discusses numerous analytical approaches including the preparation and practice of unique modification of solid-phase materials. The performance and main features of ion-imprinting polymers, carbon nanotubes, biosorbents, and nanoparticles are described, covering the period 2007-2012. The perspective and future developments in the use of these materials are illustrated.
Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
Coronavirus-like organisms have been previously identified in Arthropod ectoparasites (such as ticks and unfed cat flea). Yet, the question regarding the possible role of these arthropods as SARS-CoV-2 passive/biological transmission vectors is still poorly explored. In this study, we performed in silico structural and binding energy calculations to assess the risks associated with possible ectoparasite transmission. We found sufficient similarity between ectoparasite ACE and human ACE2 protein sequences to build good quality 3D-models of the SARS-CoV-2 Spike:ACE complex to assess the impacts of ectoparasite mutations on complex stability. For several species (e.g., water flea, deer tick, body louse), our analyses showed no significant destabilisation of the SARS-CoV-2 Spike:ACE complex, suggesting these species would bind the viral Spike protein. Our structural analyses also provide structural rationale for interactions between the viral Spike and the ectoparasite ACE proteins. Although we do not have experimental evidence of infection in these ectoparasites, the predicted stability of the complex suggests this is possible, raising concerns of a possible role in passive transmission of the virus to their human hosts.
Previous molecular dynamic simulations have reported elongation of the existing beta-sheet in prion proteins. Detailed examination has shown that these elongations do not extend beyond the proline residues flanking these beta-sheets. In addition, proline has also been suggested to possess a possible structural role in preserving protein interaction sites by preventing invasion of neighboring secondary structures. In this work, we have studied the possible structural role of the flanking proline residues by simulating mutant structures with alternate substitution of the proline residues with valine. Simulations showed a directional inhibition of elongation, with the elongation progressing in the direction of valine including evident inhibition of elongation by existing proline residues. This suggests that the flanking proline residues in prion proteins may have a containment role and would confine the beta-sheet within a specific length.
The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.
Bioassay-guided isolation protocol was performed on petroleum ether extract of Peperomia blanda (Jacq.) Kunth using column chromatographic techniques. Five compounds were isolated and their structures were elucidated via one-dimensional (1D) and two-dimensional (2D) NMR, gas chromatography mass sectroscopy (GCMS), liquid chromatography mass spectroscopy (LCMS), and ultraviolet (UV) and infrared (IR) analyses. Dindygulerione E (a new compound), and two compounds isolated from P. blanda for the first time-namely, dindygulerione A and flavokawain A-are reported herein. Antimicrobial activity was screened against selected pathogenic microbes, and minimum inhibitory concentrations (MIC) were recorded within the range of 62-250 μg/mL. Assessment of the pharmacotherapeutic potential has also been done for the isolated compounds, using the Prediction of Activity spectra for Substances (PASS) software, and different activities of compounds were predicted. Molecular docking, molecular dynamics simulation and molecular mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations have proposed the binding affinity of these compounds toward methylthioadenosine phosphorylase enzyme, which may explain their inhibitory actions.
A bioassay-guided investigation of Melicope ptelefolia Champ ex Benth (Rutaceae) resulted in the identification of an acyphloroglucinol, 2,4,6-trihydroxy-3-geranylacetophenone or tHGA, as the active principle inhibiting soybean 15-LOX. The anti-inflammatory action was also demonstrated on human leukocytes, where the compound showed prominent inhibitory activity against human PBML 5-LOX, with an IC(50) value of 0.42 μM, very close to the effect produced by the commonly used standard, NDGA. The compound concentration-dependently inhibited 5-LOX product synthesis, specifically inhibiting cysteinyl leukotriene LTC(4) with an IC(50) value of 1.80 μM, and showed no cell toxicity effects. The anti-inflammatory action does not seem to proceed via redox or metal chelating mechanism since the compound tested negative for these bioactivities. Further tests on cyclooxygenases indicated that the compound acts via a dual LOX/COX inhibitory mechanism, with greater selectivity for 5-LOX and COX-2 (IC(50) value of 0.40 μM). The molecular features that govern the 5-LOX inhibitory activity was thus explored using in silico docking experiments. The residues Ile 553 and Hie 252 were the most important residues in the interaction, each contributing significant energy values of -13.45 (electrostatic) and -5.40 kcal/mol (electrostatic and Van der Waals), respectively. The hydroxyl group of the phloroglucinol core of the compound forms a 2.56Å hydrogen bond with the side chain of the carboxylate group of Ile 553. Both Ile 553 and Hie 252 are crucial amino acid residues which chelate with the metal ion in the active site. Distorting the geometry of these ligands could be the reason for the inhibition activity shown by tHGA. The molecular simulation studies supported the bioassay results and served as a good model for understanding the way tHGA binds in the active site of human 5-LOX enzyme.
Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH2 -H4 folate substrate. NmMTHFR utilized NADH (Km = 44 μM) as an electron donor in the NAD(P)H-CH2 -H4 folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH2 -H4 folate substrate to bind. The unique topology of its NADH and CH2 -H4 folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.
Hyperpigmentation in human skin and enzymatic browning in fruits, which are caused by tyrosinase enzyme, are not desirable. Investigations in the discovery of tyrosinase enzyme inhibitors and search for improved cytotoxic agents continue to be an important line in drug discovery and development. In present work, a new series of 30 compounds bearing α,β-unsaturated carbonyl moiety was designed and synthesized following curcumin as model. All compounds were evaluated for their effects on human cancer cell lines and mushroom tyrosinase enzyme. Moreover, the structure-activity relationships of these compounds are also explained. Molecular modeling studies of these new compounds were carried out to explore interactions with tyrosinase enzyme. Synthetic curcumin-like compounds (2a-b) were identified as potent anticancer agents with 81-82% cytotoxicity. Five of these newly synthesized compounds (1a, 8a-b, 10a-b) emerged to be the potent inhibitors of mushroom tyrosinase, providing further insight into designing compounds useful in fields of food, health, and agriculture.
A cytotoxic bisindole alkaloid possessing an unprecedented structure in which two indole moieties are bridged by an aromatic spacer unit has been isolated from Alstonia angustifolia. The structure was established by analysis of the spectroscopic data and confirmed by X-ray diffraction analysis. A possible biogenetic pathway from pyrocatechuic acid and pleiocarpamine is presented.
Bisindolylmethane thiosemicarbazides 1-18 were synthesized, characterized by 1H NMR and ESI MS and evaluated for urease inhibitory potential. All analogs showed outstanding urease inhibitory potentials with IC50 values ranging between 0.14 ± 0.01 to 18.50 ± 0.90 μM when compared with the standard inhibitor thiourea having IC50 value 21.25 ± 0.90 μM. Among the series, analog 9 (0.14 ± 0.01 μM) with di-chloro substitution on phenyl ring was identified as the most potent inhibitor of urease. The structure activity relationship has been also established on the basis of binding interactions of the active analogs. These binding interactions were identified by molecular docking studies.
Two new bisindole alkaloids, bisnicalaterines B and C (1 and 2) consisting of an eburnane and a corynanthe type of skeletons, were isolated from the bark of Hunteria zeylanica. Their absolute structures were determined by combination of NMR, CD, and computational methods, and each of them was shown to be in an atropisomeric relationship. Bisnicalaterines B and C (1 and 2) showed potent vasorelaxant activity on isolated rat aorta.
Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.
This chapter describes the generation of the data in the CATH-Gene3D online resource and how it can be used to study protein domains and their evolutionary relationships. Methods will be presented for: comparing protein structures, recognizing homologs, predicting domain structures within protein sequences, and subclassifying superfamilies into functionally pure families, together with a guide on using the webpages.