Bimo Ario Tejo

Structure-based and ligand-based virtual screening of novel methyltransferase inhibitors of the dengue virus

BackgroundThe dengue virus is the most significant arthropod-borne human pathogen, and an increasing number of cases have been reported over the last few decades. Currently neither vaccines nor drugs against the dengue virus are available. NS5 methyltransferase (MTase), which is located on the surface of the dengue virus and assists in viral attachment to the host cell, is a promising antiviral target. In order to search for novel inhibitors of NS5 MTase, we performed a computer-aided virtual screening of more than 5 million commercially available chemical compounds using two approaches: i) structure-based screening using the crystal structure of NS5 MTase and ii) ligand-based screening using active ligands of NS5 MTase. Structure-based screening was performed using the LIDAEUS (LIgand Discovery At Edinburgh UniverSity) program. The ligand-based screening was carried out using the EDULISS (EDinburgh University LIgand Selection System) program.ResultsThe selection of potential inhibitors of dengue NS5 MTase was based on two criteria: the compounds must bind to NS5 MTase with a higher affinity than that of active NS5 MTase ligands, such as ribavirin triphosphate (RTP) and S-adenosyl-L-homocysteine (SAH); and the compounds must interact with residues that are catalytically important for the function of NS5 MTase. We found several compounds that bind strongly to the RNA cap site and the S-adenosyl-L-methionine (SAM) binding site of NS5 MTase with better binding affinities than that of RTP and SAH. We analyzed the mode of binding for each compound to its binding site, and our results suggest that all compounds bind to their respective binding sites by interacting with, and thus blocking, residues that are vital for maintaining the catalytic activity of NS5 MTase.ConclusionsWe discovered several potential compounds that are active against dengue virus NS5 MTase through virtual screening using structure-based and ligand-based methods. These compounds were predicted to bind into the SAM binding site and the RNA cap site with higher affinities than SAH and RTP. These compounds are commercially available and can be purchased for further biological activity tests.

An improved method for the preparations of nanostructured lipid carriers containing heat-sensitive bioactives.

Heat-sensitive bioactive compounds such as β-carotene and tocols, are widely used in the pharmaceutical and cosmetic fields. Their chemical stability in delivery systems is one of the major concerns in the production of nanostructured lipid carriers (NLCs). A previously established high-temperature high-pressure homogenisation technique involved in the preparation of NLCs can cause degradation of heat-sensitive compounds. Therefore, a novel preparation process needs to be developed to minimise the degradation of heat-sensitive active compounds during the preparation of NLCs. In this work, modified methods A and B were designed to minimise the degradation of β-carotene and tocols during the production of NLCs. These methods improved the chemical stability of heat-sensitive bioactive compounds (β-carotene and tocols) significantly compared to the previously established method. The physical stability of the formulation was maintained throughout study duration.

Bioassay-guided identification of an anti-inflammatory prenylated acylphloroglucinol from Melicope ptelefolia and molecular insights into its interaction with 5-lipoxygenase.

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.

Various polar tripeptides as asymmetric organocatalyst in direct aldol reactions in aqueous media

A series of tripeptide organocatalysts containing a secondary amine group and two amino acids with polar side chain units were developed and evaluated in the direct asymmetric intermolecular aldol reaction of 4-nitrobenzaldehyde and cyclohexanone. The effectiveness of short polar peptides as asymmetric catalysts in aldol reactions to attain high yields of enantio- and diastereoselective isomers were investigated. In a comparison, glutamic acid and histidine produced higher % ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric aldol addition reaction in aqueous media.

ICAM-1 Peptide Inhibitors of T-cell Adhesion bind to the allosteric site of LFA-1. An NMR Characterization

We have used nuclear magnetic resonance to characterize the binding site of two intercellular adhesion molecule‐1 derived cyclic peptides, cIBC and cIBR, to the I‐domain of leukocyte function‐associated antigen‐1. These peptides inhibit the leukocyte function‐associated antigen‐1/intercellular adhesion molecule‐1 interaction known to play a key role in autoimmune diseases and cancer metastasis. Perturbation of the chemical shifts and intensities of the nuclear magnetic resonance signals corresponding to a number of residues of the I‐domain of leukocyte function‐associated antigen‐1 show that both peptides bind to the I‐domain allosteric site, the binding site of I‐domain allosteric inhibitors such as lovastatin, and therefore the peptides probably also act as allosteric inhibitors of leukocyte function‐associated antigen‐1. Molecular models of the interaction of these two cyclic peptides with leukocyte function‐associated antigen‐1 I‐domain show that the binding mode of the three molecules are analogous: the hydrophobic residues of the peptides remain buried and occupy the same positions as the apolar groups of lovastatin, while the peptides regions containing the most polar residues are flexible and primarily exposed to the solvent. These results suggest an allosteric mechanism for the inhibitory effect on T‐cell adhesion displayed by both peptides, which exhibit potential as therapeutic agents.

Modeling stability and flexibility of α-Chymotrypsin in room temperature ionic liquids

We investigate the structure and dynamics of α-Chymotrypsin in five room temperature ionic liquids (RTILs) sharing a common cation, hydrated with different water percentages (w/w) (weight of water over protein). Results from molecular dynamics simulations are correlated with experimental evidences from studies on the activity of enzymes in RTILs. α-Chymotrypsin protein structure is closer to its native crystallographic structure in RTILs than in aqueous environment. We show that the structural properties of α-Chymotrypsin were affected by the water concentration assayed in a typical bell-shaped profile, which is also frequently reported for organic solvents. The protein structure was more native like at 10–20% of water (w/w) for all RTILs except for [BMIM][Cl]. We found that the fluctuations of the main chain in [BMIM][BF4] and [BMIM][TfO] were not significantly affected by the increasing amount of water. However, we were able to show that the flexible regions were the ones more hydrated, indicating that water is responsible for the flexibility of the protein. The solvation of the enzyme in water-immiscible RTILs, such as [BMIM][PF6] and [BMIM][Tf2N] lead to higher enzyme flexibility at increased water content. Enzyme solvation by [BMIM][Cl] resulted in ion penetration in the core enzyme structure, causing incremented flexibility and destabilization at low water percentages. All RTILs stripped water molecules from the protein surface, following a similar behavior also found in organic solvents. Anions formed structured arrangements around the protein, which allowed non-stripped water molecules to localize on the protein surface.

Characterization of Binding Properties of ICAM‐1 Peptides to LFA‐1: Inhibitors of T‐cell Adhesion

In the present study, we characterized the binding site of two intercellular adhesion molecule‐1‐derived cyclic peptides, cIBC and cIBR, to the LFA‐1 on the surface of T cells. These peptides had been able to inhibit LFA‐1/intercellular adhesion molecule‐1 signal by blocking the signal‐2 of immune synapse. Both peptides prefer to bind to the closed form of LFA‐1 I‐domain, indicating that two peptides act as allosteric inhibitors against intercellular adhesion molecule‐1. Binding site mapping using monoclonal antibodies proposes that cIBC binds to around residues 266–272 of LFA‐1 I‐domain where this site is adjacent to the metal ion‐dependent adhesion site. On the other hand, cIBR binds to the pocket called L‐site where is distant from metal ion‐dependent adhesion site. Cross‐inhibition mapping between two peptides show that cIBR could inhibit the binding of cIBC but not vice versa, suggesting that cIBR has some properties that allow this peptide bind to more than one site. Structural comparison between cIBC and cIBR reveals that cIBR is more flexible than cIBC, allowing this peptide bind to exposed region, such as cIBC‐binding site as well as cramped pocket like L‐site. Our findings are important for understanding the selectivity of cIBC and cIBR peptides; thus, they can be conjugated with drugs and transported specifically to the target.

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening

BackgroundRheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays.ResultsTwenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment.ConclusionThree compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.

Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system

A relationship is proposed for the interfacial partitioning of protein in poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). The relationship relates the natural logarithm of interfacial partition coefficient, ln G to the PEG concentration difference between the top and bottom phases, Δ[PEG], with the equation ln G=AΔ[PEG]+B. Results showed that this relationship provides good fits to the partition of bovine serum albumin (BSA) in ATPS which is comprised of phosphate and PEG of four different molecular weight 1450g/mol, 2000g/mol, 3350g/mol and 4000g/mol, with the tie-line length (TLL) in the range of 44-60% (w/w) at pH 7.0. The decrease of A values with the increase of PEG molecular weight indicates that the correlation between ln G and Δ[PEG] decreases with the increase in PEG molecular weight and the presence of protein-polymer hydrophobic interaction. When temperature was increased, a non-linear relationship of ln G inversely proportional to temperature was observed. The amount of proteins adsorbed at the interface increased proportionally with the amount of BSA loaded whereas the partition coefficient, K remained relatively constant. The relationship proposed could be applied to elucidate interfacial partitioning behaviour of other biomolecules in polymer-salt ATPS.

Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.