Two New Cyclic Tetrapeptides of Streptomyces rutgersensis T009 Isolated from Elaphodus davidianus Excrement

Two new cyclic tetrapeptides, cyclo(l ‐Val‐l ‐Leu‐l ‐Val‐l ‐Ile) ( 1 ) and cyclo(l ‐Leu‐l ‐Leu‐l ‐Ala‐l ‐Ala) ( 2 ), and 15 known compounds, cyclo(Gly‐l ‐Leu‐Gly‐l ‐Leu) ( 3 ), cyclo(l ‐Ser‐l ‐Phe) ( 4 ), cyclo(l ‐Leu‐l ‐Ile) ( 5 ), cyclo(l ‐Tyr‐l ‐Phe) ( 6 ), cyclo(Gly‐l ‐Trp) ( 7 ), cyclo(l ‐Leu‐l ‐Tyr) ( 8 ), cyclo(Gly‐l ‐Phe) ( 9 ), cyclo(l ‐Phe‐trans‐4‐hydroxy‐l ‐Pro) ( 10 ), cyclo(l ‐Leu‐l ‐Leu) ( 11 ), cyclo(l ‐Val‐l ‐Phe) ( 12 ), cyclo(l ‐Val‐l ‐Leu) ( 13 ), cyclo(l ‐Ile‐l ‐Ile) ( 14 ), cyclo(l ‐Tyr‐l ‐Tyr) ( 15 ), turnagainolide A ( 16 ), and bacimethrin ( 17 ) were isolated from the fermentation broth of Streptomyces rutgersensis T009 obtained from Elaphodus davidianus excrement. Their structures were identified on the basis of spectroscopic analysis. Meanwhile, the absolute configurations of the amino acid residues of compounds 1 and 2 were determined by advanced Marfey method. Compound 3 was obtained from a natural source for the first time. The X‐ray single crystal diffraction data of bacimethrin ( 17 ) were also reported for the first time. Compounds 1  –  17 exhibited no antimicrobial activities with the MICs > 100 μg/ml.     

Rose Bengal‐Sensitized Photooxidation of the Dipeptides l‐Tryptophyl‐l‐Phenylalanine,l‐Tryptophyl‐l‐Tyrosine and l‐Tryptophyl‐l‐Tryptophan: Kinetics,Mechanism and Photoproducts¶

The Rose Bengal‐sensitized photooxidations of the dipeptides l ‐tryptophyl‐l ‐phenylalanine (Trp‐Phe), l ‐tryptophyl‐l ‐tyrosine (Trp‐Tyr) and l ‐tryptophyl‐l ‐tryptophan (Trp‐Trp) have been studied in pH 7 water solution using static photolysis and time‐resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O₂(¹Δ_g)) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di‐ and polypeptides of photooxidizable amino acids. The ratio between chemical (k_r) and overall (k_t) rate constants for the interaction O₂(¹Δ_g)‐dipeptide indicates that Trp‐Phe and Trp‐Trp are good candidates to suffer photodynamic action, with k_rlk_t values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp‐Tyr, a lower k_rlk_t value (0.18) has been found, likely as a result of the high component of physical deactivation of O₂(¹Δ_g) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O₂(¹Δ_g) attack is the Trp group and suggests a much lower accumulation of kynurenine‐type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a‐oxidized hydrogenated pyrrolo[2,3‐b]indoles.     

Fast and accurate predictions of binding free energies using MM‐PBSA and MM‐GBSA

In the drug discovery process, accurate methods of computing the affinity of small molecules with a biological target are strongly needed. This is particularly true for molecular docking and virtual screening methods, which use approximated scoring functions and struggle in estimating binding energies in correlation with experimental values. Among the various methods, MM‐PBSA and MM‐GBSA are emerging as useful and effective approaches. Although these methods are typically applied to large collections of equilibrated structures of protein‐ligand complexes sampled during molecular dynamics in water, the possibility to reliably estimate ligand affinity using a single energy‐minimized structure and implicit solvation models has not been explored in sufficient detail. Herein, we thoroughly investigate this hypothesis by comparing different methods for the generation of protein‐ligand complexes and diverse methods for free energy prediction for their ability to correlate with experimental values. The methods were tested on a series of structurally diverse inhibitors of Plasmodium falciparum DHFR with known binding mode and measured affinities. The results showed that correlations between MM‐PBSA or MM‐GBSA binding free energies with experimental affinities were in most cases excellent. Importantly, we found that correlations obtained with the use of a single protein‐ligand minimized structure and with implicit solvation models were similar to those obtained after averaging over multiple MD snapshots with explicit water molecules, with consequent save of computing time without loss of accuracy. When applied to a virtual screening experiment, such an approach proved to discriminate between true binders and decoy molecules and yielded significantly better enrichment curves. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Guanitrypmycin Biosynthetic Pathways Imply Cytochrome P450 Mediated Regio‐ and Stereospecific Guaninyl‐Transfer Reactions

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential has proved to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450‐associated cyclodipeptide synthase‐containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, the C3‐guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo‐l ‐Trp‐l ‐Phe and cyclo‐l ‐Trp‐l ‐Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N‐methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio‐ and stereospecific 3α‐guaninylation at the indole ring of the tryptophanyl moiety. Isotope‐exchange experiments provided evidence for the non‐enzymatic epimerization of the biosynthetic pathway products via keto–enol tautomerism. This post‐pathway modification during cultivation further increases the structural diversity of guanitrypmycins.     

Molecular modelling prediction of ligand binding site flexibility

We have investigated the efficacy of generating multiple sidechain conformations using a rotamer library in order to find the experimentally observed ligand binding site conformation of a protein in the presence of a bound ligand. We made use of a recently published algorithm that performs an exhaustive conformational search using a rotamer library to enumerate all possible sidechain conformations in a binding site. This approach was applied to a dataset of proteins whose structures were determined by X-ray and NMR methods. All chosen proteins had two or more structures, generally involving different bound ligands. By taking one of these structures as a reference, we were able in most cases to successfully reproduce the experimentally determined conformations of the other structures, as well as to suggest alternative low-energy conformations of the binding site. In those few cases where this procedure failed, we observed that the bound ligand had induced a high-energy conformation of the binding site. These results suggest that for most proteins that exhibit limited backbone motion, ligands tend to bind to low energy conformations of their binding sites. Our results also reveal that it is possible in most cases to use a rotamer search-based approach to predict alternative low-energy protein binding site conformations that can be used by different ligands. This opens the possibility of incorporating alternative binding site conformations to improve the efficacy of docking and structure-based drug design algorithms.     

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1‐alkyl‐3‐methylimidazolium L ‐proline (L ‐Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers—dl ‐phenylalanine (dl ‐Phe), dl ‐histidine (dl ‐His), dl ‐tryptophane (dl ‐Trp), and dl ‐tyrosine (dl ‐Tyr)—were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s)=3.26–10.81 for HPLC; R_s=1.34–4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L ‐Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L ‐Pro are consequently attached to the support surface, thus inducing an ion‐exchange type of retention for the dl ‐enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand‐exchange‐based chiral separation. It also reveals the tremendous application potential of this new type of task‐specific ILs.     

Total Synthesis and Functional Evaluation of Fourteen Derivatives of Lysocin E: Importance of Cationic,Hydrophobic, and Aromatic Moieties for Antibacterial Activity

Lysocin E ( 1 ) is a structurally complex 37‐membered depsipeptide comprising 12 amino‐acid residues with an N‐methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure‐activity relationship (SAR) study by systematically changing the side‐chain structures of l ‐Thr‐1, d ‐Arg‐2, N‐Me‐d ‐Phe‐5, d ‐Arg‐7, l ‐Glu‐8, and d ‐Trp‐10. First, we achieved total synthesis of the 14 new side‐chain analogues of 1 by employing a solid‐phase strategy. We then evaluated the MK‐dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d ‐Arg‐2/7 to a neutral amide, and the C7‐acyl group of l ‐Thr‐1 to the C2 or C11 counterpart decreased the antimicrobial activities four‐ or eight‐fold. More drastically, chemical mutation of d ‐Trp‐10 to d ‐Ala‐10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side‐chain functionalities.     

Binding structures of tri‐N‐acetyl‐β‐glucosamine in hen egg white lysozyme using molecular dynamics with a polarizable force field

Lysozyme is a well‐studied enzyme that hydrolyzes the β‐(1,4)‐glycosidic linkage of N‐acetyl‐β‐glucosamine (NAG)_n oligomers. The active site of hen egg‐white lysozyme (HEWL) is believed to consist of six subsites, A‐F that can accommodate six sugar residues. We present studies exploring the use of polarizable force fields in conjunction with all‐atom molecular dynamics (MD) simulations to analyze binding structures of complexes of lysozyme and NAG trisaccharide, (NAG)₃. MD trajectories are applied to analyze structures and conformation of the complex as well as protein–ligand interactions, including the hydrogen‐bonding network in the binding pocket. Two binding modes (ABC and BCD) of (NAG)₃ are investigated independently based on a fixed‐charge model and a polarizable model. We also apply molecular mechanics with generalized born and surface area (MM‐GBSA) methods based on MD using both nonpolarizable and polarizable force fields to compute binding free energies. We also study the correlation between root‐mean‐squared deviation and binding free energies of the wildtype and W62Y mutant; we find that for this prototypical system, approaches using the MD trajectories coupled with implicit solvent models are equivalent for polarizable and fixed‐charge models. © 2012 Wiley Periodicals, Inc.     

Combining molecular docking and molecular dynamics studies for modelling Staphylococcus aureus MurD inhibitory activity

The ATP-dependent bacterial MurD enzyme catalyses the formation of the peptide bond between cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine and D-glutamic acid. This is essential for bacterial cell wall peptidoglycan synthesis in both Gram-positive and Gram-negative bacteria. MurD is recognized as an important target for the development of new antibacterial agents. In the present study we prepared the 3D-stucture of the catalytic pocket of the Staphylococcus aureus MurD enzyme by homology modelling. Extra-precision docking, binding free energy calculation by the MM–GBSA approach and a 40 ns molecular dynamics (MD) simulation of 2-thioxothiazolidin-4-one based inhibitor $1 was carried out to elucidate its inhibition potential for the S. aureus MurD enzyme. Molecular docking results showed that Lys19, Gly147, Tyr148, Lys328, Thr330 and Phe431 residues are responsible for the inhibitor–protein complex stabilization. Binding free energy calculation revealed electrostatic solvation and van der Waals energy components as major contributors for the inhibitor binding. The inhibitor-modelled S. aureus protein complex had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 40 ns in aqueous solution. We designed some molecules as potent inhibitors of S. aureus MurD, and to validate the stability of the designed molecule D1-modelled protein complex we performed a 20 ns MD simulation. Results obtained from this study can be utilized for the design of potent S. aureus MurD inhibitors.     

Crystal and NMR Structures of a Peptidomimetic β‐Turn That Provides Facile Synthesis of 13‐Membered Cyclic Tetrapeptides

Herein we report the unique conformations adopted by linear and cyclic tetrapeptides (CTPs) containing 2‐aminobenzoic acid (2‐Abz) in solution and as single crystals. The crystal structure of the linear tetrapeptide H₂N‐d ‐Leu‐d ‐Phe‐2‐Abz‐d ‐Ala‐COOH ( 1 ) reveals a novel planar peptidomimetic β‐turn stabilized by three hydrogen bonds and is in agreement with its NMR structure in solution. While CTPs are often synthetically inaccessible or cyclize in poor yield, both 1 and its N ‐Me‐d ‐Phe analogue ( 2 ) adopt pseudo‐cyclic frameworks enabling near quantitative conversion to the corresponding CTPs 3 and 4 . The crystal structure of the N ‐methylated peptide ( 4 ) is the first reported for a CTP containing 2‐Abz and reveals a distinctly planar 13‐membered ring, which is also evident in solution. The N ‐methylation of d ‐Phe results in a peptide bond inversion compared to the conformation of 3 in solution.     

Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

The glycosylation of cell‐penetrating poly(disulfide)s (CPDs) is introduced to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. With the recently developed sidechain engineering, CPDs decorated with α‐d ‐glucose (Glu), β‐d ‐galactose (Gal), d ‐trehalose (Tre), and triethyleneglycol (TEG) were readily accessible. Confocal laser scanning microscopy images of HeLa Kyoto cells incubated with the new CPDs at 2.5 μm revealed efficient uptake into cytosol and nucleoli of all glycosylated CPDs, whereas the original CPDs and TEGylated CPDs showed much precipitation into fluorescent aggregates at these high concentrations. Flow cytometry analysis identified Glu‐CPDs as most active, closely followed by Gal‐CPDs and Tre‐CPDs, and all clearly more active than non‐glycosylated CPDs. In the MTT assay, all glyco‐CPDs were non‐toxic at concentrations as high as 2.5 μm . Consistent with thiol‐mediated uptake, glycosylated CPDs remained dependent on thiols on the cell surface for dynamic covalent exchange, their removal with Ellman's reagent DTNB efficiently inhibited uptake. Multifunctionality was demonstrated by inhibition of Glu‐CPDs with d ‐glucose (IC₅₀ ca. 20 mm ). Insensitivity toward l ‐glucose and d ‐galactose and insensitivity of conventional CPDs toward d ‐glucose supported that glucose‐mediated uptake of the multifunctional Glu‐CPDs involves selective recognition by glucose receptors at the cell surface. Weaker but significant sensitivity of Gal‐CPDs toward d ‐galactose but not d ‐glucose was noted (IC₅₀ ca. 110 mm ). Biotinylation of Glu‐CPDs resulted in the efficient delivery of streptavidin together with a fluorescent model substrate. Protein delivery with Glu‐CPDs was more efficient than with conventional CPDs and remained sensitive to DTNB and d ‐glucose, i.e., multifunctional.     

Indirect reversed‐phase high‐performance liquid chromatographic and direct thin‐layer chromatographic enantioresolution of (R,S)‐Cinacalcet

Enantioresolution of the calcimimetic drug (R,S)‐Cinacalcet was achieved using both indirect and direct approaches. Six chiral variants of Marfey's reagent having l ‐Ala‐NH₂, l ‐Phe‐NH₂, l ‐Val‐NH₂, l ‐Leu‐NH₂, l ‐Met‐NH₂ and d ‐Phg‐NH₂ as chiral auxiliaries were used as derivatizing reagents under microwave irradiation. Derivatization conditions were optimized. Reversed‐phase high‐performance liquid chromatography was successful using binary mixtures of aqueous trifluoroacetic acid and acetonitrile for separation of diastereomeric pairs with detection at 340 nm. Thin silica gel layers impregnated with optically pure l ‐histidine and l ‐arginine were used for direct resolution of enantiomers. The limit of detection was found to be 60 pmol in HPLC while in TLC it was found to be in the range of 0.26–0.28 µg for each enantiomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Anti‐Adhesive,Platelet Gathering Effects of c‐RGD Modified Poly(p‐dioxanone‐co‐l‐Phe) Electrospun Membrane and Its Comprehensive Application in Intestinal Anastomosis

Intestinal resection and anastomosis are performed in over a million people with various bowel diseases annually. Excessive fibrosis and anastomotic site leakage are the main complications of anastomosis surgery, despite great improvements in operative technique and equipment in recent years. In this study, cRGD modified poly(p‐dioxanone‐co‐l ‐Phe) (PDPA) membranes are designed and applied in intestinal anastomosis to simultaneously solve the two aforementioned complications. cRGD is modified onto PDPA membranes through both physical absorption and π–π accumulation between d ‐Phe of cRGD and l ‐Phe of PDPA. Although cRGD modification enhanced the biocompatibility of PDPA membranes, cRGD modified PDPA membrane suppresses fibroblast proliferation both in vitro and in vivo as a result of degradation and subsequent release of fibroblast suppressive l ‐Phe from PDPA. Meanwhile, platelets are entrapped by cRGD modified PDPA membranes through the specific binding of cRGD and platelet GP_IIbIIIa. cRGD modified PDPA membranes are applied in rat intestinal anastomosis, and both adhesion and stenosis are successfully prevented at anastomotic sites. At the same time, bursting pressure, which represents healing intensity at anastomotic sites, is promoted. The gathering and activation of platelets on PDPA membranes induce secretion of autologous PDGF and VEGF to facilitate angiogenesis and subsequent healing of anastomotic sites.     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

Enantiomeric and Diastereomeric Self‐Assembled Multivalent Nanostructures: Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA

A family of four self‐assembling lipopeptides containing Ala‐Lys peptides attached to a C₁₆ aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C₁₆‐l ‐Ala‐l ‐Lys/C₁₆‐d ‐Ala‐d ‐Lys) and (C₁₆‐d ‐Ala‐l ‐Lys/C₁₆‐l ‐Ala‐d ‐Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self‐assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d ‐lysine ligands, with a further slight preference for attachment to l ‐alanine, with the CMC being irrelevant.     

Calculation of dissociation constants and chemical hardness of some biologically important molecules: A theoretical study

The gas‐phase geometries of neutral, protonated, and deprotonated forms of some biologically important molecules, alanine (Ala), glycine (Gly), phenylalanine (Phe), and tyrosine (Tyr), were optimized using density functional theory at B3LYP/6‐311++G(d) and the ab initio HF/6‐311++G(d) level of theories. The neutral and different stable ionic states of Ala, Gly, Phe, and Tyr have also been solvated in aqueous medium using polarizable continuum model for the determination of solvation free energies in the aqueous solution. The gas‐phase acidity constants of above four molecules have been also calculated at both levels of theories and found that the values calculated at HF/6‐311++G(d) method are in good agreement with experimental results. A thermodynamic cycle was used to determine the solvation free energies for the proton dissociation process in aqueous solution and the corresponding pK_a values of these molecules. The pK_a values calculated at B3LYP/6‐311++G(d) method are well supported by the experimental data with a mean absolute deviation 0.12 pK_a units. Additionally, the chemical hardness and the ionization potential (IP) for these molecules have been also explored at both the level of theories. The Tyr has less value of chemical hardness and IP at both levels of theories compared with other three molecules, Ala, Gly, and Phe. The calculated values of chemical hardness and IP are decreasing gradually with the substitution of the various functional groups in the side chain of the amino acids. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Inversion of Optical Activity in the Synthesis of Mercury Sulfide Nanoparticles: Role of Ligand Coordination

Optical activity in inorganic colloidal materials was controlled through interactions of chiral molecules with the nanoparticle (NP) surface. An inversion of optical activity in the synthesis of mercury sulfide (HgS) NPs was demonstrated with an intrinsically chiral crystalline system in the presence of an identical chiral capping ligand. A continuous decrease in the positive first Cotton effect and an eventual reversal of CD profile were observed upon heating the aqueous solution of HgS NPs capped with N‐acetyl‐l ‐cysteine (Ac‐l ‐Cys) at 80 °C. Ac‐l ‐Cys afforded two bidentate coordination configurations with an almost mirror image of each other using the thiolate and either of carboxylate or acetyl–carbonyl groups on the HgS core. Experiment and calculation suggest that a shift in the distribution of the NP formation with energy in response to the combinations of ligand coordination structure and chiral crystalline surface is responsible for the inversion of optical activity.     

In silico based virtual screening and mixed mode QM/MM calculation identifies caffeine scaffold for designing potential inhibitors for tyrosyl tRNA synthetase of Mycobacterium tuberculosis

Aminoacyl tRNA synthetases are novel antibacterial drug target because of their important role in protein synthesis. In this study, we performed high throughput virtual screening of 205883 compounds from Asinex ligand database to identify potential specific inhibitors for Tyrosyl tRNA synthetase of Mycobacterium tuberculosis (MtbTyrRS). Compounds are ranked based on the glide extra precision docking score. It is noted that the top ranked compounds have caffeine scaffold. The top five caffeine analogs are further evaluated for other drug‐like properties. The binding energies of caffeine analogs are estimated using mixed mode quantum mechanics/molecular mechanics calculation. The results show that these caffeine analogs have good absorption, distribution, metabolism, and excretion properties and high binding affinity to the MtbTyrRS. This suggests that caffeine could be a new scaffold for designing inhibitors against Tyrosyl tRNA synthetase of M. tuberculosis. The top five caffeine analogs are also subjected to docking calculations with human cytosolic and mitochondrial Tyrosyl tRNA synthetases to ascertain their specificities toward MtbTyrRS. The comparative docking studies indicate that the top five caffeine analogs are specific for MtbTyrRS. © 2014 Wiley Periodicals, Inc.     

Molecular modeling of Protein A affinity chromatography

The properties of the complex between fragment B of Protein A and the Fc domain of IgG were investigated adopting molecular dynamics with the intent of providing useful insight that might be exploited to design mimetic ligands with properties similar to those of Protein A. Simulations were performed both for the complex in solution and supported on an agarose surface, which was modeled as an entangled structure constituted by two agarose double chains. The energetic analysis was performed by means of the molecular mechanics Poisson Boltzmann surface area (MM/PBSA), molecular mechanics generalized Born surface area (MM/GBSA), and the linear interaction energy (LIE) approaches. An alanine scan was performed to determine the relative contribution of Protein A key amino acids to the complex interaction energy. It was found that three amino acids play a dominant role: Gln 129, Phe 132 and Lys 154, though also four other residues, Tyr 133, Leu 136, Glu 143 and Gln 151 contribute significantly to the overall binding energy. A successive molecular dynamics analysis of Protein A re-organization performed when it is not in complex with IgG has however shown that Phe 132 and Tyr 133 interact among themselves establishing a significant π–π interaction, which is disrupted upon formation of the complex with IgG and thus reduces consistently their contribution to the protein–antibody bond. The effect that adsorbing fragment B of Protein A on an agarose support has on the stability of the protein–antibody bond was investigated using a minimal molecular model and compared to a similar study performed for a synthetic ligand. It was found that the interaction with the surface does not hinder significantly the capability of Protein A to interact with IgG, while it is crucial for the synthetic ligand. These results indicate that ligand–surface interactions should be considered in the design of new synthetic affinity ligands in order to achieve results comparable to those of Protein A right from the ligand design stage.     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.