X‐ray Crystallographic and Fluorometric Analysis of the Interactions of Rhein to Human Serum Albumin

To investigate the interactions between natural drugs and human serum albumin (HSA), we performed fluorescence spectroscopy and X‐ray crystallography to gain insight into binding mechanism and behaviour of rhein to HSA. Our fluorescence results demonstrated that rhein strongly binds with HSA, and other compounds may affect binding affinity of rhein to different extent. Structural analysis revealed that rhein binds to the IIA subdomain of HSA. The carboxylate group of rhein forms hydrogen bonds with Arg218 and Lys199, as well as a salt bond with Arg222. Hydroxyl group (4) of rhein forms a hydrogen bond with His242, and hydroxyl group (5) of rhein forms a hydrogen bond with Arg257. Oxygen atom (7) of rhein forms a hydrogen bond with Arg222, and oxygen atom (6) of rhein forms a hydrogen bond with H₂O. Furthermore, hydroxyl group (4) of rhein also forms a hydrogen bond with H₂O. Our results reveal the biochemical and structural characteristics of the interaction between rhein and HSA, providing guidance for future development of rhein‐based compounds and a drug–HSA delivery system.     

Comparison of Binding Characterization of Two Antiviral Drugs to Human Serum Albumin

Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X‐ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non‐steroidal anti‐inflammatory drugs, indometacin. X‐ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin‐ and lamivudine‐based compounds and a drug‐HSA delivery system.     

Binding and Anticancer Properties of Plumbagin with Human Serum Albumin

Plumbagin has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of plumbagin with human serum albumin. Fluorescence results demonstrated that plumbagin interacts with human serum albumin, although its binding affinity may be affected to various extents by different compounds. The human serum albumin–plumbagin complex structure revealed that plumbagin binds to the hydrophobic cavity in the IIA subdomain of human serum albumin through hydrogen bonding and hydrophobic interactions. The plumbagin–human serum albumin complex enhances cytotoxicity by 2‐ to 3‐fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the human serum albumin–plumbagin complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of plumbagin‐based drugs and a drug–human serum albumin delivery system.     

Characterization of the mangiferin–human serum albumin complex by spectroscopic and molecular modeling approaches

The interactions between mangiferin and human serum albumin (HSA) were investigated by spectroscopy and molecular modeling. The results proved the formation of complex between mangiferin and HSA. Hydrophobic interaction dominated in the association reaction. Mangiferin statically quenched the fluorescence of HSA in a concentration dependent manner positively deviating from the linear Scatchard equation. The binding of mangiferin to HSA lead to changes in the conformation of HSA according to synchronous fluorescence spectra, FT-IR, UV–vis and CD data. The presence of amino acids and metal ion affected the binding constant of mangiferin–HSA complex. Computational mapping of the possible binding sites of mangiferin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

Interaction of daunomycin antibiotic with human serum albumin: investigation by resonant mirror biosensor technique, fluorescence spectroscopy and molecular modeling methods

Daunomycin (DM) is a clinically used antitumor anthracycline antibiotic, which is transported primarily by human serum albumin (HSA) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of DM. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of DM with HSA at different temperatures and the affinity constants were obtained. The HSA-DM interaction is exothermic with having favorable enthalpy and entropy followed by the integrated van't Hoff equation analysis. Fluorescence studies showed that DM has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure according to the Stern-Volmer equation and DM displays a pH-dependent binding affinity to HSA. Molecular modeling calculations showed that the DM binds HSA to a non-classical drug binding site and further analysis of the binding site of DM within the HSA molecule suggested that hydrophobic contacts, hydrogen bond formation and electrostatic interactions account for the binding of DM.     

Regulation of Amantadine Hydrochloride Binding with IIA Subdomain of Human Serum Albumin by Fatty Acid Chains

Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X‐ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.     

Probing the binding of the flavonoid,quercetin to human serum albumin by circular dichroism,electronic absorption spectroscopy and molecular modelling methods

The plant derived flavonoid compound quercetin, possesses wide range of biological activities in the human body by interacting with nucleic acids, enzymes and other proteins. As has recently been shown this molecule of polyphenolic type extensively binds to human serum albumin (HSA), the most abundant carrier protein in the blood. Electronic absorption, circular dichroism (CD) spectroscopy and molecular modelling methods were used to characterize optical properties of the quercetin-HSA complex, and to gain information on the binding mechanism at molecular level. The red shift and hypochromism of the longest-wavelength absorption band of quercetin relative to the spectral properties in ethanol suggests that one or more phenolic OH groups of the bound ligand is ionized and that the exocyclic phenyl ring is not coplanar with the benzopyrone moiety. It was found that quercetin shows extrinsic optical activity on interaction with HSA. The induced CD spectra were utilized to calculate the association constant at 37 degrees (1.46+/-0.21 x 10(4)M(-1)) and to probe the ligand binding site. Results of the CD displacement experiments performed with palmitic acid and salicylate were interpreted together with the findings of molecular modelling calculation performed on the quercetin-HSA complex. Computational mapping of possible binding sites of quercetin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA. The protein microenvironment of this site was found to be rich in polar (basic) amino acid residues which are able to help to stabilize the negatively charged ligand bound in non-planar conformation. Additionally, the position of quercetin within the binding pocket allows simultaneous binding of other ligands such as warfarin, or sodium salycilate.     

Molecular dynamics simulation of the antimicrobial salivary peptide histatin‐5 in water and in trifluoroethanol: a microscopic description of the water destructuring effect

Abstract: The results of 520 ps molecular dynamics simulation of histatin‐5, a small peptide present in human saliva and possessing antimicrobial activity, dissolved in water and in 2,2,2‐trifluoroethanol, are reported. The simulations indicate that histatin‐5 is destabilized in water and begins to unfold after 250 ps, while in organic solvent it maintains a regular secondary structure throughout the trajectory. Analysis of the peptide?solvent hydrogen bonds indicates that 2,2,2‐trifluoroethanol is a poorer proton acceptor than water. The fluorine atom of the alcohol is almost never engaged in a hydrogen bond and the organic solvent interacts mainly with the peptide through its hydroxyl group. For some residues analysis of the solvent residence time indicated longer values for 2,2,2‐trifluoroethanol than for water. The most striking difference is related to the number of times the solvent enters and leaves the first coordination shell of the peptide. This value was more than one order of magnitude higher for water than for the alcohol, suggesting that this may be the main cause of α‐helix destabilization perpetrated by water.     

Synthesis,X‐ray crystallographic structures of thio substituted N‐acetyl N′‐methylamide alanine and evaluation of sp2 sulfur parameters of the CFF91 force field

Abstract Acetyl thioalanine N‐methyl (Ac‐Alat‐NHMe) and thioacetyl alanine N‐methyl (Act‐Ala‐NHMe) were synthesized, crystallized and their X‐ray diffraction structures determined for the first time. Both molecules adopted β‐sheet conformations and showed similar hydrogen bonding patterns with one molecular surface forming two oxo hydrogen bonds and the other forming two thio hydrogen bonds. The crystal structure data for the two thioamides provided a validation of the thioamide parameters for the newly derived CFF91 force field because the observed crystal (φ, ψ) angles were situated in the global minimum regions of the theoretical (φ, ψ) map predicted using the parameters. In addition, the parameters were further validated because conformational energy minimization of the crystal structure produced low deviations in unit cell dimensions, bond lengths, bond angles and torsional angles, and a 120‐ps molecular dynamics simulation also gave a low deviation for the most probable N‐H^…S=C bond distance.     

Pharmacophore, 3D‐QSAR,and Bayesian Model Analysis for Ligands Binding at the Benzodiazepine Site of GABAA Receptors: the Key Roles of Amino Group and Hydrophobic Sites

Ligands binding at the benzodiazepine site of GABA_A receptor play important pharmacological roles in clinical application. In this study, ligand‐based pharmacophore modeling, 3D‐QSAR analysis, and Bayesian model studies have been performed on a set of 84 diverse ligands binding at the benzodiazepine site. The results showed the best pharmacophore hypothesis AADHR.4, which included two hydrogen acceptors (A), one hydrogen donor (D), one hydrophobic group (H), and one aromatic ring (R). Atom‐based 3D‐QSAR model was built, and it showed good statistical significance (R² = 0.936) and excellent predictive ability (Q² = 0.821). Moreover, Bayesian model was developed and used to identify the key molecular features which are good or bad for the ligand binding activity. All the results from the pharmacophore, 3D‐QSAR, and Bayesian modeling studies revealed that a hydrogen‐bond donor (e.g., N‐H) and a hydrophobic group (e.g., Br) are critical structural features for the ligands binding at the benzodiazepine site.     

Synthesis,Evaluation and Molecular Docking of Prolyl‐Fluoropyrrolidine Derivatives as Dipeptidyl Peptidase IV Inhibitors

A series of prolyl‐fluoropyrrolidine derivatives were designed, synthesized and screened for in vitro inhibition of dipeptidyl peptidase IV. The SAR study revealed the influence of substituted chemical modifications on dipeptidyl peptidase IV inhibitory activity. Among all the compounds screened, compound 9 (IC₅₀ = 0.83 μm ) and 10 (IC₅₀ = 0.43 μm ) possessing aryl substituted piperazine with acetamide linker resulted as most potent dipeptidyl peptidase IV inhibitors. Both the compounds 9 and 10 resulted significant reduction in glucose excursion during oral glucose tolerance test in streptozotocin‐induced diabetic rat model at single dose of 10 mg/kg. Molecular docking studies were performed to illustrate the probable binding mode and interactions of prolyl‐fluoropyrrolidine nucleus and its derivatives at binding site of receptor. The fluoropyrrolidine moiety of prolyl‐fluoropyrrolidine derivatives occupied S1 pocket as observed in the crystal structure (PDB id: 2FJP). The compounds 9 and 10 were observed to occupy S2 binding pocket and were observed to have interaction with Arg125, Tyr547 and Ser630 acquired through hydrogen bond. The aryl moiety at piperazine ring was found to extend into the cavity and interacted with Arg358. The observed interactions signalled that occupancy of the highly hydrophobic S2 pocket is very crucial for dipeptidyl peptidase IV inhibitory activity.     

Fragment‐ and negative image‐based screening of phosphodiesterase 10A inhibitors

A novel virtual screening methodology called fragment‐ and negative image‐based (F‐NiB) screening is introduced and tested experimentally using phosphodiesterase 10A (PDE10A) as a case study. Potent PDE10A‐specific small‐molecule inhibitors are actively sought after for their antipsychotic and neuroprotective effects. The F‐NiB combines features from both fragment‐based drug discovery and negative image‐based (NIB) screening methodologies to facilitate rational drug discovery. The selected structural parts of protein‐bound ligand(s) are seamlessly combined with the negative image of the target's ligand‐binding cavity. This cavity‐ and fragment‐based hybrid model, namely its shape and electrostatics, is used directly in the rigid docking of ab initio generated ligand 3D conformers. In total, 14 compounds were acquired using the F‐NiB methodology, 3D quantitative structure–activity relationship modeling, and pharmacophore modeling. Three of the small molecules inhibited PDE10A at ~27 to ~67 μM range in a radiometric assay. In a larger context, the study shows that the F‐NiB provides a flexible way to incorporate small‐molecule fragments into the drug discovery.     

TAK1 Inhibition in the DFG‐Out Conformation

The first example of an inhibitor of the kinase TAK1 that binds in the DFG‐out conformation is disclosed. These preliminary studies used kinase‐targeted screening and structure‐based drug design to create a molecule with dual pharmacological inhibition of p38 and TAK1 that demonstrated significant activity in a cell‐based, anti‐inflammatory assay.     

Synthesis and X‐ray crystal structure study of the hydroxyurea and hydantoin derivatives of l‐valine

Abstract: The novel hydroxyurea 5 derivative of L ‐valine was prepared by aminolysis of N‐(1‐benzotriazolecarbonyl)‐L ‐valine cyclohexanemethylamide 4 with hydroxylamine. The corresponding hydantoin derivative 6 was synthesized by base catalyzed cyclization of the amide 4 . The exact stereostructure of hydantoin derivative 6 has been determined by X‐ray crystal structure analysis. The chiral atom of the hydantoin ring in 6 has S configuration what is in agreement with its configuration in the starting L ‐valine. The molecules of 6 are joined into infinite chains by N–H?O intermolecular hydrogen bond. The infinite chains are additionally linked by two C–H?O hydrogen bonds, thus forming two‐dimensional network. The hydantoin derivative of L ‐valine 6 and its L ‐leucine analogue LH have similar packing arrangements, so they are homostructural.     

Comparative Analysis of Pharmacophore Features and Quantitative Structure–Activity Relationships for CD38 Covalent and Non‐covalent Inhibitors

In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non‐covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure‐based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure–activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non‐covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non‐bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross‐validated correlation coefficient values (q²) of 0.564 and 0.571, and non‐cross‐validated values (r²) of 0.967 and 0.971, respectively. The CD38 non‐covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non‐covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross‐validated correlation coefficient values (q²) of 0.469 and 0.454, and non‐cross‐validated values (r²) of 0.814 and 0.819, respectively.     

Identification of Type‐II Inhibitors Using Kinase Structures

Spleen tyrosine kinase is a non‐receptor tyrosine kinase, overactivation of which is thought to contribute to autoimmune diseases as well as allergy and asthma. Protein kinases have a highly conserved ATP binding site, thus making challenging the design of selective small molecule inhibitors. It has been well documented that some protein kinases can be stabilized in their inactive conformations (Type‐II inhibitors). Herein, we describe a protein structure/ligand‐based approach to successfully identify ligands that bind to novel conformations of spleen tyrosine kinase. By utilizing kinase protein crystal structures both in the public domain (RCSB) and within Pfizer’s protein crystal database, we report the discovery of the first spleen tyrosine kinase Type‐II ligands. Compounds 1 and 3 were found to bind to the DFG‐out conformation of spleen tyrosine kinase, while compound 2 binds to a DFG‐in, C‐Helix‐out conformation. In this instance, the C‐helix moved significantly to create a large hydrophobic pocket rarely seen in kinase protein crystal structures.     

Acetylcholinesterase Inhibitory Activity and Molecular Docking Study of 1‐Nitro‐2‐Phenylethane,the Main Constituent of Aniba canelilla Essential Oil

The odoriferous principle of Aniba canelilla (H.B.K.) Mez is due 1‐nitro‐2‐phenylethane, the main constituent of its essential oil and also responsible for the plant's cinnamon scent. This nitroderivative was previously reported by their antioxidant, antinociception, cardiovascular, and vasorelaxant properties, and now it was tested as the inhibitor of acetylcholinesterase using bioautography on TLC plates. The oil and a purified fraction containing 1‐nitro‐2‐phenylethane were analyzed by GC and GC‐MS. The percentage content of 1‐nitro‐2‐phenylethane in the oil and after fractionation was 70.2% and 98.0%, respectively. The results showed that the oil and 1‐nitro‐2‐phenylethane are strong acetylcholinesterase inhibitors with the detection limit of 0.01 ng, equivalent to physostigmine used as the positive control. A molecular docking study was used to determine the position and conformation of the 1‐nitro‐2‐phenylethane inhibitor in the receptor‐binding pocket of the acetylcholinesterase enzyme. The nitrogroup of 1‐nitro‐2‐phenylethane was positioned near of the catalytic serine residue of acetylcholinesterase, forming strong hydrogen bond with its hydroxyl group. Therefore, the electronegative character of 1‐nitro‐2‐phenylethane may explain the interaction that occurs with the catalytic serine residue and its significant inhibitory activity of acetylcholinesterase.     

Structure‐based pharmacophore models to probe anticancer activity of inhibitors of protein kinase B‐beta (PKB β)

Protein kinase B ‐ beta (PKBβ/Akt2) is a non‐receptor kinase that has attracted a great deal of attention as a promising cancer therapy drug target. In mammalian cells, hyperactivation of Akt2 exclusively facilitates the survival of solid tumors by interfering with cell cycle progression. This definite function of Akt2 in tumor survival/maintenance provides the basis for the development of its antagonists with the aim of desensitizing cell proliferation. In order to find novel and potent Akt2 inhibitors, structure ‐ based pharmacophore models have been developed and validated by the test set prediction. The final pharmacophore model was used for hits identification using public chemical databases. The hits were further prioritized using drug ‐ like filters which revealed 14 potential hit compounds having novel chemical scaffolds. Our results elucidate the importance of three hydrogen bond acceptors (A), one hydrogen bond donor (D), one hydrophobic group (H), and one positive ionic charge (P) toward inhibition of the Ak2. One of our selected hits showed 68% cell apoptosis at 8 μg/ml concentration. We proposed various chemical scaffolds including benzamide, carboxamide, and methyl benzimidazole targeting Akt2 and thus may act as potential leads for the further development of new anticancer agents.