Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays.

The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-beta-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL(pro) were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure-activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.

Focused combinatorial library design based on structural diversity, druglikeness and binding affinity score.

The advent of focused library and virtual screening has reduced the disadvantage of combinatorial chemistry and changed it to a realizable and cost-effective tool in drug discovery. Usually, genetic algorithms (GAs) are used to quickly finding high-scoring molecules by sampling a small subset of the total combinatorial space. Therefore, scoring functions play essential roles in focused library design. Reported here is our initial attempt to establish a new approach for generating a target-focused library using the combination of the scores of structural diversity and binding affinity with our newly improved drug-likeness scoring functions. Meanwhile, a software package, named LD1.0, was developed on the basis of the new approach. One test on a cyclooxygenase (COX)2-focused library successfully reproduced the structures that have been experimentally studied as COX2-selective inhibitors. Another test is on a peroxisome proliferator-activated receptors gamma-focused library design, which not only reproduces the key fragments in the approved (thiazolidinedione) TZD drugs, but also generates some new structures that are more active than the approved drugs or published ligands. Both of the two tests took approximately 15% of the running time of the ordinary molecular docking method. Thus, our new approach is an effective, reliable, and practical way for building up a properly sized focused library with a high hit rate, novel structure, and good ADME/T profile.

QSAR analyses on ginkgolides and their analogues using CoMFA, CoMSIA, and HQSAR.

Ginkgolides, isolated from ginkgo balba leaves, were found to be powerful as natural antagonists of human platelet activating factor (PAF) in treatment of some diseases such as acute inflammation, tissue rejection, asthma, and ischemic injury. Ginkgolides have a cage skeleton consisting of six five-membered rings, therefore, are very tough to be synthesized. For finding new powerful substitutes of the natural ginkgolides for treating those diseases, three methods, viz. CoMFA, CoMSIA, and HQSAR, were used to investigate the relationship between 117 ginkgolide analogues with great structural diversity and their bioactivities against PAF receptor. The high q2 released from the different QSAR methods, ranging from 0.583 to 0.684, suggests that three rational and predictive QSAR models were successfully built. These models also show clearly how steric, electrostatic, hydrophobicity, and individual atom affect molecular bioactivity as antagonists of PAF. These results could also be used to account for the unusually higher bioactivity of ginkgolide B than other ginkgolides. The possible binding mechanism between ginkgolides and human PAF receptor was also deduced based on the QSAR models. Therefore, this study should be very helpful in discovering new drugs as PAF antagonists in fighting against various diseases related to PAF and PAF receptor.

Elucidating inhibitory models of the inhibitors of epidermal growth factor receptor by docking and 3D-QSAR.

Epidermal growth factor receptor (EGFR) protein tyrosine kinases (PTKs) are attractive targets for anti-tumor drug design. Although thousands of their ligands have been studied as potential inhibitors against PTKs, there is no QSAR study that covers different kinds of inhibitors with observable structural diversity. However, by using this approach, we could mine far more useful information. Hence in order to better understand the binding model and the relationship between the physicochemical properties and the inhibitory activities of different kind of various inhibitors, molecular docking and 3D-QSAR, viz. CoMFA and CoMSIA, were combined to study 124 reported inhibitors with different scaffolds. Based on the docked binding conformations, highly reliable and predictive 3D-QSAR models were derived, which reveal how steric, electrostatic, and hydrophobic interactions contribute to inhibitors' bioactivities. This result also demonstrates that it is possible to include different kinds of inhibitors with observable structural diversity into one 3D-QSAR study. Therefore, this study not only casts light on binding mechanism between EGFR and its inhibitors, but also provides new hints for de novo design of new EGFR inhibitors with observable structural diversity.

N-methylformamide-benzene complex as a prototypical peptide N-H...pi hydrogen-bonded system: density functional theory and MP2 studies.

Although the existence of peptide N-H...pi hydrogen bonds recently has been reported in protein structures, little is known about their strength and binding nature and, therefore, the relative importance of the interaction. To shed light on this binding, the N-methylformamide-benzene complex, as a model of the peptide N-H...pi hydrogen bonding, was studied by using density functional theory and Møller-Plesset second-order perturbation (MP2) methods. The geometry of the complex was fully optimized at the B3LYP/6-31G(d,p) and MP2/6-31G(d,p) levels. The optimized interaction distances are about 3.6 and 3.2 A, respectively, at the two levels. The binding energy corrected by basis set superposition error with the MP2/cc-pVTZ method based on the MP2/6-31G geometry is -4.37 kcal/mol, which is as strong as the conventional hydrogen bonding. The calculated results suggest that the peptide N-H...pi hydrogen bonding is of sufficient strength to play an important role in the stabilization of protein structures. These results are helpful to better understand the characteristics and nature of the peptide N-H...pi interaction as well as to modify current force fields to better represent this special interaction.