Chum Mok Puah

Binding interaction of quercetin -3 -β -galactoside and its synthetic derivatives with SARS-CoV 3CLpro : Structure-activity relationship studies reveal salient pharmacophore features

Abstract The 3C-like protease (3CLpro) 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-β-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 3CLpro 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.

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

ABSTRACT 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.

Understanding the regulation mechanisms of PAF receptor by agonists and antagonists: Molecular modeling and molecular dynamics simulation studies

Platelet‐activating factor receptor (PAFR) is a member of G‐protein coupled receptor (GPCR) superfamily. Understanding the regulation mechanisms of PAFR by its agonists and antagonists at the atomic level is essential for designing PAFR antagonists as drug candidates for treating PAF‐mediated diseases. In this study, a 3D model of PAFR was constructed by a hierarchical approach integrating homology modeling, molecular docking and molecular dynamics (MD) simulations. Based on the 3D model, regulation mechanisms of PAFR by agonists and antagonists were investigated via three 8‐ns MD simulations on the systems of apo‐PAFR, PAFR‐PAF and PAFR‐GB. The simulations revealed that binding of PAF to PAFR triggers the straightening process of the kinked helix VI, leading to its activated state. In contrast, binding of GB to PAFR locks PAFR in its inactive state. Proteins 2007.

The interaction model between metal cation and tropylium: a quantum chemistry predication

Abstract The aromatic cation tropylium, C 7 H 7 + , predicted at the MP2/6-31G** level, is capable of binding with metal cations Be 2+ or Mg 2+ , forming M 2+ –C 7 H 7 + complexes. The obstacle for their binding is almost electrostatic repulsion, and the binding is from polarization and charge transfer. The orbital interaction between the M 2+ and C 7 H 7 + is mainly the s–π and p–π interactions. Interestingly, Be 2+ is possible to pass through the ring of C 7 H 7 + , while Mg 2+ is not. The intrinsic IR band of the M 2+ –C 7 H 7 + complex is below 600 cm −1 , which results from the vibration of the M 2+ along the normal axis of C 7 H 7 + .

Density functional theory (DFT) study on the interaction of ammonium (NH4+) and aromatic nitrogen heterocyclics

A DFT calculation was performed at the B3LYP/6-31G* level on the complexes formed by NH4+ and aromatic nitrogen heterocyclics, viz. pyrrole, imidazole, pyridine and indole, in order to investigate the mechanism and complexity of the interaction between the ammonium group and the aromatic heterocyclic in biomacromolecules. The optimized geometries suggested that there are two different types of complexes: one is a cation–π complex and the other is a hydrogen bond complex. A cation–π complex will be formed if the heteroatom has no localized lone-pair electrons. A hydrogen bond complex will be formed by proton transfer from NH4+ to the heteroatom if the heteroatom has localized lone-pair electrons. In the case of the cation–π complex, the predicted geometries, atomic charges and thermodynamic parameters revealed that ammonium binds more strongly to heterocyclics than it binds to benzene. The calculated orbital coefficient and the optimized structures implied that NH4+ interacts with the π electrons of the CC bond of heterocyclics to form a cation–π complex mainly through one hydrogen atom. Regarding the hydrogen bond complex, although the calculated binding strength is similar to that for the cation–π complex, the ΔH of the whole reaction process suggested that the formation of the hydrogen bond complex is favorable to the stability of the whole system. Calculated IR spectra showed that three groups of new bands appear when NH4+ binds to heterocyclics. Normal mode analysis showed that these new bands are all related to the relative motion of the two parts in the formed complexes. All these results suggest that the NH4+–heterocyclic system is a better model for studying the nature and complexity of the interaction between the ammonium group and the aromatic ring structure in biomolecules.

Hydrogen Peroxide Enhances Enterokinase-Catalysed Proteolytic Cleavage of Fusion Protein

The effects of hydrogen peroxide on enterokinase catalysis were studied using several fusion proteins recombinantly produced from E. coli. It was demonstrated that hydrogen peroxide enhanced the rate of enterokinase cleavage reaction, leading to a faster release of the target peptide as discussed in patent WO07149053. Among the conditions tested, we observed that hydrogen peroxide could exert its effect on the cleavage of fusion proteins over a wide range of pH and temperature. This finding might provide a simple solution for the accelerated enterokinase cleavage of thermolabile fusion proteins at low temperature.

A micro-fluidic level sensing and dispensing system for automation of cell cultivation experimentations

Embryonic stem cells, ESC are the foundation for all the tissue and organ, in the body. They are unique and have the ability to differentiate into any type of cell in the human body. Large number of experiments is usually required in order to establish how stem cells can be effectively grown outside the body before they can be effectively used in regenerative medicine to cure life-style related diseases. These can range from the determination of the optimum growth factors to the cell differentiation mechanism. Such experiments if they were to be truly exhaustive are not only costly in terms of the scientists effort but require large sterilised incubation space, particularly if they were to be carried out manually using Petri dishes based on 10 reagents - requiring a combination of 10! In order to comprehensively explore all the possible combination of the reagent and growth factors required, high throughput automated dispensing and high density micro-wells are critically required. The viability of the cells for large scale experiments depends on many factors. The main aim of the research describe in this paper is to establish the key factors to ensure cell viability. This include the determination of most effective number of wells per plate; its ideal size, diameter and depth; amount of fluid to contain; effective number of cells in a colony; level of humidity; salinity and evaporation rates of the medium. It is through these research findings, that a micro- fiuidic level sensing and dispensing system for cell cultivation is developed as a base for high throughput experiments [1].

A micro-fluidic level sensing and dispensing system for large-scale stem cell experimentations

Embryonic stem cells, ESC are the foundation for all the tissue and organ, in the body. They are unique and have the ability to differentiate into any type of cell in the human body. Large number of experiments is usually required in order to establish how stem cells can be effectively grown outside the body before they can be effectively used in regenerative medicine to cure life-style related diseases. These can range from the determination of the optimum growth factors to the cell differentiation mechanism. Such experiments if they were to be truly exhaustive are not only costly in terms of the scientists effort but require large sterilised incubation space, particularly if they were to be carried out manually using Petri dishes based on 10 reagents - requiring a combination of 10! In order to comprehensively explore all the possible combination of the reagent and growth factors required, high throughput automated dispensing and high density micro-wells are critically required. The viability of the cells for large scale experiments depends on many factors. The main aim of the research describe in this paper is to establish the key factors to ensure cell viability. This include the determination of most effective number of wells per plate; its ideal size, diameter and depth; amount of fluid to contain; effective number of cells in a colony; level of humidity; salinity and evaporation rates of the medium. It is through these research findings, that a micro-fluidic level sensing and dispensing system for cell cultivation is developed as a base for high throughput experiments.

STRUCTURAL FEATURE OF AChE INHIBITOR HUPERZINE B IN NATURE AND IN THE BINDING SITE OF AChE: DENSITY FUNCTIONAL THEORY STUDY COMBINED WITH IR DETERMINATION

Quantum chemical DFT-B3LYP/6-31G method and IR spectrometry have been used to investigate the natural and binding structures of Huperzine B (HupB) in order to better understand the interaction nature between acetylcholinesterase (AChE) and its inhibitor, with the view of designing new AChE inhibitors. The predicted and experimental results reveal that both the natural state and binding form of HupB adopt the chair conformation. Furthermore, the B3LYP/6-31G results suggest that structure S1 should be the dominant form of the two possible chair structures (S1 and S2, Fig. 2). The calculated results also show that the condensed ring structure composing of rings A, B and C is very rigid. Therefore, its flexibility does not need to be considered when we try to dock this structure to its target. Indeed, this supposition is conrmed by the excellent alignment of the binding structure produced from our recent X-ray crystallographic structure of the HupB-AChE complex with the B3LYP/6-31G predicted geometry. Among all the 111 predicted vibrational bands, the mode 110, which is resulted from the stretching of the bond N2{H and having the second highest frequency, is essential for the geometrical identication. The dierence between our predicted strongest absorption band and experimental IR spectrum suggests

Quantum chemical HF/4-31G calculations on buckminsterfullerene intermediates

Quantum chemical ab initio (U)HF/4-31G investigation on buckminsterfullerene and some proposed intermediates in its formation is carried out in this study with a view to better understanding how small carbon species carry out self-assembly to form fullerenes. The calculations on 19 carefully designed fullerene intermediates reveal that the core of an intermediate, rather than the number of its dangling bonds or abutting pentagon rings, has an intrinsic effect on its energy. The computational results show that hexagonal-core structures have lower energies than pentagonal-core structures. In addition, the pentagonal core enclosed completely by hexagonal rings has the highest energy. The UHF/4-31G results also suggest that some intermediates such as C18, C21 and C30 with hexagonal cores have unusually low energies in comparison with their isomers or neighbours. Based on these calculated results, we outline the possible pathways from precursor to intermediates to fullerenes, subject to synthesis conditions and raw materials. These pathways support some existing proposals, such as medium monocyclic ring stacking and small ring polymerization mechanisms. However, our results do not suggest that the numbers of dangling bonds or abutting pentagonal rings have the highest impact on fullerene formation. The calculated thermodynamic parameters of the dimerization and addition reactions between two bowl-shaped intermediates suggest that these reactions are favorable to fullerene formation, and that the concentration of bowl-shaped fullerene intermediates should be very low in all detectable carbon species.