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5-Amino-8-hydroxyquinoline (5A8HQ), an amino derivative of 8-hydroxyquinoline, has become a potential anticancer candidate because of its promising proteasome inhibitory activity to overcome and yet synergize bortezomib for fighting cancers. Therefore, in this study, its physicochemical properties and interaction activities with serum protein have extensively been elucidated by both in vitro and in silico approaches to fulfill the pharmacokinetic and pharmacodynamic gaps. 5A8HQ exhibited the drug-likeness properties, where oral administration seems to be a route of choice owing to its high-water solubility and intestinal absorptivity. Multi-spectroscopic investigations suggested that 5A8HQ tended to associate with bovine serum albumin (BSA), a representative of serum protein, via the ground-state complexation. It apparently bound in a protein cleft between subdomains IIA and IIIA of BSA as suggested by the molecular docking and molecular dynamics simulations. The binding was mainly driven by hydrogen bonding and electrostatic interactions with a moderate binding constant at 104 M−1, conforming with the predicted free fraction in serum at 0.484. Therefore, 5A8HQ seems to display a good bioavailability in plasma to reach target sites and exerts its potent pharmacological activity. Likewise, serum albumin is a good candidate to be reservoir and transporter of 5A8HQ in the circulatory system.

Clioquinol has recently been proposed for the treatment of Alzheimer's disease. It is able to diminish β-amyloid protein aggregation and to restore cognition of Alzheimer's mice. However, its therapeutic benefits for Alzheimer's disease in human remain controversy and need further confirmation. Herein, we have explored the interaction mechanism of clioquinol toward bovine serum albumin (BSA) by means of multi-spectroscopic and docking simulation approaches. Clioquinol interacts with BSA by a combined mechanism of static and dynamic processes. Application of the Hill's equation to fluorescence quenching experiment revealed that the binding constant of the BSA-clioquinol complex is extremely high at 108 M−1 level. Competitive displacement and docking analysis consistently suggested that there are the multiple binding modes of clioquinol toward BSA. Competitive binding study showed that clioquinol shares the binding sites with ibuprofen and digitoxin on albumin, referring to be site II and site III binding compounds. Besides, partial binding in site I was also observed. Docking simulation confirmed that clioquinol favors to bind in site I, site II, site III, fatty acid binding site 5, and the protein cleft between subdomain IB and IIIB of the BSA. Due to its small size and electric dipole property, clioquinol may easily fit in multiple pockets of the BSA. Our finding suggests the potential role of BSA as a clioquinol carrier in the vascular system. Nonetheless, clioquinol-induced BSA aggregation has been observed by the three-dimensional fluorescence technique. This phenomenon may not only impair the BSA, but may also affect other endogenous proteins, which eventually causes adverse effects to human. Therefore, the redesigned or modified molecular structure of clioquinol may reduce its toxicity and improve its bioavailability.

Cloxyquin is a potential therapeutic compound possessing various bioactivities, especially antibacterial, antifungal, cardioprotective, and pain relief activities. Herein, the interaction mechanism between cloxyquin and bovine serum albumin (BSA) has been elucidated in order to fulfill its pharmacokinetic and pharmacodynamic gaps essential for further development as a therapeutic drug. Multi-spectroscopic and biophysical model analysis suggested that cloxyquin interacts with BSA via a static process by ground-state complex formation. Its binding behavior emerged as a biphasic fashion with a moderate binding constant at the level of 104 M&minus, 1. Thermodynamic analysis and molecular docking simulation concurrently revealed that hydrophobic interaction is a major driving force for BSA&ndash, cloxyquin complexation. Binding of cloxyquin tends to slightly enlarge the monomeric size of BSA without a significant increase of aggregate fraction. Cloxyquin preferentially binds into the fatty acid binding site 5 (FA5) of the BSA via hydrophobic interaction amongst its quinoline scaffold and Phe550, Leu531, and Leu574 residues of BSA. The quinoline ring and hydroxyl moiety of cloxyquin also form the &pi, &ndash, &pi, interaction and the hydrogen bond with Phe506. Our data indicate a potential function of serum albumin as a carrier of cloxyquin in blood circulation.

Estrogen is an important component for the sustenance of normal physiological functions of the mammary glands, particularly for growth and differentiation. Approximately, two-thirds of breast cancers are positive for estrogen receptor (ERs), which is a predisposing factor for the growth of breast cancer cells. As such, ERα represents a lucrative therapeutic target for breast cancer that has attracted wide interest in the search for inhibitory agents. However, the conventional laboratory processes are cost- and time-consuming. Thus, it is highly desirable to develop alternative methods such as quantitative structure–activity relationship (QSAR) models for predicting ER-mediated endocrine agitation as to simplify their prioritization for future screening. In this study, we compiled and curated a large, non-redundant data set of 1231 compounds with ERα inhibitory activity (pIC50). Using comprehensive validation tests, it was clearly observed that the model utilizing the substructure count as descriptors, performed well considering two objectives: using less descriptors for model development and achieving high predictive performance (RTr2 = 0.94, QCV2 = 0.73, and QExt2 = 0.73). It is anticipated that our proposed QSAR model may become a useful high-throughput tool for identifying novel inhibitors against ERα.

Estrogen is an important component for the sustenance of normal physiological functions of the mammary glands, particularly for growth and differentiation. Approximately, two-thirds of breast cancers are positive for estrogen receptor (ERs), which is a predisposing factor for the growth of breast cancer cells. As such, ERα represents a lucrative therapeutic target for breast cancer that has attracted wide interest in the search for inhibitory agents. However, the conventional laboratory processes are cost- and time-consuming. Thus, it is highly desirable to develop alternative methods such as quantitative structure-activity relationship (QSAR) models for predicting ER-mediated endocrine agitation as to simplify their prioritization for future screening. In this study, we compiled and curated a large, non-redundant data set of 1231 compounds with ERα inhibitory activity (pIC

A series of isomeric α- and β-(1-adamantylthio)pyridines were previously documented to possess interesting antimicrobial and antimalarial activities. In this study, the antioxidant and cytotoxic potentials of 1-adamantylthio-3-methyl and 2-,3-,4-phenylpyridines (1–10) were investigated. The tested compounds were shown to exhibit interesting superoxide (SOD)- and free radical (DPPH)-scavenging activities as well as cytotoxic activities. Particularly, β-(1-adamantylthio)-4-phenylpyridine (8) was shown to be the most potent antioxidant and cytotoxic compound. QSAR studies revealed that dipole moment (μ) and electrophilic index (ωi) were the most important descriptors accounting for the observed SOD activities. Compounds with high μ and ωi values were observed to display high SOD activity. Inversely, compounds with the lowest atomic polarizability (MATS4p) exhibited the highest DPPH activity. Other quantum chemical descriptors such as atomic masses (GATS4m), ωi, and LUMO energy were also well correlated with cytotoxicity. The findings demonstrated that thiopyridine 8 is a potential lead compound that should be further investigated in drug discovery efforts. The QSAR results offer good prospect for the rational design of novel compounds with robust bioactivities.

The genes encoding human manganese superoxide dismutase (MnSOD) and Vitreoscilla hemoglobin (VHb) were fused in-frame to generate a bifunctional enzyme that possessed MnSOD and peroxidase-like activities. At neutral pH, the coupling of the SOD and peroxidase reactions revealed that the bifunctional enzyme exhibited a 2.5 times shorter transient period and a 1.67 times higher reaction rate at steady-state conditions. Furthermore, the catalytic rate of the bifunctional enzyme was not affected as much by the external H₂O₂ scavenger catalase. This indicates that the bifunctional protein possesses a greater antioxidant capability, which is possibly due to the close proximity between the active site of MnSOD and the heme moiety of VHb. Our findings not only provide insight into the synergistic functions of SOD and peroxidase but also could potentially be used to develop novel therapeutic agents with more efficient O₂ carrying capability.

Ischemic heart disease (IHD) is predominantly the leading cause of death worldwide. Early detection of IHD may effectively prevent severity and reduce mortality rate. Recently, magnetocardiography (MCG) has been developed for the detection of heart malfunction. Although MCG is capable of monitoring the abnormal patterns of magnetic field as emitted by physiologically defective heart, data interpretation is time-consuming and requires highly trained professional. Hence, we propose an automatic method for the interpretation of IHD pattern of MCG recordings using machine learning approaches. Two types of machine learning techniques, namely back-propagation neural network (BNN) and direct kernel self-organizing map (DK-SOM), were applied to explore the IHD pattern recorded by MCG. Data sets were obtained by sequential measurement of magnetic field emitted by cardiac muscle of 125 individuals. Data were divided into training set and testing set of 74 cases and 51 cases, respectively. Predictive performance was obtained by both machine learning approaches. The BNN exhibited sensitivity of 89.7%, specificity of 54.5% and accuracy of 74.5%, while the DK-SOM provided relatively higher prediction performance with a sensitivity, specificity and accuracy of 86.2%, 72.7% and 80.4%, respectively. This finding suggests a high potential of applying machine learning approaches for high-throughput detection of IHD from MCG data.

A quantitative structure-activity relationship (QSAR) study was performed to model the lactonolysis activity of N-acyl-homoserine lactone lactonase. A data set comprising of 20 homoserine lactones and related compounds was taken from the work of Wang et al. Quantum chemical descriptors were calculated using the semiempirical AM1 method. Partial least squares regression was utilized to construct a predictive model. This computational approach reliably reproduced the lactonolysis activity with high accuracy as illustrated by the correlation coefficient in excess of 0.9. It is demonstrated that the combined use of quantum chemical descriptors with partial least squares regression are suitable for modeling the AHL lactonolysis activity.

Superoxide radical (O2*-) is a toxic byproduct of oxidative metabolism that extensively damages cellular macromolecules and organelles. Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide (H2O2) and molecular oxygen (O2) thus providing a biological defense against oxygen toxicity. The structural gene of human manganese superoxide dismutase (hMnSOD) was successfully cloned into the pET46Ek/LIC by using a Ligation Independent Cloning (LIC) technique. The recombinant human MnSOD was expressed in E. coli strain BL21(DE3)pLysS and purified to homogeneity by Ni2+ -NTA. Supplementation of Mn2+ in the bacterial growth media was proven to be crucial for production of enzymatically active hMnSOD. The recombinant enzyme revealed a specific activity up to 2,857 U mg(-1) as measured by inhibition of photoreduction of nitroblue tetrazolium (NBT). The molecular weight of each subunit was estimated to be 22 kDa by SDS-PAGE. More interestingly, E. coli expressing hMnSOD provides resistance against oxidative stress induced by the herbicide paraquat up to 1.2 mM. These findings gain insights into the biochemical characterization and significant roles of oxidative-protection of the hMnSOD in biological systems.

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His6GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His6GFPuv/H148G and His6GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 Å, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn2+ and Cu2+ than those of the template protein (His6GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (Kd) to metal ions were in the order of 4.88×10-6 M, 16.67×10-6 M, 25×10-6 M, and 33.33×10-6 M for His6GFPuv/F165G, His6GFPuv, His6GFPuv/H148G/F165G and His6GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.

The fight against the emergence of mutant influenza strains has led to the screening of an increasing number of compounds for inhibitory activity against influenza neuraminidase. This study explores the chemical space of neuraminidase inhibitors (NAIs), which provides an opportunity to obtain further molecular insights regarding the underlying basis of their bioactivity. In particular, a large set of 347 and 175 NAIs against influenza A and B, respectively, was compiled from the literature. Molecular and quantum chemical descriptors were obtained from low-energy conformational structures geometrically optimized at the B3LYP/6-31G(d) level. The bioactivities of NAIs were classified as active or inactive according to their half maximum inhibitory concentration (IC50) value in which IC50 < 1 μM and > 10 μM were defined as active and inactive compounds, respectively. Interpretable decision rules were derived from a quantitative structure-activity relationship (QSAR) model established using a set of 13 descriptors via decision tree analysis. Good predictive performance was achieved as deduced from 10-fold cross-validation, in which an accuracy and MCC of 82.46% and 0.649, respectively, were obtained for influenza A NAIs while values of 80.00% and 0.553 were obtained for influenza B NAIs. Both univariate and multivariate analyses revealed the importance of the lowest unoccupied molecular orbital, number of hydrogen bond donors and number of hydrogen bond acceptors in the predictive model of NAIs against influenza A while the number of hydrogen bond acceptors, molecular energy and the energy gap between the highest occupied and lowest unoccupied molecular orbitals were important in the predictive model for influenza B NAIs. Molecular scaffold analysis was performed on both data sets for discriminating important structural features among active and inactive NAIs. Furthermore, molecular docking was employed to investigate the binding modes and their moiety preferences of active NAIs against both influenza A and B neuraminidase. Moreover, novel NAIs with robust binding fitness towards influenza A and B neuraminidase were generated via combinatorial library enumeration and their binding fitness was on par or better than FDA-approved drugs. The results from this study are anticipated to be beneficial for guiding the rational drug design of novel NAIs for treating influenza infections.

A simple, inexpensive and field applicable metal determination system would be a powerful tool for the efficient control of metal ion contamination in various sources e.g. drinking-water, water reservoir and waste discharges. In this study, we developed a cell-based metal sensor for specific and real-time detection of copper ions. E. coli expressing metal-sensing green fluorescent protein (designated as TG1/(CG)6GFP and TG1/H6CdBP4GFP) were constructed and served as a metal analytical system. Copper ions were found to exert a fluorescence quenching effect, while zinc and cadmium ions caused minor fluorescence enhancement in the engineered bacterial suspension. To construct a user-friendly and reagentless metal detection system, TG1/H6CdBP4GFP and TG1/(CG)6GFP were encapsulated in polyacrylamide hydrogels that were subsequently immobilized on an optical fiber equipped with a fluorescence detection module. The sensor could be applied to measure metal ions by simply dipping the encapsulated bacteria into a metal solution and monitoring fluorescence changes in real time as a function of the metal concentration in solution. The sensor system demonstrated high specificity toward copper ions. The fluorescence intensities of the encapsulated TG1/(CG)6GFP and TG1/H6CdBP4GFP were quenched by approximately 70 % and 80 % by a high-dose of copper ions (50 mM), respectively. The level of fluorescence quenching exhibited a direct correlation with the copper concentration, with a linear correlation coefficient (r) of 0.99. The cell-based metal sensor was able to efficiently monitor copper concentrations ranging between 5 M and 50 mM, encompassing the maximum allowed copper contamination in drinking water (31.15 M) established by the WHO. Furthermore, the cell-based metal sensor could undergo prolonged storage for at least 2 weeks without significantly influencing the copper sensitivity.

In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 microM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10-20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore's ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195-196 nm in combination with a broad negative deflection around 215-216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 microM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.

The molecular mechanism of ethylenediaminetetraacetic acid (EDTA)-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixtures of DPPC and metal-chelating lipids, such as N(alpha),N(alpha)-Bis[carboxymethyl]-N(epsilon)-[(dioctadecylamino)succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His(6)GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His(6)GFPuv/H148G and His(6)GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 A, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn(2+) and Cu(2+) than those of the template protein (His(6)GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K(d)) to metal ions were in the order of 4.88 x 10(-6) M, 16.67 x 10(-6) M, 25 x 10(-6) M, and 33.33 x 10(-6) M for His(6)GFPuv/F165G, His(6)GFPuv, His(6)GFPuv/H148G/F165G and His(6)GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.