Structural and biochemical analyses of the tetrameric carboxypeptidase S9Cfn from Fusobacterium nucleatum.

As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass-spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6 Šresolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site-directed mutagenesis and enzymatic activity assays, revealed a substrate-entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity.

Organic arsenicals target thioredoxin reductase followed by oxidative stress and mitochondrial dysfunction resulting in apoptosis.

Considering the vital role of cellular redox state, more and more researches focus on the design of drugs targeting thioredoxin reductase (TrxR), an important enzyme in maintaining the balance of cellular redox. Here two organic arsenicals, 2-(((4-(1,3,2-dithiarsinan-2-yl) phenyl) imino) methyl) phenol (PIM-PAO-PDT) and N-(4-(1,3,2-dithiarsinan-2-yl) phenyl)-2-hydroxybenzamide (PAM-PAO-PDT), bearing the S-As-S chemical scaffold and different linking groups have been synthesized, and both of them show the better inhibitory activity and selectivity towards HL-60 cells. Importantly, it is illustrated that they can target TrxR selectively and inhibit its activity via the disturbance for Cys83 and Cys88 located in conserved active sites. Afterwards, the cells suffer from the burst of ROS, consumption of antioxidants and high sensitivity for oxidants, which further damage the mitochondria leading to dysfunction including the collapse of membrane potential, ATP level decline, mitochondrial membrane swelling, MPTP opening, Ca2+ and cytochrome c release. Then the mitochondria-dependent apoptosis is triggered by PIM-PAO-PDT and PAM-PAO-PDT, which can also be deterred in the presence of NAC, DTT or LA. Although the organic arsenicals can suppress TrxR activity, the following oxidative stress and mitochondrial dysfunction are the main causes for apoptosis.

Discovery of Cdc25A Lead Inhibitors with a Novel Chemotype by Virtual Screening: Application of Pharmacophore Modeling Based on a Training Set with a Limited Number of Unique Components.

Cdc25 phosphatase was studied as an attractive target for cancer therapy. Multiple pharmacophore models with the unique core features of classic quinone inhibitors and those of novel inhibitors were used to discover a novel lead inhibitor. A total of 21 compounds with qualified physical properties were screened from the Maybridge HitFinder database containing 14 400 compounds by pharmacophore models. Four compounds were found to inhibit Cdc25A activity by more than 50 % at a concentration of 100 µm. Among these compounds, KM10389 (N-{2-[(furan-2-ylmethyl)thio]ethyl}-2-[(4-hydroxy-6-propylpyrimidin-2-yl)thio]acetamide) showed high inhibitory activity with an IC50 value of 7.9 µm. Selective cytotoxicity toward HeLa cells was observed with an IC50 value of 66.3 µm, whereas the IC50 value for HEK293 cells was higher than 100 µm. Blocking of the G1/S transition was also observed for HeLa cells in the presence of the compound by increasing the G1 phase by 16.15 %. Together with compounds HTS02435 and HTS01205, a novel lead inhibitor structure was identified and analyzed by a molecular docking study. The implication of virtual screening by using different pharmacophore models representing the different features is fully discussed.

Identification of Binding Modes for Amino Naphthalene 2-Cyanoacrylate (ANCA) Probes to Amyloid Fibrils from Molecular Dynamics Simulations.

The amino naphthalene 2-cyanoacrylate (ANCA) probe is a kind of fluorescent amyloid binding probe that can report different fluorescence emissions when bound to various amyloid deposits in tissue, while their interactions with amyloid fibrils remain unclear due to the insoluble nature of amyloid fibrils. Here, all-atom molecular dynamics simulations were used to investigate the interaction between ANCA probes with three different amyloid fibrils. Two common binding modes of ANCA probes on Aß40 amyloid fibrils were identified by cluster analysis of multiple simulations. The van der Waals and electrostatic interactions were found to be major driving forces for the binding. Atomic contacts analysis and binding free energy decomposition results suggested that the hydrophobic part of ANCA mainly interacts with aromatic side chains on the fibril surface and the hydrophilic part mainly interacts with positive charged residues in the ß-sheet region. By comparing the binding modes with different fibrils, we can find that ANCA adopts different conformations while interacting with residues of different hydrophobicity, aromaticity, and electrochemical properties in the ß-sheet region, which accounts for its selective mechanism toward different amyloid fibrils.

Spectroscopic and Microscopic Studies on the Mechanism of Mitochondrial Toxicity Induced by CdTe QDs Modified with Different Ligands.

Quantum dots (QDs) are increasingly applied in sensing, drug delivery, biomedical imaging, electronics industries, etc. Consequently, it is urgently required to examine their potential threat to humans and the environment. In the present work, the toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with two different ligands (MPA and BSA) to mitochondria was investigated using flow cytometry, spectroscopic, and microscopic methods. The results showed that QDs induced mitochondrial permeability transition (MPT), which resulted in mitochondrial swelling, collapse of the membrane potential, inner membrane permeability to H(+) and K(+), the increase of membrane fluidity, depression of respiration, alterations of ultrastructure, and the release of cytochrome c. Furthermore, the protective effects of CsA and EDTA confirmed QDs might be able to induce MPT via a Ca(2+)-dependent domain. However, the difference between the influence of CdTe QDs and that of Cd(2+) on mitochondrial membrane fluidity indicated the release of Cd(2+) was not the sole reason that QDs induced mitochondrial dysfunction, which might be related to the nanoscale effect of QDs. Compared with MPA-CdTe QDs, BSA-CdTe QDs had a greater effect on the mitochondrial swelling, membrane fluidity, and permeabilization to H(+) and K(+) by mitochondrial inner membrane, which was caused the fact that BSA was more lipophilic than MPA. This study provides an important basis for understanding the mechanism of the toxicity of CdTe QDs to mitochondria, and valuable information for safe use of QDs in the future.

Multi-spectroscopic analysis and molecular modeling on the interaction of curcumin and its derivatives with human serum albumin: a comparative study.

The comparative study about the interaction between curcumin and its derivatives (demothxycurcumin and bisdeoxycurcumin) with human serum albumin (HSA) has been carried out using multi-spectroscopic analysis and molecular modeling method. The characteristic of fluorescence quenching and the thermodynamic parameters have been studied by state emission fluorescence experiments under different temperatures with an interval of 6 K. Curcumin shows largest quenching constant and bisdeoxycurcumin shows the smallest at the temperature of 298 K. However, the quenching constant of curcumin drops quickly with the increase of temperature. Demothxycurcumin gives the largest quenching efficiency at the temperature of 310 K. An average distance of 6.7 nm for energy transfer has been determined based on förster resonance energy theory (FRET). The site competitive replacement experiments illustrate three compounds mainly binding on site I (Subdomain IIA) of the protein, and show tendency of binding on site II (Subdomain IIIA) with the removing of methoxyl groups. Circular dichroism spectra and Fourier transform infrared spectroscopy (FTIR) have been used to investigate the influence on protein secondary structure. Content of the α-helix increases at low concentrations of the compounds, while unfolding occurs at high concentrations. Docking simulation reveals possible mechanism for different quenching behavior and binding sites preferred by three compounds. The binding modes have effectively supported the conclusion of the experiments.

Synthesis of F16 conjugated with 5-fluorouracil and biophysical investigation of its interaction with bovine serum albumin by a spectroscopic and molecular modeling approach.

5-Fluorouracil (5-FU) has been widely used as a chemotherapy agent in the treatment of many types of solid tumors. Investigation of its antimetabolites led to the development of an entire class of fluorinated pyrimidines. However, the toxicity profile associated with 5-FU is significant and includes diarrhea, mucositis, hand-foot syndrome and myelosuppression. In aiming at reducing of the side effects of 5-FU, we have designed and synthesized delocalized lipophilic cations (DLCs) as a vehicle for the delivery of 5-FU. DLCs accumulate selectively in the mitochondria of cancer cells because of the high mitochondrial transmembrane potential (ΔΨm). Many DLCs exhibited anti-cancer efficacy and were explored as potential anti-cancer drugs based on their selective accumulation in the mitochondria of cancer cells. F16, the DLC we used as a vehicle, is a small molecule that selectively inhibits tumor cell growth and dissipates mitochondrial membrane potential. The binding of the conjugate F16-5-FU to bovine serum albumin (BSA) was investigated using spectroscopic and molecular modeling approaches. Fluorescence quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between F16-5-FU and BSA. The activation energy of the interaction between F16-5-FU and BSA was calculated and the unusually high value was discussed in terms of the special structural block indicated by the molecular modeling approach. Molecular modeling showed that F16-5-FU binds to human serum albumin in site II, which is consistent with the results of site-competitive replacement experiments. It is suggested that hydrophobic and polar forces played important roles in the binding reaction, in accordance with the results of thermodynamic experiments.

Spectroscopic studies on the interactions between CdTe quantum dots coated with different ligands and human serum albumin.

This paper investigates the interactions between human serum albumin (HSA) and CdTe quantum dots (QDs) with nearly identical hydrodynamic size, but capped with four different ligands (MPA, NAC, and GSH are negatively charged; CA is positively charged) under physiological conditions. The investigation was carried out using fluorescence spectroscopy, circular dichroism (CD) spectra, UV-vis spectroscopy, and dynamic light scattering (DLS). The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of HSA and negatively charged QDs (MPA-CdTe, NAC-CdTe, and GSH-CdTe), which was also reconfirmed by the increasing of the hydrodynamic radius of QDs. The K(a) values of the three negatively charged QDs are of the same order of magnitude, indicating that the interactions are related to the nanoparticle itself rather than the ligands. ΔH<0 and ΔS>0 implied that the electrostatic interactions play predominant roles in the adsorption process. Furthermore, it was also proven that QDs can induce the conformational changes of HSA from the CD spectra and the three-dimensional fluorescence spectra of HSA. However, our results demonstrate that the interaction mechanism between the positively charged QDs (CA-CdTe) and HSA is significantly different from negatively charged QDs. For CA-CdTe QDs, both the static and dynamic quenching occur within the investigated range of concentrations. According to the DLS results, some large-size agglomeration also emerged.

Synthesis of three novel anionic gemini surfactants and comparative studies of their assemble behavior in the presence of bovine serum albumin.

Three novel anionic sulfonate gemini surfactants, sodium 4,4'-(10,19-dioxo-9,11,18,20-tetraazaoctacosane-9,20-diyl) dibenzenesulfonate (Surfactant I), sodium 4,4'-(12,21-dioxo-11,13,20,22-tetraazadotriacontane-11,22-diyl) dibenzenesulfonate (Surfactant II), and sodium 4,4'-(14,23-dioxo-13,15,22,24-tetraazahezatriacontane-13,24-diyl) dibenzenesulfonate (Surfactant III), with different lengths of hydrophobic tail have been synthesized, and their assembly behavior in the presence of bovine serum albumin (BSA) has been studied using spectral methods and molecular modeling methods at physiological pH and 298 K. Critical micelle concentrations (CMCs) of the three surfactants have been determined by surface tension measurements. Despite the obvious decrease of CMC with the increase of tail length, fluorescence spectra have shown much closer CAC in the presence of BSA. Surfactant II shows the highest CAC of 3.19 × 10(-5) mol L(-1) compared with the other two. The polarity of the microenvironment in BSA-surfactant systems has been investigated using pyrene as the probe. In addition, far-UV CD spectra studied the change of the secondary structure content of BSA caused by the three surfactants. The features of the assembly behavior were discussed by three concentration regions. Surfactant II could unfold the protein much more efficiently than the other two surfactants at low concentration, but at high concentration, the change of the secondary structure and the formation of hydrophobic microenvironment show a direct relationship to the length of the hydrophobic tail with the increase of the surfactant concentration.

Spectroscopic, structural and thermodynamic properties of chlorpyrifos bound to serum albumin: A comparative study between BSA and HSA.

Chlorpyrifos (CPF) is a widely used organophosphate insecticide which could bind with human serum albumin (HSA) and bovine serum albumin (BSA). The binding behavior was studied employing fluorescence, three-dimensional fluorescence, Circular dichroism (CD) spectroscopy, UV-vis absorption spectroscopy, electrochemistry and molecular modeling methods. The fluorescence spectra revealed that CPF causes the quenching of the fluorescence emission of serum albumin. Stern-Volmer plots were made and quenching constants were thus obtained. The results suggested the formation of the complexes of CPF with serum albumins, which were in good agreement with the results from electrochemical experiments. Association constants at 25°C were 3.039 × 10(5) mol L(-1) for HSA, and 0.3307 × 10(5) mol L(-1) for BSA, which could affect the distribution, metabolism, and excretion of pesticide. The alterations of protein secondary structure in the presence of CPF were confirmed by the evidences from UV and CD spectra. Site competitive experiments also suggested that the primary binding site for CPF on serum albumin is close to tryptophan residues 214 of HSA and 212 of BSA, which was further confirmed by molecular modeling.

Binding of fullerol to human serum albumin: spectroscopic and electrochemical approach.

The potential impact of human exposure to carbonaceous nanomaterials in the environment becomes a concerning issue. Here we report on the interaction of fullerol with human serum albumin (HSA) using spectroscopic and electrochemical methods. The water-soluble fullerene derivative (fullerol) was synthesized and characterized by IR, (1)H NMR, TG-DSC, XRD, HR-TEM, etc. The spectroscopic methods show that the fluorescence quenching of HSA by fullerol is the result of the formation of an HSA-fullerol complex. Binding parameters such as ΔG, ΔH and ΔS were calculated, and the quenching constant K(a) at different temperatures was determined using the modified Stern-Volmer equation. The electrochemical experiments further confirmed the conclusions. In addition, the influences of coexisting heavy metal ions have also been studied in the present system. The circular dichroism spectra (CD), 3D fluorescence spectra and FT-IR spectra results suggest that the secondary structure of HSA was changed by fullerol. Based on the site marker competitive experiments, we can predict the possible binding position of fullerol on the HSA was located at the site of sub domain II A. Furthermore, the distance r between donor (HSA) and acceptor (fullerol) was obtained according to the famous fluorescence resonance energy transfer (FRET) mechanism.

Mitochondrial permeability transition induced by different concentrations of zinc.

Zinc is one of the required trace elements in animals, and it serves an important role in biological systems. However, high levels of zinc are poisonous to organisms. So far, there exist conflicting reports about zinc ions-induced mitochondrial permeability transition (MPT). We analyzed the effects of Zn²âº on MPT by monitoring mitochondrial swelling with the ultraviolet-visible light absorption spectrum, characterizing the fluidity of the membrane with fluorescence anisotropy, detecting the transmembrane potential (Δψ) with fluorescence intensity, and observing mitochondrial ultrastructure with transmission electron microscopy. Data reveal that low concentrations of zinc ions can trigger MPT while high levels of zinc ions cannot, which implies that zinc ions' toxicity cannot be the result of a single simple mechanism.

A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil adduct with antioxidant and anticancer properties.

A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil (VE-5-FU) adduct with antioxidant and anticancer properties for antioxidant-based cancer chemoprevention was synthesized and utilized for selective drug release in the stomach.

Spectroscopic and molecular modeling studies on the interaction between a fluorine-containing triazole derivative and human serum albumin.

The interaction between 4-(4-fluorobenzylideneamino)-5-propyl-4H-1,2,4-triazole-3-thiol (FBTZ) and human serum albumin (HSA) under simulative physiological conditions was investigated by fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling method. Fluorescence spectroscopic data showed that the fluorescence quenching of HSA was a result of the formation of FBTZ-HSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K (a)) of FBTZ to HSA were obtained at three different temperatures. The enthalpy change (ΔH) and entropy change (ΔS) were calculated on the basis of van't Hoff equation, and the results showed that hydrogen-bonding and van der Waals forces were the dominant intermolecular forces to stabilize the complex. Site marker competitive replacement experiments demonstrated that the binding of FBTZ to HSA primarily took place in sub-domain IIA (Sudlow's site I). The binding distance (r) between FBTZ and the tryptophan residue of HSA was estimated according to the theory of fluorescence resonance energy transfer. The conformational investigation showed that the presence of FBTZ induced some changes of secondary structure of HSA. Molecular modeling study further confirmed the binding mode obtained by experimental study.

Biophysical studies on the interactions of a classic mitochondrial uncoupler with bovine serum albumin by spectroscopic, isothermal titration calorimetric and molecular modeling methods.

The interaction between a classic uncoupler (2,4-dinitrophenol, DNP) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy under the physiological conditions. The fluorescence quenching constants were calculated by the Stern-Volmer equation, and based upon the temperature dependence of quenching constants, it was proved that DNP caused a static quenching of the intrinsic fluorescence of BSA. Owing to the static quenching mechanism, different associative binding constants at various temperatures were determined and thus the thermodynamic parameters, namely enthalpy (ΔH=-21.12 kJ mol(-1)) and entropy changes (ΔS=23.51 J mol(-1) K(-1)) could be calculated based on the binding constants. Moreover, the enthalpy and entropy changes are consistent with the "Enthalpy-Entropy Compensation" equation obtained from our previous work. The negative enthalpy and positive entropy indicated that the electrostatic interactions played a major role in DNP-BSA binding process. Site marker competitive displacement experiments were carried out by using fluorescence and isothermal titration calorimetry (ITC) methods. These results showed that DNP bound with high affinity to Sudlow's site I (subdomain IIA) of BSA. The distance (r=3.78 nm) between donor (BSA) and acceptor (DNP) was obtained according to the mechanism of fluorescence resonance energy transfer (FRET). Furthermore, the results of synchronous fluorescence and circular dichroism (CD) spectroscopic studies indicated that the microenvironment and the secondary conformation of BSA were altered. The above results were supported by theoretical molecular modeling methods.

Synthesis of a novel hydrazone derivative and biophysical studies of its interactions with bovine serum albumin by spectroscopic, electrochemical, and molecular docking methods.

Hydrazone derivatives possess potential antitumor activities based on modulation of the iron metabolism in cancer cell. A novel hydrazone, N'-(2,4-dimethoxybenzylidene)-2-hydroxybenzohydrazide (DBH), has been synthesized and characterized, which is an analogue of 311 possessing potent anticancer activity. The interactions between DBH and bovine serum albumin (BSA) have been investigated systematically by fluorescence, molecular docking, circular dichroism (CD), UV-vis absorption, and electrochemical impedance spectroscopy (EIS) methods under physiological conditions. The fluorescence quenching observed is attributed to the formation of a complex between BSA and DBH, and the reverse temperature effect of the fluorescence quenching has been found and discussed. The primary binding pattern is determined by hydrophobic interaction occurring in Sudlow's site I of BSA. DBH could slightly change the secondary structure and induce unfolding of the polypeptides of protein. An average binding distance of ~4.0 nm has been determined on the basis of the Förster resonance energy theory (FRET). The effects of iron on the system of DBH-BSA have also been investigated. It is found that iron could compete against BSA to bind DBH. All of these results are supported by a docking study using a BSA crystal model. It is shown that DBH can efficiently bind with BSA and be transported to the focuses needed. Subsequent antitumor test and detailed anticancer mechanism are undergoing in our lab.