Isothermal Titration Calorimetry Techniques Research Articles

Chiral electrochemical sensing of propranolol enantiomers on D/L-Cys modified gold nanoparticles

The present study aims to explore the interaction mechanisms between propranolol (PRNL) enantiomers and bare and L/D-Cysteine modified gold nanoparticles (L/D-Cys−AuNPs) surfaces, using electrochemical (cyclic voltammetry, differential pulse voltammetry, electrochemical impedance spectroscopy) and microcalorimetric (isothermal titration calorimetry) techniques. In the absence of a chiral environment, PRNL enantiomers are strongly physisorbed on the AuNPs modified surface, yet no significant difference was recorded in the oxidation peak potential (+0.778 V R(+)-PRNL versus +0.78 V for S(-)-PRNL). Upon decorating AuNPs with L-Cys, an ∼40 mV anodic shift is recorded for S(-)-PRNL due to a more favored orientation of its diastereoisomer on the electrode's surface towards electron transfer. Interestingly, by altering the conformation of the diastereoisomeric chiral selector (replacing L-Cys with D-Cys), the enantioselective nature of the recorded electrochemical signals fades out. Additionally, the potential synergistic effects of a dual chiral system, based on the covalently bound L-/D-Cys−AuNPs and β-cyclodextrin (β-CD), a free, host-guest type chiral selector, added to the electrolyte, has also been studied.

Investigation of steric hindrance effect on the interactions between four alkaloids and HSA by isothermal titration calorimetry and molecular docking.

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA.

Application of a modern theoretical approach to the study of the interaction of KR-12 peptides derived from human cathelicidins with Cu(II) ions.

The human cationic antimicrobial protein (hCAP) corresponding to the overlapping sequences of 151-162 of hCAP named KR-12 peptide is the smallest portion of the only type of human Cathelicidin, which has been shown to be modifiable into a more effective antimicrobial. In this study, an in silico analysis, supported by potentiometric titration and isothermal titration calorimetry techniques, was performed to identify potential Cu(II) binding sites of KR-12. The analysis of the presented data at the given theoretical level (GFN2-xTB/ALPB) revealed which peptide chain fragments are involved in the most favourable KR-12-Cu(II) binding mode. Based on a quantum chemical approach, the most favourable coordination modes of Cu(II) to peptides are proposed together with the discussion of the chemical nature of the interactions. The presented results demonstrated that KR-12 interacts with metal ions mostly via the main chain's oxygen atoms; however, the two types of amino acids that are expected to be vital for the interaction of Cu(II) are D (aspartic acid) and R29 (arginine). It was demonstrated that in order to explain the complexity of the interaction process in peptide-metal ion systems, the use of theoretical methods is sometimes necessary to explain the details of the experimental results and provide an in-depth understanding of these dynamic systems.

The study of excess enthalpy for {ionic liquid + ethanol} binary systems as new working fluids in absorption refrigeration technology

Binary systems composed of phosphonium – based ionic liquid and water, or short-chain alcohols exhibit properties which render them suitable as candidates for working fluids in absorption refrigeration technology. This work presents the study of the excess enthalpy (HE) of mixing in ethanolic solutions of the following ionic liquids: 1-ethyl-1-methylpiperidinium dimethyl phosphate, [C1C2PIP][DMP], 1-ethyl-1-methyl-pyrrolidinium dimethyl phosphate, [C1C2PYR][DMP], N,N,N-triethyl-N-methylammonium dimethyl phosphate, [N1,2,2,2][DMP],1-hydroxyethyl-1-methylpyrrolidinium dimethyl phosphate, [C1C2OHPYR][DMP], 1-ehtyl-1-methylmorpholinium diethyl phosphate, [C1C2MOR][DEP], 1-ethyl-1-methylpyrrolidinium diethyl phosphate, [C1C2PYR][DEP], and 1-ethyl-3-methylimidazolium diethyl phosphate, [C1C2IM][DEP] is presented. The experiment was performed at temperature T = 298.15 K and under ambient pressure using isothermal titration calorimetry technique. This work presents the effect of the core structure of the cation and the effect of the carbon chain length in the anion of the ionic liquid on the HE value of the systems studied. Experimental HE values were correlated using Redlich – Kister type equation. The standard deviations for the correlation vary from σ = 9 J∙mol−1 for {[C1C2OHPYR][DMP] + ethanol} to 28 J∙mol−1 for {[C1C2IM][DMP] + ethanol} showing significant agreement between experimental and calculated values. The negative excess enthalpy of mixing over the entire composition range was found in each system which indicate stronger (IL - ethanol) interactions associated with hydrogen bond formation in this system, compared to (IL - IL) and (ethanol - ethanol) interactions. For each system, the minimum of the excess enthalpy of mixing was determined, which increases in the following series: [N1,2,2,2][DMP] (HE = −2645.9 J·mol−1) < [C1C2PYR] [DMP] < [C1C2IM][DEP] < [C1C2PYR][DEP] < [C1C2PIP][DMP] < [C1C2MOR][DMP] < [C1C2OHPYR][DMP] (HE = −881.5 J·mol−1). This characteristic is desirable from the point of view of the possibility of using these systems in absorption refrigeration technology.

Eukaryotic Expression of the Cytochrome c Oxidase Subunit I of Sitophilus zeamais and Its Interaction with Allyl Isothiocyanate.

Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is a destructive pest of stored grains around the world. Allyl isothiocyanate (AITC) was shown to have good bioactivity in the control of S. zeamais. In this study, the interaction of AITC on cytochrome c oxidase core subunits I (COX I) and their binding mechanism were determined using spectroscopic, isothermal titration calorimetry and molecular docking techniques. The results indicate the binding constant (Ka) of AITC and COX I was 6.742 × 103 L/mol. Analysis of spectroscopic revealed that the binding of COX I to reduced Cyt c induced conformational changes of reduced Cyt c, while AITC could competitively bind and inhibit the activity of the COX I protein. Moreover, molecular docking results suggested a sulfur atom in the AITC structure could form a hydrogen bond having a length of 3.3 Å with the Gly- 27 of COX I.

In vitro studies on the selective cytotoxic effect of luminescent Ru(II)-p-cymene complexes of imidazo-pyridine and imidazo quinoline ligands.

In recent years, Ru(II) complexes have gained high importance in medicinal chemistry due to their significant anti-cancer activities, which are directly related to their DNA binding ability. In this report, the chemistry and cytotoxicity of two new Ru(II) complexes containing imidazole pyridine (Ru-1) and imidazole quinoline (Ru-2) have been studied. The prepared compounds were characterized using infrared (IR), nuclear magnetic resonance (NMR), mass spectrometry (MS), isothermal titration calorimetry (ITC), UV-Vis, and fluorescence spectral techniques. The structural analyses show that the Ru(II) complexes exhibit a 'piano stool' coordination geometry and they are composed of one bound arene, two sigma bonded benzil nitrogen atoms, and labile chlorine linked to Ru(II). The photo-physical properties of these complexes were examined, and they exhibit absorption peaks at 260 nm and 380 nm, which are due to the involvement of intra-ligand charge transitions (ILCT) and metal-to-ligand charge transitions (MLCT), respectively. The binding process of the Ru(II) complexes with DNA and BSA is non-covalent in nature and the binding constants of Ru-1 and Ru-2 complexes with DNA and BSA were found to be 1 × 105 M-1 and 1 × 103 M-1, respectively. In the presence of the Ru(II) complexes, ethidium bromide (EtBr) is competitively displaced from DNA by intercalation of the Ru(II) complexes in DNA and it is well corroborated by viscosity and in silico studies. Both the ligands and Ru(II) complexes were carefully investigated in vitro for cytotoxicity against HeLa, MCF-7, and MDA-MB-231 cells. Surprisingly, both Ru(II) complexes exhibit superior cytotoxicity to cisplatin with a low LD50 value against the examined cancer cells. Besides, an insignificant effect on HEK normal cells (LD50 > 140 μM) was observed.

Influence of zinc doping on the molecular biocompatibility of cadmium-based quantum dots: Insights from the interaction with trypsin

Doping quantum dots (QDs) with extra element presents a promising future for their applications in the fields of environmental monitoring, commercial products and biomedical sciences. However, it remains unknown for the influence of doping on the molecular biocompatibility of QDs and the underlying mechanisms of the interaction between doped-QDs and protein molecules. Using the “one-pot” method, we synthesized N-acetyl-l-cysteine capped CdTe: Zn2+ QDs with higher fluorescence quantum yield, improved stability and better molecular biocompatibility compared with undoped CdTe QDs. Using digestive enzyme trypsin (TRY) as the protein model, the interactions of undoped QDs and Zn-doped QDs with TRY as well as the underlying mechanisms were investigated using multi-spectroscopy, isothermal titration calorimetry and dialysis techniques. Van der Waals forces and hydrogen bonds are the major driving forces in the interaction of both QDs with TRY, which leading to the loosening of protein skeleton and tertiary structural changes. Compared with undoped QDs, Zn-doped QDs bind less amount of TRY with a higher affinity and then release higher amount of Cd. Zn-doped QDs have a less stimulating impact on TRY activity by decreasing TRY binding and reducing Cd binding to TRY. Taken all together, Zn-doped QDs offer a safer alternative for the applications of QDs by reducing unwanted interactions with proteins and improving biocompatibility at the molecular level.

SARS‐CoV‐2 main protease inhibitory efficacy of natural peptidic α‐hydroxy amides for therapeutic applications of COVID‐19

Background: COVID‐19, a disease caused by a novel coronavirus, is a major global human threat that has turned into a pandemic. Identification and development of drugs/vaccines is urgently needed for prevention and therapy of the COVID‐19 virus (SARS‐CoV‐2) infection. SARS‐CoV‐2 main protease (Mpro,) plays a key role in viral protein processing for its replication and inhibition of Mpro leads to prevention of viral replication. The SARS‐CoV‐2 Mpro is considered a promising drug target, as it is dissimilar to human proteases. Several peptidic protease inhibitors have been developed as SARS‐CoV‐2 Mpro inhibitors for COVID‐19 treatment. In this study, we screened naturally occurring peptidic α‐hydroxy amides (PHAs) (phebestin, probestin, bestatin) for SARS‐CoV‐2 Mpro inhibition.Materials and methods: In vitro COVID‐19 Mpro fluorometric assay was performed to evaluate its inhibitory efficacy by PHAs. Isothermal titration calorimetry technique was used to demonstrate the binding affinity between COVID‐19 Mpro and PHAs. Circular dichroism technique was used to determine the structural changes in secondary structure of COVID‐19 Mpro induced by PHAs. Molecular docking studies were performed to demonstrate the molecular level mechanism behind COVID‐19 Mpro inhibitory efficacy of PHAs. In addition, cytotoxic effects of PHAs were evaluated on lung epithelial cells of mice for its therapeutic applications for COVID‐19 through nasal delivery.Results and discussion: Phebestin (IC50 = 17.98 µM) Probestin (IC50 = 48.31 µM) and Bestatin (IC50 > 100 µM) significantly inhibited the COVID‐19 Mpro activity. The KD (dissociation constant) values of phebestin and probestin for COVID‐19 Mpro were 31.23 and 43.51 µM, respectively. CD spectrum analysis demonstrated that phebestin and probestin (10 µM) induced changes in the secondary structure of COVID‐19 Mpro. Molecular docking studies demonstrated that Interactions of phebestin and probestin with active site amino acids. Phebestin and probestin did not show any cytotoxicity on normal mouse lung epithelial cells up to 100 µM.Conclusion: Preliminary in vitro studies demonstrated that naturally occurring PHAs derivatives phebestin and probestin are SARS‐CoV‐2 Mpro inhibitors and warranted further studies to develop as drugs for COVID‐19.

The Behavior of Carbonized Polymer Dots at the Nano‐Bio Interface and Their Luminescent Mechanism: A Physical Chemistry Perspective

AbstractCarbonized polymer dots (CPDs) have been widely applied in biomedical fields, such as imaging, diagnosis and drug delivery. Since the complex, non‐equilibrated and dynamic nature of biological systems inevitably affect the predesigned properties of CPDs, then efficiency and ultimate outcome of CPDs in biological identity will be transformed by the ubiquitous nano‐bio interactions. Herein, our recent progress about elucidating the behavior of CPDs at nano‐bio interface from the perspective of physical chemistry has been summarized in the review, mainly at the bio‐macromolecular, cellular membrane and cellular levels, which is crucial for characterize their relative cytotoxicity and clinical transformation. Moreover, we mainly focused on the quantitative relationship of nano‐bio interactions between CPDs with biological identity and related thermodynamics parameters during this process is also obtained from advanced isothermal titration calorimetry technique. Finally, our recent study about the photoluminescence origin is also included in this review, which favors modulating the photoluminescence of CPDs.What is the most favorite and original chemistry developed in your research group?Chemical thermodynamic nature of chemical and biological process.How do you get into this specific field? Could you please share some experiences with our readers?In 2003, several articles from Science, Nature and Nature Biotechnology discussed the potential side effects of nanomaterials on the human health and environment. At the same time, Prof. Daiwen Pang developed QDs for biolabling, and I (Prof. Yi Liu) began to pay attention to the effect of QDs on living things. In 2006, we obtained the Innovation Research Groups of NSFC (20621502), and in this project, I was responsible for the biological effect of nanoprobe.How do you keep balance between research and family?90% of my time for research work, and 10% of my time for family things.How do you supervise your students?First, according to the students’ interests, I will give them some new and important papers to read, and then discuss with them about their research field. We will discuss the research progress at any time, and we have seminar every week.What is the most important personality for scientific research?Curiosity, power of understanding.What's your hobbies? What's your favorite book(s)?My hobbies are reading and badminton. My favorite books are Physical Chemistry (Edited by Xiancai Fu) and Organic Chemistry (4th Edited by David Klein).

Study of the effect of ascorbic acid on Aβ42 peptide aggregation

AbstractBackgroundAscorbic acid (AA) is an antioxidant molecule found at high concentrations in the brain. A negative correlation was found in Alzheimer’s disease (AD) patients between the decrease in AA brain levels and the accumulation of amyloid plaques indicating that AA plays some function in amyloid aggregation, however, no studies have elucidated its mechanism of action so far, which can greatly contribute to the development of new therapeutic strategies for AD.MethodTo investigate AA as inhibitor of amyloid aggregation, amyloid‐beta 1‐42 (Aβ42) samples containing or not AA were incubated in pro‐aggregation conditions and analyzed by atomic force microscopy (AFM) and fluorescence spectroscopy using Thioflavin T. The mechanism of action presented by AA was evaluated using isothermal titration calorimetry (ITC) technique to investigate its binding to Aβ42 monomers.ResultFluorescence analyses pointed out to a decrease in fibril formation for samples incubated with AA at a concentration found in brains of healthy patients. After 72 h, these samples presented almost 80% fewer β‐sheet structures than samples without the antioxidant molecule. The fluorescence results were corroborated by AFM images, indicating that AA inhibited the amyloid aggregation in the fibrillar form. ITC measurements showed a negative cooperative binding between AA and Aβ42 monomers. In this interaction model for multiple sites, the binding of AA molecule in the first site of Aβ42 decreases the affinity constant (Kd) for the binding in the second site (Kd1&gt;Kd2).ConclusionOur study revealed that ascorbic acid is able to inhibit the aggregation of Aβ42 peptides into fibrils through the direct binding to the monomers. Based on the thermodynamic parameters, we observed that the interaction is spontaneous and exothermic. This finding highlights ascorbic acid as a potential candidate for Alzheimer’s disease treatment and the mechanism of action provides novel insights for the inhibition of amyloid aggregation.

Thermodynamic and electrochemical study of tailor-made crown ethers for redox-switchable (pseudo)rotaxanes.

Crown ethers are common building blocks in supramolecular chemistry and are frequently applied as cation sensors or as subunits in synthetic molecular machines. Developing switchable and specifically designed crown ethers enables the implementation of function into molecular assemblies. Seven tailor-made redox-active crown ethers incorporating tetrathiafulvalene (TTF) or naphthalene diimide (NDI) as redox-switchable building blocks are described with regard to their potential to form redox-switchable rotaxanes. A combination of isothermal titration calorimetry and voltammetric techniques reveals correlations between the binding energies and redox-switching properties of the corresponding pseudorotaxanes with secondary ammonium ions. For two different weakly coordinating anions, a surprising relation between the enthalpic and entropic binding contributions of the pseudorotaxanes was discovered. These findings were applied to the synthesis of an NDI-[2]rotaxane, which retains similar spectroelectrochemical properties compared to the corresponding free macrocycle. The detailed understanding of the thermodynamic and electrochemical properties of the tailor-made crown ethers lays the foundation for the construction of new types of molecular redox switches with emergent properties.

Thermodynamics, in vitro release and cytotoxity studies on doxorubicin–toluidine blue O combination drugs co-loaded in aptamer-tethered DNA nanostructures

DNA nanostructures-based chemotherapy-phototherapy (CTPT) combination therapy can achieve targeted release and effectively overcome multidrug resistance (MDR). The therapeutic effect of CTPT combination drugs loaded in DNA nanostructures is related to the interactions among them. Herein, based on our previous research on AS1411NTrs loading with a single drug of DOX, the interactions between a single drug (toluidine blue O, TBO) or combination drugs (doxorubicin and toluidine blue O, DOX + TBO) and AS1411 aptamer-tethered DNA nanotrains (AS1411NTrs) were investigated. By fluorescence spectroscopy and isothermal titration calorimetry technique, the corresponding thermodynamic parameters were obtained, including the number of binding sites, binding constant, enthalpy change and entropy change. By combining with the differential scanning calorimetry and ferrocyanide ion quenching analysis, the binding mode-thermodynamic parameters were correlated. In addition, the effect of the binding affinity of DOX and TBO to AS1411NTrs on the drugs release rate was studied. Finally, the in vitro cytotoxicity and confocal laser scanning microscopy imaging of AS1411NTrs + TBO + DOX was investigated. The results showed that this drug delivery system could increase its in vitro cytotoxicity and overcome drug resistance in DOX-resistant human breast cancer cells (MCF-7/ADR). The work would provide important information for the combination therapy of DOX and TBO to overcome MDR effectively.

Side-Chain Proline-Based Polymers as Effective Inhibitors for In Vitro Aggregation of Insulin.

Insulin fibril formation is considered as the hallmark of several debilitating pathological conditions. To develop effective therapeutics that are able to control the amyloidogenesis process and inhibit fibril formation, herein we have designed a side-chain proline (Pro)-based homopolymer and block copolymers through the reversible addition-fragmentation chain transfer (RAFT) polymerization technique and further explored their obligatory role in the in vitro insulin fibrillation process. Using a variety of biophysical tools, including turbidity measurements, thioflavin T (ThT) fluorescence kinetics, tyrosine (Tyr) fluorescence study, Nile red (NR) fluorescence assay, dynamic light scattering (DLS) study, circular dichroism (CD) measurements, and isothermal titration calorimetry (ITC) techniques, we demonstrated that Pro-based polymers can significantly inhibit the insulin fibrillation process. Among them, the Pro-based homopolymer acts as the most potent inhibitor of insulin fibrillation as confirmed by ThT assay, CD study, and transmission electron microscopic (TEM) analysis. Tyrosine fluorescence measurements and NR fluorescence assay revealed that hydrophobic interactions are the crucial factor that mainly controls the inhibition process. Apart from hydrophobic interactions, polar interactions may also be responsible for the inhibition process as evaluated by ITC study.

VCCI:Chemokine Interactions: Experimental Biochemistry Meets Computational Prediction

Inflammation plays a major role in many pathologies including asthma, arthritis, atherosclerosis and traumatic brain injury. As such, controlling inflammation is an important goal. While many biological pathways are involved in inflammation, an appealing target is the chemokine pathway. Chemokines are small immune system proteins that mediate chemotaxis of leukocytes bearing cognate chemokine receptors to the site of infection or inflammation. Many viruses have evolved strategies to counter the chemokine system, including the production of chemokine binding proteins. In particular, poxviruses encode vCCI (viral CC chemokine Inhibitor; also called p35), a protein that binds members of the CC class of chemokines. vCCI has been shown to bind many CC chemokines with high affinity and as such vCCI could be a potent tool as part of an anti‐inflammation strategy.The mechanistic details of how vCCI is able to bind dozens of CC chemokines with nanomolar (or sub‐nanomolar) affinity is still being elucidated. Structures of vCCI alone and in complex have revealed a beta sandwich composed of two beta sheets that binds chemokines using one face of the sandwich in conjunction with a long, highly acidic loop after the second beta strand. Experimental work shows that this highly negatively charged loop in vCCI can act as a lid on the bound chemokine and interacts with positive charges on the chemokine.A particularly puzzling vCCI‐chemokine interaction is in the area at the edge of the negatively charged loop in vCCI, where a conserved tyrosine (Y80) is close to K48 of the chemokine. It was hypothesized that mutating Y80 to Ala in vCCI would allow more room for the chemokine to bind, but experimentally the opposite has been shown to be the case: the Y80A variant in vCCI has a greatly decreased ability to bind chemokines.We report a collaborative effort to understand the role of Y80 and the action of the acidic loop in vCCI to bind chemokines. Molecular dynamics simulations suggest that rather than being a hindrance to the function of the loop, the role of Y80 is actually to prop the loop in the “open” conformation so that the chemokine can bind. When Y80 is replaced with Ala in simulations, the loop closes and blocks the chemokine binding site. This intriguing and unexpected hypothesis from in silico work has been tested experimentally using NMR, isothermal titration calorimetry and other biophysical techniques. We therefore report a collaborative set of experiments that includes computation and experimentation to aid in the understanding of the vCCI:chemokine interactionSupport or Funding InformationSupport provided by UC Merced summer graduate funding.

Thermodynamic and kinetic insights into the interaction of kynurenic acid with human serum albumin: Spectroscopic and calorimetric approaches

The interaction of neuroactive kynurenic acid (KYNA) with human serum albumin (HSA) was studied from both thermodynamic and structural point of view. This protein-drug interaction has high relevance in neuroscience, because the serum albumin-based KYNA-containing composites may act as a potential drug delivery system. Reversible binding of KYNA on HSA has been investigated under physiological conditions at various temperatures. The overall results of isothermal titration calorimetry (ITC), fluorescence spectroscopy and two-dimensional surface plasmon resonance (SPR) techniques showed similar trend for the temperature dependence of the estimated association constant (KA) values. Moreover, the enthalpy- (ΔH0), entropy- (ΔS0) and heat capacity changes (ΔCp) of KYNA-HSA complex formation were also calculated and interpreted according to the integrated van't Hoff equation. The results indicate low-affinity complex formation between the protein and the drug molecule, with two independent binding sites. The presence of the drug in the protein caused significant structural change with an α-helix content decrease from 69.7% to 51.2% proved by circular dichroism (CD) measurements. Theoretical calculations also support the experimentally suggested two independent binding sites theory stabilized by salt bridges and aromatic π-π stacking in the proposed binding positions.

A novel view of the separate and simultaneous binding effects of docetaxel and anastrozole with calf thymus DNA: Experimental and in silico approaches.

DNA stands as the primary purpose of many anticancer drugs and according to the performed research on this field, some certain changes contain crucial functionalities in the regulated transcription of DNA. Therefore, the interaction between anticancer drugs and DNA play an important role in understanding their function and also provide a better groundwork for producing more efficient and newer drugs. Here, the interaction between Docetaxel (DO) and calf thymus DNA (ct DNA), in the presence and absence of Anastrozole (AN), has been examined through the usage of different methods that include isothermal titration calorimetry, multi-spectroscopic, viscometry, and molecular docking techniques. Interaction studies have been performed by preparing different molar ratios of DO with the constant ct DNA and AN concentration at pH = 6.8. The binding constants have been calculated to be 7.93 × 104 M-1 and 6.27 × 104 M-1, which indicate the strong binding of DO with ct DNA double helix in the absence and presence of AN, respectively. Thermodynamic parameters, which were obtained from fluorescence spectroscopy and isothermal titration calorimetry, have suggested that the binding of DO and AN to ct DNA as binary and ternary systems have been mainly driven by the electrostatic interactions. The relative viscosity of ct DNA has increased upon the addition of DO and AN, which confirms the interaction mode. A competitive binding study has reported that the enhanced emission intensity of ethidium bromide (EB) and acridine orange (AO), in the presence of ct DNA, have been quenched through the addition of DO and Anastrozole as binary and ternary systems. As it is indicated by these findings, DO is capable of displacing EB and AO from their binding site in ct DNA; hence, it can be concluded that DO and AN are able to intercalate into the base pairs of ct DNA in binary and ternary systems. Molecular docking studies have corroborated the mentioned experimental results.

Probing the binding selected metal ions and biologically active substances to the antimicrobial peptide LL-37 using DSC, ITC measurements and calculations

Differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) techniques have been used to describe physicochemical properties of polypeptide LL-37. LL-37 is a 37-residue, amphipathic, helical peptide, the only cathelicidin-derived antimicrobial peptide found in the human organism. Thermal stability of the LL-37 peptide in a water solution was measured by DSC over the 288.15–333.15 K range. Furthermore, the ITC method supported by theoretical calculations (peptide–ligand docking) were used to study the interactions between LL-37 and some divalent metal ions, namely Cu2+, Zn2+, and Ni2+ ions as well as acetylsalicylic acid, ascorbic acid, and caffeine molecules. It has been proven that under experimental conditions, the LL-37 peptide reveals affinity only toward Cu2+ and Zn2+ ions. The stoichiometry, conditional binding constants, logKITC, and thermodynamic parameters (∆ITCG, ∆ITCH, T∆ITCS) of the resulting Cu(II) and Zn(II) complexes were determined and discussed.

Development of an on-line analytical platform to screen haptens in shuxuening injection based on high-performance liquid chromatography coupled with ion-trap multistage mass spectrometry and human serum albumin fluorescence detection

Shuxuening injection (SXNI), one of the traditional Chinese medicine injections (TCMI), is widely used for the treatment of cardiovascular diseases in the clinic. However, its allergic reactions have impeded the clinical applications of SXNI, such adverse reactions have not been well understood due to the lack of methods for detecting haptens. In this study, a high-performance liquid chromatography-diode–array detector–multi-stage mass spectrometry–human serum albumin–fluorescence detector (HPLC–DAD–MSn–HSA–FLD) system was established to identify and screen haptens for the first time. Flavones, flavonols and their glycosides in SXNI showed strong HSA binding ability in different degrees. Fifteen of these compounds were used to study the association of HSA binding ability and sensitizability using isothermal titration calorimetry (ITC) and fluorescence techniques, furthermore, RBL-2H3 cell experiments were conducted to verify the results. It was found that ginkgolides showed no sensitizability, while flavones and flavonol aglycones showed stronger sensitizability than their glycosides. The system was proven to be precise, stable and reproducible, which lays a foundation for screening haptens in SXNI and relevant samples.

Spectroscopic, calorimetric and molecular docking insight into the interaction of Alzheimer’s drug donepezil with human transferrin: implications of Alzheimer’s drug

Donepezil, a highly selective and reversible inhibitor, is amongst the widely used drugs for the treatment of Alzheimer’s disease (AD) across the globe. Clinically significant human transferrin (hTf) is a key player involved in iron metabolism. The current study targets to have an insight into the interaction between donepezil and this plasma protein, hTf using UV–vis absorbance, fluorescence, isothermal titration calorimetry, circular dichroism (CD) and molecular docking techniques. UV spectroscopy of hTf in the presence of donepezil revealed the occurrence of hyperchromism coupled with blue shift in indicating complex formation. Binding constant obtained from UV–vis absorption measurements was of the order 0.9 × 104 M−1 implicative of strong binding. Fluorescence spectroscopy shows that donepezil quenches fluorescence intensity of the native hTf in dynamic manner with a shift in wavelength maxima. Donepezil binds to hTf at 310 K with a binding constant of 1.3 × 104 M−1 suggestive of strong binding. ITC results clearly suggest the reaction to be spontaneous and thermodynamically favorable. CD spectroscopy also showed a shift in the spectrum of the native hTf in the presence of donepezil suggesting formation of complex between protein and drug. Furthermore, molecular docking experiments showed Pro 91, Phe 94 and His 207 to be key players involved in the complex formation. This study gives an insight into the molecular basis of interaction between donepezil and hTf thereby aiding in understanding the activity and mechanism of protein and drug binding. This will serve as a boon for drug designing industries which are working day and night for improved treatment of existing disorders across the world.Communicated by Ramaswamy H. Sarma

Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, β-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and β-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 μg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring β-thujaplicin and the α- and β-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 μM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.