Isothermal Titration Calorimetry Methods Research Articles

A Systematic Study on the Physicochemical Interactions Between Polymeric Micelles and Mucin: Toward the Development of Optimal Drug Delivery Nanocarriers

AbstractThe optimal performance of drug delivery formulations, including polymeric nanoparticles, relies on particle distribution throughout the body and the interactions with biological barriers, particularly mucosal layers, which often limit their potential. A systematic and comprehensive study is presented through a multidisciplinary approach combining conventional and novel techniques for in vitro studies to understand the key molecular interactions between polymeric micelles and mucin. The results shows that polymeric micelles are integrates within the mucin layer, mirroring its viscoelastic properties, evidenced as a dissipation difference of 0.1 ± 0.44, measured by quartz crystal microbalance with dissipation. Surface‐enhanced Raman scattering reveals predominant hydrogen bonding within the mucin's hydrophilic core, while the isothermal titration calorimetry method confirms multiple non‐specific binding sites on the protein backbone. By performing the periodic acid‐Schiff stain assay, a binding amount of 0.20 mg of mucin per milligram of nanoparticles is quantified. Furthermore, motility studies show the surface binding of mucin on the polymeric nanoparticles influencing their Brownian motion. This study sheds light toward the improvement for a better drug delivery formulation and fabrication of optimal nanoparticle colloidal systems, which can advance translational drug delivery technologies into clinical application while enriching the field of surface and colloidal chemistry.

Biophysical and docking study on the interaction of anticancer drugs encorafenib and binimetinib with human serum albumin

The utilization of BRAF and MEK inhibitors in combination therapy has demonstrated superior outcomes in the treatment of melanoma as compared to monotherapy. In the present scenario, the combination therapy of Encorafenib (ENC), a BRAF inhibitor, and Binimetinib (BINI), a MEK inhibitor, has been identified as one of the most efficacious treatment modalities for this malignancy. Investigations of protein binding, particularly with human serum albumin (HSA), are essential to understand drug performance and enhance therapeutic outcomes. The investigation of the interplay between small molecule drugs and HSA is of paramount importance, given that such interactions can exert a substantial influence on the pharmacokinetics of these therapeutic agents. The present study aims to bridge these lacunae by implementing a comprehensive approach that integrates fluorescence spectroscopy (FS), isothermal titration calorimetry (ITC), far-ultraviolet circular dichroism (far-UV CD), and molecular simulations. Through analysis of the fluorescence quenching of HSA at three distinct temperatures, it was ascertained that the association constants for the complexes formed between drugs and HSA were of the magnitude of 104 M−1. This suggests that the interactions between the compounds and albumin were moderate and comparable. Simultaneously, the investigation of fluorescence indicated a contrasting binding mechanism for the two inhibitors: ENC predominantly binds to HSA through enthalpic interaction, while BINI/HSA is stabilized by entropic contributions. The data obtained was confirmed through experimental procedures conducted using the ITC method. The results of ligand-competitive displacement experiments indicate that ENC and BINI can bind to HSA within subdomain IIA, specifically Sudlow site I. However, far-UV CD studies show that there are no notable alterations in the structure of HSA upon binding with either of the two inhibitors. Ultimately, the results were supported by computational molecular analysis, which identified the key interactions that contribute to the stabilization of the two ligand/HSA complexes.

A novel multi-hyphenated approach to screen and character the xanthine oxidase inhibitors from saffron floral bio-residues

Recently, the incidence of hyperuricemia increased with patient rejuvenation, searching for new xanthine oxidase (XOD) inhibitors from natural products becomes important. In our previous work, a flavonoid extract of saffron floral bio-residues (SFB) was found to alleviate hyperuricemia via inhibiting XOD. In this study, an integrated approach combining two-dimensional liquid chromatography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was developed to online screen and character the potential XOD inhibitors from SFB. The two-dimensional liquid chromatography consisted of affinity chromatography and reverse phase chromatography (2D-AR), in which an XOD column, an inactive XOD column, and a control column were used in the first dimensional liquid chromatography to avoid phenomena of “false positive” and “missing screen of compounds with weak affinity to XOD” that often occur in the screening process, and a C18 column was used in the second dimensional liquid chromatography to separate the mixed XOD binders. Four flavonoid glycosides, i.e., quercetin-3-O-sophoroside (QS), kaempferol-3-O-sophoroside (KS), kaempferol-3-O-rutinoside (KR), and kaempferol-3-O-glucoside (KG), were thus successfully screened and identified from SFB extract by the 2D-AR method. The affinity of QS, KS, KR, KG, kaempferol (aglycone of KS, KR and KG), and quercetin (aglycone of QS) binding to XOD was investigated using SPR method, with KD ranged from 4.8 μM to 47.6 μM. The inhibitor constant (KI) of KS, KR, KG, quercetin and kaempferol were 4.92 mM, 1.11 mM, 0.294 mM, 4.93 μM and 3.27 μM, respectively, determined using ITC method. Finally, the anti-XOD activities of KS, the most abundant flavonoid in SFB extract, and kaempferol in hyperuricemia mice were verified, which suggested that the multi-hyphenated approach established herein can be applied for screen and character the XOD inhibitors in natural products.

Analysis of Aptamer-Small Molecule Binding Interactions Using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a technique where the heat given off, or absorbed, during a binding event is measured and used to determine the binding thermodynamics and affinity associated with binding. This protocol focuses on ITC applications for studying aptamer interactions with small molecule ligands where ITC has the advantage of being a label-free solution-based technique. The limitation of ITC using a relatively large amount of material compared to other analytical techniques is not applicable here as large amounts of nucleic acids, especially DNA, can be readily obtained. In this chapter we describe how to use ITC methods to measure the thermodynamics and affinity of binding using the interaction of quinine with a DNA cocaine-binding aptamer as an example.

Structural and Thermodynamic Model for the Activation of Cardiac Troponin.

Cardiac troponin is a regulatory protein complex located on the sarcomere that regulates the engagement of myosin on actin filaments. Low-molecular weight modulators of troponin that bind allosterically with the calcium ion have the potential to improve cardiac contractility in patients with reduced cardiac function. Here we propose an approach to the rational design of troponin modulators through the combined use of solution nuclear magnetic resonance and isothermal titration calorimetry methods. In contrast to traditional approaches limited to calcium and activator-bound troponin structures, here we analyzed the structural and thermodynamic impact of an activator in the context of the troponin functional cycle. This led us to propose a rationale for developing an efficacious troponin activator.

DNA binding by the antimalarial compound artemisinin

Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

A robust colorimetric aptasensor for the label-free detection of marine toxins based on tyrosine-capped gold nanoparticles.

PbTx-2 and okadaic acid (OA) are two typical marine toxins that are highly toxic and harmful to human health. The approach based on citrate-capped gold nanoparticles (Cit-AuNPs) and specific aptamers to construct label-free colorimetric sensors is a widely used method for marine toxin detection. However, the potential interactions between Cit-AuNPs and target molecules have always been ignored, which may result in wrong analytical results due to shortcomings in the Cit-AuNPs. To overcome these shortfalls, in this work, AuNPs were synthesized using tyrosine as a reducing and capping agent, and a robust colorimetric aptasensor based on tyrosine-capped AuNPs (Tyr-AuNPs) was constructed for the label-free detection of marine toxins. Tyr-AuNPs presented better stability compared to Cit-AuNPs due to the stronger binding of amine groups on tyrosine to AuNPs through the Au-N bond. Interactions between Tyr-AuNPs and PbTx-2 were analyzed through UV-vis and isothermal titration calorimetry methods and the results validated the robustness of the Tyr-AuNPs. Colorimetric aptasensors were established for PbTx-2 and OA detection with a linear range of 0.05-4 ppm and limits of detection of 2.25 ppb and 5.19 ppb, respectively. These results demonstrate that the developed colorimetric aptasensor can be a robust and promising method for marine toxin detection.

Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC.

There is growing interest in using isothermal titration calorimetry (ITC) to characterize enzyme kinetics by measuring the heat produced or absorbed by catalysis in real time. Since virtually all chemical reactions are associated with changes in enthalpy, ITC represents a robust and nearly universal experimental approach. Nevertheless, there are technical challenges that limit ITC's applicability. For instance, the full kinetic characterization of enzymes with two substrates (bi-substrate enzymes), which comprise the majority of known examples, requires a series of experiments to be performed as the concentrations of both substrates are varied. This is a time-consuming and expensive process using current ITC methods since many (>5) individual experiments must be performed independently to obtain a sufficient quantity of data. We have developed a new ITC method, which we term 2D-ITC, which maps the reaction velocity as a function of two substrate concentrations in a single, roughly 2 h long experiment. This method provides a level of detail that rivals or exceeds any existing enzyme assay, as a single experiment generates on the order of 7000 catalytic rate measurements. In a proof-of-principle application to rabbit muscle pyruvate kinase (rMPK), the method correctly identified the enzyme's random sequential mechanism and allosteric catalytic suppression by the amino acid phenylalanine (Phe). Unexpectedly, we found that while Phe reduces affinity for the substrate phosphoenolpyruvate, a known phenomenon, it also alleviates inhibition by the reaction product ATP, which had not been reported previously. Given the relative abundance of ATP in the cell, this opposing effect is expected to have a substantial impact on rMPK activity.

Weak Binding of Levamisole by the Cocaine-Binding Aptamer Does Not Interfere with an Aptamer-Based Detection Assay.

Levamisole is a common and harmful adulterant of street samples of cocaine and can cause electrochemical tests for cocaine to give false negative results. To see if levamisole would interfere with aptamer-based bioassays, we analyzed the binding of levamisole to the cocaine-binding DNA aptamer. At low aptamer concentrations (0.5 to 20 μM) using isothermal titration calorimetry methods and thermal stability measurements, no binding of levamisole to the cocaine-binding aptamer was observed. At higher levamisole concentrations (500 μM), weak binding to the cocaine-binding aptamer was detected using nuclear magnetic resonance (NMR) spectroscopy chemical shift perturbations. NMR-detected titrations show that levamisole binding is competitive with cocaine binding, indicating that both ligands share a common binding site. Finally, we show that the presence of levamisole does not interfere with the photochrome aptamer switch binding assay for cocaine. We conclude that assays using low concentrations of cocaine, and consequently low concentration of levamisole as an adulterant, should be unaffected by the weak binding of levamisole.

Characterizing the binding interactions of sodium benzoate with lysozyme at the molecular level using multi-spectroscopy, ITC and modeling methods

In this paper, we mainly study the interaction mechanism between food additives and antioxidant enzymes. Spectral methods were used to study the effect of sodium benzoate on the structure and function of lysozyme at the molecular level. Multi-spectroscopic results showed that sodium benzoate statically quenched the intrinsic fluorescence of lysozyme, formed complexes with lysozyme, increased the polarity of the aromatic amino acid, effected the molecular skeleton of lysozyme and stretched the secondary structure. The molecular docking and isothermal titration calorimetry (ITC) results showed that sodium benzoate entered the depression of the surface of lysozyme molecule both through hydrophobic interaction and hydrogen bond. Sodium benzoate was linked to tryptophan (Trp-63) by a hydrogen bond with a bond length of 2.48 Å. Thermodynamic studies showed that the combination was spontaneous, as the values of the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be 12.558 kJmol−1 and 25 kJmol-1k−1, respectively. Enzyme activity determination showed that Sodium benzoate increased lysozyme activity by 22.31%. This study can provide experimental support for evaluating the edible safety of sodium benzoate.

Thermodynamic analysis of cooperative ligand binding by the ATP-binding DNA aptamer indicates a population-shift binding mechanism

The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods. This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer. Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. We have determined the binding affinity and thermodynamics for both ligand-binding sites. By separating the ligand-binding sites by an additional four base pairs, we engineered a variant of this aptamer that binds two adenosine ligands in an independent manner. Together with NMR and thermal stability experiments, these data indicate that the ATP-binding DNA aptamer follows a population-shift binding mechanism that is the source of the positive binding cooperativity by the aptamer.

Aliphatic chain-containing macrocycles as diazonamide A analogs

Aliphatic alkyl chain-containing 12–14-membered macrocycles have been designed as structural analogs of antimitotic natural product diazonamide A. Macrocycles were synthesized from 5-bromooxazole in 7 to 9 linear steps using Ru-catalyzed ring-closing metathesis as the key transformation. Heat effect of binding to α,β-tubulin tetramer (T4-RB3 complex) has been measured for the synthesized macrocycles by isothermal titration calorimetry method.

Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase.

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme’s physiological substrates. The methods described herein will enable further studies on EpFAs’ metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.

Modeling of Multivalent Ligand-Receptor Binding Measured by kinITC

In addition to the conventional Isothermal Titration Calorimetry (ITC), kinetic ITC (kinITC) not only gains thermodynamic information, but also kinetic data from a biochemical binding process. Moreover, kinITC gives insights into reactions consisting of two separate kinetic steps, such as protein folding or sequential binding processes. The ITC method alone cannot deliver kinetic parameters, especially not for multivalent bindings. This paper describes how to solve the problem using kinITC and an invariant subspace projection. The algorithm is tested for multivalent systems with different valencies.

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.

Studies on the reversible enzyme reaction of rabbit muscle glycogen phosphorylase b using isothermal titration calorimetry

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (Pi, phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/Pi, respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the KM values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by Ki of 23 ± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. Ki values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the Ki values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism.The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

Insights into the saponification process of di(2-ethylhexyl) phosphoric acid extractant: Thermodynamics and structural aspects

The saponification process of di(2-ethylhexyl)phosphoric acid extractant (D2EHPA) was investigated by the isothermal titration calorimetry (ITC), attenuated total reflection infrared (ATR-IR), and small-angle X-ray scattering (SAXS) spectroscopies. The effects of the temperature, types of saponifier, and diluent on the saponification process of D2EHPA were disclosed by ITC method. The saponification reaction is an exothermic process, but the contribution of temperature on the maximum saponification degree of D2EHPA is slight. The theoretical maximum saponification degree of D2EHPA is about 40%. Moreover, the maximum effective saponification degree of D2EHPA is the same at the condition of different saponifiers because the two systems release the same amount of heat. The effective saponification degree of D2EHPA in octane is larger than that in toluene at the same content of saponifier. The IR spectra indicate that the structure of organic phases varies significantly with the increase of saponification degree. The most water-in-oil microemulsion in the organic phase is formed at the 40% theoretical saponification degree. The water content and SAXS data of organic phase further verify this phenomenon. Solvent extraction results show that the saponified extractant can obviously improve extraction efficiency of cobalt. These results will be helpful for optimizing the saponification process of D2EHPA during metal extraction.

Comparative study of the bindings between 3-phenyl-1H-indazole and five proteins by isothermal titration calorimetry, spectroscopy and docking methods

In this study, the interaction between 3-phenyl-1H-indazole (1a) and the fat mass and obesity-associated (FTO) protein was confirmed by isothermal titration calorimetry (ITC). The structure feature of 1a was different from our previously reported FTO inhibitors (radicicol, N-CDPCB and CHTB); the Cl and diol group in structure motif is critical for inhibitors to bind to FTO. In order to test whether there is specificity for the interaction between FTO and 1a, the interactions between 1a and four important proteins (human serum albumin (HSA), pepsin, catalase and trypsin) were investigated by ITC, spectroscopy and molecular docking methods. ITC results showed spontaneous exothermic reactions occurring between 1a and the proteins except trypsin under investigated conditions. The order of the binding affinity of 3-phenyl-1H-indazole is catalase > HSA > FTO > pepsin. Comparison between ITC and spectral results was made. This work will provide the basis for the design of novel inhibitors for FTO.AbbreviationsCATcatalaseDMSOdimethyl sulfoxideFTOfat mass and obesity-associated proteinHSAhuman serum albuminPeppepsinTrytrypsinCommunicated by Ramaswamy H. Sarma

Isothermal Titration Calorimetry for the Screening of Aflatoxin B1 Surface-Enhanced Raman Scattering Sensor Affinity Agents.

In this work, isothermal titration calorimetry (ITC) is employed as an affinity agent screening method for the surface-enhanced Raman scattering (SERS) detection of aflatoxin B1 (AFB1). AFB1, a potent carcinogen produced by a fungus that infects crops, is an important target due to the monitoring required based on its FDA regulation. Polymer affinity agents, like those studied here, have the potential to enable separation and detection of relevant small molecules such as pesticides, drugs, and biological toxins, like AFB1, especially when paired with a vibrational spectroscopy technique such as SERS. Herein, seven homopolymers were synthesized to be evaluated as AFB1 affinity agents based on hypothetical hydrogen bonding interactions. Nitrogen-inclusive poly( N-(2-aminoethyl) methacrylamide) (pAEMA) polymers and their oxygen analogs, poly(2-hydroxyethyl methacrylate) (pHEMA) were evaluated. ITC was demonstrated as an effective method for rapid screening among the polymer affinity agents. Chain lengths between seven and 39 repeat units were synthesized to study length-based variance in affinity agent performance. An ITC method was optimized and used for the rapid screening of polymer affinity agents. The results were compared to those generated by SERS. Good agreement between the ITC results and follow-up SERS sensing experiments showcased ITC's screening potential for analytical applications such as separation and detection.

Nanomolar binding affinity of quinine-based antimalarial compounds by the cocaine-binding aptamer.

An unusual feature of the cocaine-binding aptamer is that it binds quinine much tighter than the ligand it was selected for, cocaine. Here we expand the repertoire of ligands that this aptamer binds to include the quinine-based antimalarial compounds amodiaquine, mefloquine, chloroquine and primaquine. Using isothermal titration calorimetry (ITC) we show that amodiaquine is bound by the cocaine-binding aptamer with an affinity of (7 ± 4) nM, one of the tightest aptamer-small molecule affinities currently known. Amodiaquine, mefloquine and chloroquine binding are driven by both a favorable entropy and enthalpy of binding, while primaquine, quinine and cocaine binding are enthalpy driven with unfavorable binding entropy. Using nuclear magnetic resonance (NMR) and ITC methods we show that these ligands compete for the same binding sites in the aptamer. Our identification of such a tight binding ligand for this aptamer should prove useful in developing new biosensor techniques and applications using the cocaine-binding aptamer as a model system.