Isothermal Titration Calorimetry Research Articles

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1–2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated “GE” motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

Adsorption behavior of soybean protein isolate-soyasaponins mixed system at saliva interface and influence mechanism of saponin bitterness presentation

The bitterness of soyasaponins (Ssa) is one of the main causes of undesirable flavors in soy products. In this study, a Ssa-soybean protein isolate (SPI) binary system was constructed to simulate the adsorption behavior of soybean products at the oral-salivary interface and the mechanism of bitter taste development was investigated. The concentration of Ssa with was determined to be the minimum bitterness value approximately 0.5 mg/mL by sensory evaluation and electronic tongue determination. The nonspecific interactions were confirmed by isothermal titration calorimetry (ITC) and endogenous fluorescence measurements. Ssa formed aggregates with SPI that desorbed from the interface into the bulk phase, resulting in a subsequent increase in particle size and turbidity. This study demonstrated the ability of SPI with Ssa at specific concentrations to attenuate the bitter flavor of Ssa, thus providing important implications for increasing the acceptability of soy products.

Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.

A new Eosin Y-based ‘turn[sbnd]on’ fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer β-sheets, and β-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 μM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5’-ACCCCAATTACGAAAATTTTT-3’), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development

Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine. Enzymatic assays showed that these compounds can inhibit FLN proteolytic activity. Their interaction with FLN was explored systematically by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with lowered FLN expression demonstrated statistically significant increases in susceptibility toward MK-4815, MMV000848, and several quinolines. Importantly, the hydrophobic pocket of FLN appears amenable to inhibition and the structures reported here can guide the development of novel drugs against malaria.

In vitro antifungal activity, cytotoxicity and binding analysis of imidazolium based ionic liquids with fluconazole: DFT and spectroscopic study

Fungal infections represent a significant challenge in various living organisms, including humans, animals, and plants. Fungi are versatile organisms capable of thriving in diverse environments, which contributes to their widespread presence and potential to cause infections. Recently, imidazolium-based ionic liquids emerged as good antimicrobial agent in either monomeric or polymeric form. Therefore, herein, we have reporting the in-vitro antifungal activity, cytotoxicity, and binding analysis of 1-decyl-3-methylimidazolium chloride, [C10Mim][Cl] and 1-hexyl-3-methylimidazolium chloride, [C6Mim][Cl] ionic liquids with fluconazole (Flz). The antifungal activity of ILs and ILs with Flz against candidiasis species (C. albicans) in terms of minimum inhibitory concentration (MIC) was assessed. The antifungal activity results showed that [C10Mim][Cl]-Flz exhibit good antifungal activity then [C10Mim][Cl] and Flz alone. The cytotoxicity of ILs with Flz was checked against human embryonic kidney (HEK293) cell line regarding cell viability and result showed minute cytotoxicity of the combinations at higher concentrations. Further, the binding of ILs with Flz was also studied using various techniques such as UV–visible, fluorescence, Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and conductivity method. It was found that [C10Mim][Cl] shows more binding affinity then [C6Mim][Cl] with Flz. In-silico molecular docking analysis of Flz-[C10Mim][Cl] complex show a higher binding affinity with CYP51 which in turn enhanced its ability to inhibit the ergosterol biosynthesis as compared to alone [C10Mim][Cl] and Flz. The classical density functional theory (DFT) model was also utilized which further supports the spectroscopic results.

Seco-cyclic phorbol derivatives and their anti-HIV-1 activities

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (Kd) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 μmol·L-1 and a CC50 of 153.92 μmol·L-1, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Polymers as candidates for broad‐spectrum antivirals—in vitro inhibition of Zika virus with sodium polyanethole sulfonate

AbstractZika virus (ZIKV), belonging to the family of flaviviruses, is an emerging mosquito‐borne pathogen causing microcephaly and other congenital abnormalities in newborns as well as various neurologic pathologies in adults. The climate change and the lifestyle of current societies, involving global mobility, result in a rapid broadening of ZIKV geographic distribution. There are no vaccines or drugs available to protect against or cure the diseases caused by that virus. Our in vitro studies have demonstrated that sodium polyanethole sulfonate (PASNa), a degradable polymer used in various biomedical applications, acts as an efficient ZIKV inhibitor, both in animal and human cells. The mechanistic studies indicated that PASNa bound to the ZIKV particles thus hindering their attachment to the host cells and also interfered in the late steps of viral replication. PASNa is characterized by very low cytotoxicity and its antiviral activity can be achieved at a concentration much lower than that at which its anticoagulant activity has to be considered. Isothermal titration calorimetry (ITC) studies have shown that the interaction of PASNa with human serum albumin (HSA), the most abundant blood protein, is the endothermic, entropy‐driven process resulting in the formation of stoichiometric (1:1) PASNa‐HSA complexes with a hydrodynamic diameter of 8.8 ± 0.8 nm and zeta potential (ζ) equal to −21.3 mV. The analysis of CD spectra indicated that PASNa did not significantly affect the HSA secondary structure on binding to that protein.

Co-assembly of quinoa protein isolate and shellac as nano-vehicles for naringenin: Characterization, formation mechanism, release behavior and cellular uptake

Naringenin (Nar) is a natural flavonoid with diverse health-promoting benefits. However, the bioavailability of Nar is significantly restricted due to its poor water-solubility, instability under physiological conditions, and limited absorption profile. In this study, a shellac-quinoa protein isolate (QPI) binary nanocomplex (SQP) was fabricated using the pH-shifting method for effectively delivering Nar. Isothermal titration calorimetry (ITC) and analysis of inner interactive forces revealed that the complexation between QPI and shellac was primarily driven by electrostatic and hydrophobic forces. Subsequently, Nar was successfully encapsulated within the stable and uniform SQP nanocomplex (Nar@SQP), achieving a high encapsulation capacity (94.52 % encapsulation efficiency and 14.68 % loading capacity). Meanwhile, the Nar@SQP nanoparticle exhibited excellent stability against pH, ionic strength, and long-term storage. Moreover, it also demonstrated sustained and slow-release characteristics during simulated gastrointestinal digestion. Compared to free Nar, the safe Nar@SQP nanoparticle effectively facilitated the cellular uptake of encapsulated Nar by Caco-2 cells, suggesting that Nar@SQP has a good potential to improve the bioavailability of Nar. Hence, these findings indicated that the developed Nar@SQP nanoparticle might hold significant promise as a nano-delivery system for hydrophobic bioactive nutrients, like Nar.

Can the heptapeptide ASSIVSF of the β2-adrenoceptor recognize ephedrine and pseudoephedrine epimers in a complex system?

Epimer separation is crucial in the field of analytical chemistry, separation science, and the pharmaceutical industry. No reported methods could separate simultaneously epimers or even isomers and remove other unwanted, co-existing, interfering substances from complex systems like herbal extracts. Herein, we prepared a heptapeptide-modified stationary phase for the separation of 1R,2S-(-)-ephedrine [(-)-Ephe] and 1S,2S-(+)-pseudoephedrine [(+)-Pse] epimers from Ephedra sinica Stapf extract and blood samples. The heptapeptide stationary phase was comprehensively characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The separation efficiency of the heptapeptide column was compared with an affinity column packed with full-length β2-AR functionalized silica gel (β2-AR column). The binding affinity of the heptapeptide with (+)-Pse was 3-fold greater than that with (-)-Ephe. Their binding mechanisms were extensively characterized by chromatographic analysis, ultraviolet spectra, circular dichroism analysis, isothermal titration calorimetry, and molecule docking. An enhanced hydrogen bonding was clearly observed in the heptapeptide-(+)-Pse complex. Such results demonstrated that the heptapeptide can recognize (+)-Pse and (-)-Ephe epimers in a complex system. This work, we believe, was the first report to simultaneously separate epimers and remove non-specific interfering substances from complex samples. The method was potentially applicable to more challenging sample separation, such as chiral separation from complex systems.

Structural characterization and mechanisms of macrophage immunomodulatory activity of a novel polysaccharide with a galactose backbone from the processed Polygonati Rhizoma

A purified polysaccharide with a galactose backbone (SPR-1, Mw 3,622 Da) was isolated from processed Polygonati Rhizoma with black beans (PRWB) and characterized its chemical properties. The backbone of SPR-1 consisted of [(4)-β-D-Galp-(1]9 → 4,6)-β-D-Galp-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-Glcp-(1 → 4,6)-α-D-Glcp-(1 → 4)-α/β-D-Glcp, with a branch chain of R1: β-D-Galp-(1 → 3)-β-D-Galp-(1→ connected to the →4,6)-β-D-Galp-(1→ via O-6, and a branch chain of R2: α-D-Glcp-(1 → 6)-α-D-Glcp-(1→ connected to the →4,6)-α-D-Glcp-(1→ via O-6. Immunomodulatory assays showed that the SPR-1 significantly activated macrophages, and increased secretion of NO and cytokines (i.e., IL-1β and TNF-α), as well as promoted the phagocytic activities of cells. Furthermore, isothermal titration calorimetry (ITC) analysis and molecular docking results indicated high-affinity binding between SPR-1 and MD2 with the equilibrium dissociation constant (KD) of 18.8 μM. It was suggested that SPR-1 activated the immune response through Toll-like receptor 4 (TLR4) signaling and downstream responses. Our research demonstrated that the SPR-1 has a promising candidate from PRWB for the TLR4 agonist to induce immune response, and also provided an easily accessible way that can be used for PR deep processing.

Covalent conjugate of pea protein induced by cyanidin-3-O-glucoside quinone: The structural formation and functional properties

The unique structural of anthocyanins makes them vulnerable to degradation within food processing environments. Proteins commonly coexist with polyphenols in natural food matrices on a non-covalent and covalent basis, serving to stabilize protection for polyphenols. Here, three natural pea-extracted proteins, including albumin, vicilin, and legumin were bound to cyanidin-3-O-glucoside (C3G), and binding modes, conjugate structures and functions were characterized. Isothermal titration calorimetry showed a strongly exothermic interaction between C3G and proteins. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fourier transform infrared spectroscopy revealed that the C3G-quinone (C3GQ) formed by C3G oxidation established covalent cross-links with C–S and C–N in the amino acid residues of proteins, and could form more large molecular weight aggregates at an addition amount of 1 mM. The free sulfhydryl and amino contents of the three protein conjugates decreased with the addition of C3GQ. Due to the presence of 4–5 times more sulfhydryl groups in albumin as compared to globulin, albumin showed a 20% higher binding capacity for C3GQ than globulin. This consequently led to the highest antioxidant activity in the C3GQ-albumin conjugate. In contrast to native pea proteins, the introduction of covalent modifications induces changes in the secondary structure of the pea proteins. Specifically, the structure of albumin tends to exhibit increased orderliness, while the molecular flexibility of globular proteins was augmented. Thus, the conjugates have enhanced thermal stability. Our results affirmed that the stability of C3G can be more effectively improved through designing it to covalently interact with proteins relative to non-covalent interactions. This study aimed at offering novel theoretical underpinnings for the functional utilization of C3G and pea proteins in the food industry.

Thermodynamics of doxorubicin - bile salt association: An investigation based on isothermal titration calorimetry

Association of doxorubicin (DOX), a positively charged anthracycline, with bile salts (sodium cholate, NaC; sodium deoxycholate, NaDC, and sodium taurodeoxy cholate, NaTDC) has been studied by isothermal titration calorimetry (ITC). These bile salts form micelles in physiological conditions, and subsequent micellization parameters vary in accordance to their hydrophobicity. In this study, we show that thermodynamics of micellization of bile salts can be modulated via incorporation of DOX and changes in solution temperature. DOX incorporation was accompanied with reduction in critical micelle concentration (CMC) of bile salts. This was mediated through expulsion of structured water from the hydrophobic part of bile salt, and it was verifiable from the appearance of additional thermodynamic events in demicellization process. Interestingly, free energy of bile salts increased upon adding DOX which is suggestive of loss in the spontaneity of their micellization following this association. We assert that micelle formation in case of bile salt – DOX complex may not have occurred instantly. Instead, it was preceded by a bile salt - DOX equilibrium as a result of changes in the interfacial characteristics of bile salts in question.

Specific Binding of Alzheimer's Aβ Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aβ42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aβ42 is detectable in the plasma, a phenomenon thought to result from Aβ becoming more aggregated in the brain and less Aβ42 and Aβ40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse "cargos" of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aβ42 and Aβ40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aβ and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aβ42 and Aβ40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aβ incubation with EVs overnight yielded larger amounts of bound Aβ peptide that was fibrillar in structure. These findings point to a specific amyloid-EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

Effect and mechanism of pectin for modulating bitter taste of soyasaponin

Soyasaponins (Ssa) have various biological benefits for the human body. However, they are one of the main factors contributing to the bitter taste of certain soybean-based foods. In this study, the case interaction between pectin and Ssa was investigated by us, as well as the impact of pectin addition on the bitter taste of Ssa. The findings revealed that the thermal stability of Ssa–pectin complexes was higher than that of Ssa–mucin complexes. Fluorescence spectroscopy and isothermal titration calorimetry revealed that pectin interferes with the binding of Ssa to mucin by interacting with Ssa, thereby disrupting the Ssa binding process. As the proportion of pectin increases, the comprehensive score also increases. This confirms that pectin can effectively reduce the bitterness and astringency of Ssa, signifying its importance in further exploration of the mechanism by which Ssa–pectin complexes influence the presentation of bitterness.

Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin.

Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 μmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.

Elucidation of binding mechanisms of bovine serum albumin and 1-alkylsulfonates with different hydrophobic chain lengths

In this article, the binding interactions between bovine serum albumin (BSA) and three 1-alkylsulfonates, namely sodium 1-dodecanesulfonate, sodium 1-decanesulfonate, and sodium 1-octanesulfonate, have been thoroughly investigated. The study employed various experimental techniques such as isothermal titration calorimetry (ITC), steady-state fluorescence spectroscopy (SF), circular dichroism spectroscopy (CD), and molecular dynamics-based simulations. The objective was to understand the influence of the alkyl chain length of the investigated ligands on several aspects, including the strength of the interaction, the stoichiometry of the resulting complexes, the number of BSA binding sites, and the underlying mechanisms of binding. Notably, the study also demonstrated that sodium dodecyl sulfate (S12S) can serve as an effective site marker for BSA when studying ligands with similar structural and topological features. These findings may have significant implications for enhancing our understanding of the interactions between small amphiphilic molecules and proteins.

Profiling the Interaction between Human Serum Albumin and Clinically Relevant HIV Reverse Transcriptase Inhibitors.

Tenofovir (TFV) is the active form of the prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), both clinically prescribed as HIV reverse transcriptase inhibitors. The biophysical interactions between these compounds and human serum albumin (HSA), the primary carrier of exogenous compounds in the human bloodstream, have not yet been thoroughly characterized. Thus, the present study reports the interaction profile between HSA and TFV, TDF, and TAF via UV-Vis, steady-state, and time-resolved fluorescence techniques combined with isothermal titration calorimetry (ITC) and in silico calculations. A spontaneous interaction in the ground state, which does not perturb the microenvironment close to the Trp-214 residue, is classified as weak. In the case of HSA/TFV and HSA/TDF, the binding is both enthalpically and entropically driven, while for HSA/TAF, the binding is only entropically dominated. The binding constant (Ka) and thermodynamic parameters obtained via ITC assays agree with those obtained using steady-state fluorescence quenching measurements, reinforcing the reliability of the data. The small internal cavity known as site I is probably the main binding pocket for TFV due to the low steric volume of the drug. In contrast, most external sites (II and III) can better accommodate TAF due to the high steric volume of this prodrug. The cross-docking approach corroborated experimental drug-displacement assays, indicating that the binding affinity of TFV and TAF might be impacted by the presence of different compounds bound to albumin. Overall, the weak binding capacity of albumin to TFV, TDF, and TAF is one of the main factors for the low residence time of these antiretrovirals in the human bloodstream; however, positive cooperativity for TAF and TDF was detected in the presence of some drugs, which might improve their residence time (pharmacokinetic profile).

Entropy Drives Interpolymer Association in Water: Insights into Molecular Mechanisms.

Interpolymer association in aqueous solutions is essential for many industrial processes, new materials design, and the biochemistry of life. However, our understanding of the association mechanism is limited. Classical theories do not provide molecular details, creating a need for detailed mechanistic insights. This work consolidates previous literature with complementary isothermal titration calorimetry (ITC) measurements and molecular dynamics (MD) simulations to investigate molecular mechanisms to provide such insights. The large body of ITC data shows that intermolecular bonds, such as ionic or hydrogen bonds, cannot drive association. Instead, polymer association is entropy-driven due to the reorganization of water and ions. We propose a unifying entropy-driven association mechanism by generalizing previously suggested polyion association principles to include nonionic polymers, here termed polydipoles. In this mechanism, complementary charge densities of the polymers are the common denominators of association, for both polyions and polydipoles. The association of the polymers results mainly from two processes: charge exchange and amphiphilic association. MD simulations indicate that the amphiphilic assembly alone is enough for the initial association. Our proposed mechanism is a step toward a molecular understanding of the formation of complexes between synthetic and biological polymers under ambient or biological conditions.