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1.  Solvated Crystalline Forms of Nevirapine: Thermoanalytical and Spectroscopic Studies 
AAPS PharmSciTech  2010;11(3):1328-1339.
The study is aimed at exploring the utility of thermoanalytical methods in the solid-state characterization of various crystalline forms of nevirapine. The different forms obtained by recrystallization of nevirapine from various solvents were identified using differential scanning calorimetry and thermogravimetric analysis (TGA). The appearance of desolvation peak accompanied by weight loss in TGA indicated the formation of solvates: hemi-ethanolate (Form I), hemi-acetonitrilate (Form II), hemi-chloroformate (Form III), hemi-THF solvate (Form IV), mixed hemi-ethanolate hemi-hydrate (Form V), and hemi-toluenate (Form VI). The higher desolvation temperatures of all the solvates except toluenate than their respective boiling point indicate tighter binding of solvent. Emphasis has been laid on the determination of heat capacity and heat of solution utilizing microreaction calorimeter to further distinguish the various forms. The enthalpy of solution (ΔHsol), an indirect measure of the lattice energy of a solid, was well correlated with the crystallinity of all the solid forms obtained. The magnitude of ΔHsol was found to be −14.14 kJ/mol for Form I and −2.83 kJ/mol for Form V in phosphate buffer of pH 2, exhibiting maximum ease of molecular release from the lattice in Form I. The heat capacity for solvation (ΔCp) was found to be positive, providing information about the state of solvent molecules in the host lattice. The solubility and dissolution rate of the forms were also found to be in agreement with their enthalpy of solution. Form (I), being the most exothermic, was found to be the most soluble of all the forms.
PMCID: PMC2974108  PMID: 20737259
calorimetry; enthalpy of solution; heat capacity; recrystallization; solvates
2.  Characterisation and evaluation of pharmaceutical solvates of Atorvastatin calcium by thermoanalytical and spectroscopic studies 
Atorvastatin calcium (ATC), an anti-lipid biopharmaceutical class II drug, is widely prescribed as a cholesterol-lowering agent and is presently the world’s best-selling medicine. A large number of crystalline forms of ATC have been published in patents. A variety of solid forms may give rise to different physical properties. Therefore, the discovery of new forms of this unusual molecule, ATC, may still provide an opportunity for further improvement of advantageous properties.
In the present work, eight new solvates (Solvate I-VIII) have been discovered by recrystallization method. Thermal behaviour of ATC and its solvates studied by DSC and TGA indicate similar pattern suggesting similar mode of entrapment of solvent molecules. The type of solvent present in the crystal lattice of the solvates is identified by GC-MS analysis and the stoichiometric ratio of the solvents is confirmed by 1HNMR. The high positive value of binding energy determined from thermochemical parameters indicates deep inclusion of the solvent molecules into the host cavity. The XRPD patterns point towards the differences in their crystallanity, however, after desolvation solvate II, III, IV, V and VIII transform to isostructral amorphous desolvated solvates. The order of crystallinity was confirmed by solution calorimetric technique as the enthalpy of solution is an indirect measure of lattice energy. All the solvates behaved endothermically following the order solvate-VIII (1-butanol solvate) < solvate-I (isoproplyate) < solvate-V (methanol solvate) < solvate-III (ethonalate) < solvate-VI (acetone ethanol solvate) < solvate-IV (t-butanol solvate) < solvate-II (THF solvate) < solvate-VII (mixed hemi-ethanol hydrate). The positive value of the heat capacity of the solvate formation provides information about the state of solvent molecules in the host lattice. The solvents molecules incorporated in the crystal lattice induced local chemical environment changes in the drug molecules which are observed in 13CP/MAS NMR spectral changes.
Aqueous solubility of solvate-VIII was found to be maximum, however, solvate-I and VIII showed better reduction in total cholesterol and triglyceride levels as compared to atorvastatin against triton-induced dyslipidemia.
PMCID: PMC3547732  PMID: 23039933
Solvate; Recrystalliztion; Heat capacity; Calorimetry; 13C /MAS solid state NMR
3.  Thermoanalytical Investigation of Terazosin Hydrochloride 
Advanced Pharmaceutical Bulletin  2013;3(1):147-152.
Purpose: Thermal analysis (TGA, DTG and DTA) and differential scanning calorimetry (DSC) have been used to study the thermal behavior of terazosin hydrochloride (TER). Methods: Thermogravimetric analysis (TGA/DTG), differential thermal analysis (DTA) and differential scanning calorimetry (DSC) were used to determine the thermal behavior and purity of the used drug. Thermodynamic parameters such as activation energy (E*), enthalpy (∆H*), entropy (∆S*) and Gibbs free energy change of the decomposition (∆G*) were calculated using different kinetic models. Results: The purity of the used drug was determined by differential scanning calorimetry (99.97%) and specialized official method (99.85%) indicating to satisfactory values of the degree of purity. Thermal analysis technique gave satisfactory results to obtain quality control parameters such as melting point (273 ºC), water content (7.49%) and ash content (zero) in comparison to what were obtained using official method: (272 ºC), (8.0%) and (0.02%) for melting point, water content and ash content, respectively. Conclusion: Thermal analysis justifies its application in quality control of pharmaceutical compounds due to its simplicity, sensitivity and low operational costs. DSC data indicated that the degree of purity of terazosin hydrochloride is similar to that found by official method.
PMCID: PMC3846028  PMID: 24312828
Terazosin hydrochloride; Thermal analysis; Differential scanning calorimetry; Purity
4.  Sublimation characterization and vapor pressure estimation of an HIV nonnucleoside reverse transcriptase inhibitor using thermogravimetric analysis 
AAPS PharmSciTech  2003;4(2):99-108.
The purpose of this research is to investigate the sublimation process of DPC 963, a second-generation nonnucleoside reverse transcriptase inhibitor for HIV-1 retrovirus, and to better understant the effect of sublimation during active pharmaceutical ingredient (API) manufacture and formulation development, especially the drying processes. Sublimation of DPC 963 at 150°C and above was determined by thermogravimetric analysis-Fourier transform infrared (TGA-FTIR). The rates of sublimation at different temperatures were measured using isothermal TGA. Condensed material was collected and analyzed by differential scanning calorimetry (DSC), x-ray powder diffraction (XRPD), and infrared (IR) spectrometry. Benzoic acid was used as a reference standard to derive a linear logarithmic relationship between sublimation/evaporation rate and vapor pressure specific to the TGA system used in this study. Sublimation and evaporation of DPC 963 were found to follow apparent zero-order kinetics. Using the Eyring equation, the enthalpy and entropy of the sublimation and evaporation processes were obtained. The enthalpies of sublimation and evaporation were found to be 29 and 22 kcal/mol, respectively. The condensed material from the vapor phase was found to exist in 2 physical forms, amorphous and crystalline. Using benzoic acid as a reference standard, vapor pressure of DPC 963 at different temperatures was calculated using the linear logarithmic relationship obtained. DPC 963 undergoes sublimation at appreciable rates at 150°C and above but this is not likely to pose a serious issue during the manufacturing process. Vapor pressure estimation using thermogravimetric analysis provided sufficient accuracy to be used as a fast, simple, and safe alternative to the traditional methods of vapor pressure determination.
PMCID: PMC2750601  PMID: 12916905
sublimation; vapor pressure; thermogravimetric analysis-infrared (TGA-IR)
5.  Influence of Position and Size of Substituents on the Mechanism of Partitioning: A Thermodynamic Study on Acetaminophens, Hydroxybenzoic Acids, and Parabens 
AAPS PharmSciTech  2008;9(1):205-216.
The objective of the present investigation was to study the influence of size, nature, and topology of substituents on the thermodynamic characteristics of sublimation, fusion, solubility, solvation, and partitioning processes of some drug and druglike molecules. Thermodynamic functions of sublimation process 2-acetaminophen and 3-acetaminophen were obtained on the basis of temperature dependencies of vapor pressure by the transpiration method. Thermodynamic characteristics of solubility processes in water, n-octanol, and n-hexane were calculated from the temperature dependencies of solubility using the solubility saturation method. For evaluation of fusion parameters, differential scanning calorimetry was used. A new approach to distinguishing specific and nonspecific energetic terms in the crystal lattice was developed. Specific and nonspecific solvation terms were distinguished using the transfer from the “inert” n-hexane to the other solvents. For the acetaminophen compounds and for some related drug molecules, the correlation between melting points and a parameter describing the ratio between specific and nonspecific interaction in the crystal lattices was obtained. A diagram enabling analysis of the mechanism of the partitioning process was applied. It was found that for isomers of benzoic acids and for acetaminophens, the position of substituents affects the mechanism of the partitioning process but not the extent of partitioning (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta G_{{\text{tr}}}^{\text{0}}$$\end{document} values). In contrast to this, an increased size of substituents (parabens) leads to essential changes in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta G_{{\text{tr}}}^{\text{0}}$$\end{document} values, but the mechanism of the partitioning process stays the same.
PMCID: PMC2976908  PMID: 18446483
partitioning; solubility; solvation; sublimation; thermodynamics; transfer
6.  Vapor pressures and sublimation enthalpies of seven heteroatomic aromatic hydrocarbons measured using the Knudsen effusion technique 
The vapor pressures of seven heteroatom-containing cyclic aromatic hydrocarbons, ranging in molecular weight from (168.19 to 208.21) grams⊕mol−1 were measured over the temperature range of (301 to 486) Kelvin using the isothermal Knudsen effusion technique. The compounds measured include: anthraquinone, 9-fluorenone, 9-fluorenone oxime, phenoxazine, phenoxathiin and 9H-pyrido[3,4-b]indole. These solid-state sublimation measurements provided values that are compared to vapor pressures of parent aromatic compounds (anthracene and fluorene) and to others with substituent groups in order to examine the effects of alcohol, ketone, pyridine, and pyrrole functionality on this property. The enthalpies and entropies of sublimation for each compound were determined from the Clausius-Clapeyron equation. Though there is no consistent trend in terms of the effects of substitutions on changes in the enthalpy or entropy of sublimation, we note that the prevalence of enthalpic or entropic driving forces on vapor pressure depend on molecule-specific factors and not merely molecular weight of the substituents.
PMCID: PMC2856104  PMID: 20414454
7.  Towards Accurate Microscopic Calculation of Solvation Entropies: Extending the Restraint Release Approach to Studies of Solvation Effects 
The journal of physical chemistry. B  2009;113(20):7372-7382.
The evaluation of the solvation entropies is a major conceptual and practical challenge. On the one hand, it is interesting to quantify the factors that are responsible for the solvation entropies in solutions, while on the other, it is essential to be able to assess the contributions of the solvation entropies to the binding free energies and related properties. In fact, the solvation entropies are neglected in almost all the studies of the binding entropies. The main problem is that widely used approaches, such as the quasiharmonic (QH) approximation do not provide reliable results particularly, in cases of shallow potential and multidimensional surfaces while brute force evaluations of the entropic effects by simulating temperature dependence of the free energy converges very slowly. This paper addresses the above issue by starting with an analysis of the factors that are responsible for the negative solvation entropy of ions, showing that it is not due to the change in the solvent vibration modes or to the solvent force constant but to the changes in the solvent configurational space upon change in the solute charges. We begin by clarifying that when one deals with aqueous solutions, it is easy to evaluate the corresponding entropic effect by the Langevin dipole(LD) treatment. However, in this work we are interested in developing a general microscopic tool that can be used to study similar effects in the proteins. To this end, we explore the ability of our restraint release (RR) approach to evaluate the solvation entropy. We start this analysis by reviewing the foundation of this approach and in particular, the requirements of minimizing the enthalpy contribution to the RR free energy. We then establish that our approach is not a specialized harmonic treatment but a rather powerful approach. Moving to the main topic of this work, we demonstrate that the RR approach provides quantitative results for the solvation entropies of monovalent and divalent ions and effectively captures the physics of these entropic effects. The success of the current approach indicates that it should be applicable to the studies of the solvation entropies in the proteins and also, in examining hydrophobic effects. Thus, we believe that the RR approach provides a powerful tool for evaluating the corresponding contributions to the binding entropies and eventually, to the binding free energies. This holds promise for extending the information theory modeling to proteins and protein-ligand complexes in aqueous solutions and consequently, facilitating computer-aided drug design.
PMCID: PMC2738853  PMID: 19402609
Langevin dipole; Restraint release; Molecular dynamics; Quasiharmonic approximation; Drug design
8.  Drug-binding energetics of human α-1-acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations 
This study utilizes sensitive, modern isothermal titration calorimetric (ITC) methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics were characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (ΔH°) and favorable (positive) entropic (ΔS°) contributions to the Gibbs free energy (ΔG°). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug-serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity.
PMCID: PMC3513776  PMID: 23192962
human α-1-acid glycoprotein; thermodynamics; drug binding
9.  Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90 
PLoS ONE  2012;7(5):e36899.
The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors.
PMCID: PMC3360036  PMID: 22655030
10.  DNA Plasticity Is a Key Determinant of the Energetics of Binding of Jun-Fos Heterodimeric Transcription Factor to Genetic Variants of TGACGTCA Motif† 
Biochemistry  2009;48(51):12213-12222.
The Jun-Fos heterodimeric transcription factor is a target of a diverse array of signaling cascades that initiate at the cell surface and converge in the nucleus and ultimately result in the expression of genes involved in a multitude of cellular processes central to health and disease. Here, using isothermal titration calorimetry in conjunction with circular dichroism, we report the effect of introducing single nucleotide variations within the TGACGTCA canonical motif on the binding of bZIP domains of Jun-Fos heterodimer to DNA. Our data reveal that the Jun-Fos heterodimer exhibits differential energetics in binding to such genetic variants in the physiologically relevant micromolar-submicromolar range with the TGACGTCA canonical motif affording the highest affinity. Although binding energetics are largely favored by enthalpic forces and accompanied by entropic penalty, neither the favorable enthalpy nor the unfavorable entropy correlate with overall free energy of binding in agreement with enthalpy-entropy compensation phenomenon widely observed in biological systems. However, a number of variants including the TGACGTCA canonical motif bind to the Jun-Fos heterodimer with high affinity through having overcome such enthalpy-entropy compensation barrier, arguing strongly that better understanding of the underlying invisible forces driving macromolecular interactions may be the key to future drug design. Our data also suggest that the Jun-Fos heterodimer has a preference for binding to TGACGTCA variants with higher AT content, implying that the DNA plasticity may be an important determinant of protein-DNA interactions. This notion is further corroborated by the observation that the introduction of genetic variations within the TGACGTCA motif allows it to sample a much greater conformational space. Taken together, these new findings further our understanding of the role of DNA sequence and conformation on protein-DNA interactions in thermodynamic terms.
PMCID: PMC2807364  PMID: 19921846
AP1-DNA thermodynamics; Jun-Fos heterodimer; bZIP domain; Single nucleotide variants; DNA plasticity
11.  Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction 
PLoS ONE  2013;8(7):e70510.
The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.
Methodology/Principal Findings
Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.
From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.
PMCID: PMC3722294  PMID: 23894663
12.  The Thermochemistry of London Dispersion-Driven Transition Metal Reactions: Getting the ‘Right Answer for the Right Reason’ 
ChemistryOpen  2014;3(5):177-189.
Reliable thermochemical measurements and theoretical predictions for reactions involving large transition metal complexes in which long-range intramolecular London dispersion interactions contribute significantly to their stabilization are still a challenge, particularly for reactions in solution. As an illustrative and chemically important example, two reactions are investigated where a large dipalladium complex is quenched by bulky phosphane ligands (triphenylphosphane and tricyclohexylphosphane). Reaction enthalpies and Gibbs free energies were measured by isotherm titration calorimetry (ITC) and theoretically ‘back-corrected’ to yield 0 K gas-phase reaction energies (ΔE). It is shown that the Gibbs free solvation energy calculated with continuum models represents the largest source of error in theoretical thermochemistry protocols. The (‘back-corrected’) experimental reaction energies were used to benchmark (dispersion-corrected) density functional and wave function theory methods. Particularly, we investigated whether the atom-pairwise D3 dispersion correction is also accurate for transition metal chemistry, and how accurately recently developed local coupled-cluster methods describe the important long-range electron correlation contributions. Both, modern dispersion-corrected density functions (e.g., PW6B95-D3(BJ) or B3LYP-NL), as well as the now possible DLPNO-CCSD(T) calculations, are within the ‘experimental’ gas phase reference value. The remaining uncertainties of 2–3 kcal mol−1 can be essentially attributed to the solvation models. Hence, the future for accurate theoretical thermochemistry of large transition metal reactions in solution is very promising.
PMCID: PMC4234214  PMID: 25478313
density functional theory; isothermal titration calorimetry; local coupled cluster; London dispersion interactions; transition metal reactions
13.  Prediction of cyclin-dependent kinase 2 inhibitor potency using the fragment molecular orbital method 
The reliable and robust estimation of ligand binding affinity continues to be a challenge in drug design. Many current methods rely on molecular mechanics (MM) calculations which do not fully explain complex molecular interactions. Full quantum mechanical (QM) computation of the electronic state of protein-ligand complexes has recently become possible by the latest advances in the development of linear-scaling QM methods such as the ab initio fragment molecular orbital (FMO) method. This approximate molecular orbital method is sufficiently fast that it can be incorporated into the development cycle during structure-based drug design for the reliable estimation of ligand binding affinity. Additionally, the FMO method can be combined with approximations for entropy and solvation to make it applicable for binding affinity prediction for a broad range of target and chemotypes.
We applied this method to examine the binding affinity for a series of published cyclin-dependent kinase 2 (CDK2) inhibitors. We calculated the binding affinity for 28 CDK2 inhibitors using the ab initio FMO method based on a number of X-ray crystal structures. The sum of the pair interaction energies (PIE) was calculated and used to explain the gas-phase enthalpic contribution to binding. The correlation of the ligand potencies to the protein-ligand interaction energies gained from FMO was examined and was seen to give a good correlation which outperformed three MM force field based scoring functions used to appoximate the free energy of binding. Although the FMO calculation allows for the enthalpic component of binding interactions to be understood at the quantum level, as it is an in vacuo single point calculation, the entropic component and solvation terms are neglected. For this reason a more accurate and predictive estimate for binding free energy was desired. Therefore, additional terms used to describe the protein-ligand interactions were then calculated to improve the correlation of the FMO derived values to experimental free energies of binding. These terms were used to account for the polar and non-polar solvation of the molecule estimated by the Poisson-Boltzmann equation and the solvent accessible surface area (SASA), respectively, as well as a correction term for ligand entropy. A quantitative structure-activity relationship (QSAR) model obtained by Partial Least Squares projection to latent structures (PLS) analysis of the ligand potencies and the calculated terms showed a strong correlation (r2 = 0.939, q2 = 0.896) for the 14 molecule test set which had a Pearson rank order correlation of 0.97. A training set of a further 14 molecules was well predicted (r2 = 0.842), and could be used to obtain meaningful estimations of the binding free energy.
Our results show that binding energies calculated with the FMO method correlate well with published data. Analysis of the terms used to derive the FMO energies adds greater understanding to the binding interactions than can be gained by MM methods. Combining this information with additional terms and creating a scaled model to describe the data results in more accurate predictions of ligand potencies than the absolute values obtained by FMO alone.
PMCID: PMC3032746  PMID: 21219630
14.  Development of CHARMM Polarizable Force Field for Nucleic Acid Bases Based on the Classical Drude Oscillator Model 
A polarizable force field for nucleic acid bases based on the classical Drude oscillator model is presented. Parameter optimization was performed to reproduce crystallographic geometries, crystal unit cell parameters, heats of sublimation, vibrational frequencies and assignments, dipole moments, molecular polarizabilities and quantum mechanical base-base and base-water interaction energies. The training and validation data included crystals of unsubstituted and alkyl-substituted adenine, guanine, cytosine, uracil, and thymine bases, hydrated crystals, and hydrogen bonded base pairs. Across all compounds, the RMSD in the calculated heats of sublimation is 4.1%. This equates to an improvement of more than 2.5 kcal/mol in accuracy compared to the non-polarizable CHARMM27 force field. However, the level of agreement with experimental molecular volume decreased from 1.7% to 2.1% upon moving from the non-polarizable to the polarizable model. The representation of dipole moments is significantly improved with the Drude polarizable force field. Unlike in additive force fields, there is no requirement for the gas-phase dipole moments to be overestimated, illustrating the ability of the Drude polarizable force field to treat accurately differently dielectric environments and indicating the improvements in the electrostatic model. Validation of the model was performed based on the calculation of the gas phase binding enthalpies of base pairs obtained via potential of mean force calculations; the additive and polarizable models both performed satisfactorily with average differences of 0.2 and 0.9 kcal/mol, respectively, and RMS differences of 1.3 and 1.7 kcal/mol, respectively. Overall, considering the number of significant improvements versus the additive CHARMM force field, the incorporation of explicit polarizability into the force field for nucleic acid bases represents an additional step toward accurate computational modeling of biological systems.
PMCID: PMC3166616  PMID: 21166469
Molecular modeling; empirical force field; molecular dynamics simulation; potential of mean force; biomolecules; DNA; RNA
15.  Interaction of the Anticancer Plant Alkaloid Sanguinarine with Bovine Serum Albumin 
PLoS ONE  2011;6(4):e18333.
Interaction of the iminium and alkanolamine forms of sanguinarine with bovine serum albumin (BSA) was characterized by spectroscopic and calorimetric techniques.
Methodology/Principal Findings
Formation of strong complexes of BSA with both iminium and alkanolamine forms was revealed from fluorescence quenching of sanguinarine. Binding parameters calculated from Stern-Volmer quenching method revealed that the neutral alkanolamine had higher affinity to BSA compared to the charged iminium form. Specific binding distances of 3.37 and 2.38 nm between Trp 212 (donor) and iminium and alkanolamine forms (acceptor), respectively, were obtained from Forster resonance energy transfer studies. Competitive binding using the site markers warfarin and ibuprofen, having definite binding sites, demonstrated that both forms of sanguinarine bind to site I (subdomain IIA) on BSA. Sanguinarine binding alters protein conformation by reducing the α-helical organization and increasing the coiled structure, indicating a small but definitive partial unfolding of the protein. Thermodynamic parameters evaluated from isothermal titration calorimetry suggested that the binding was enthalpy driven for the iminium form but favoured by negative enthalpy and strong favourable entropy contributions for the alkanolamine form, revealing the involvement of different molecular forces in the complexation.
The results suggest that the neutral alkanolamine form binds to the protein more favourably compared to the charged iminium, in stark contrast to the reported DNA binding preference of sanguinarine.
PMCID: PMC3071820  PMID: 21494677
16.  Some Binding-Related Drug Properties are Dependent on Thermodynamic Signature 
Chemical biology & drug design  2011;77(3):161-165.
The binding affinity is determined by the Gibbs energy of binding (ΔG) which is the sum of enthalpic (ΔH) and entropic (-TΔS) contributions. Because the enthalpy and entropy contribute in an additive way to the binding energy, the same binding affinity can be achieved by many different combinations of enthalpic and entropic contributions; however, do compounds with similar binding affinities but different thermodynamic signatures (i.e. different ΔH, -TΔS combinations) exhibit the same functional effects? Are there characteristics of compounds that can be modulated by modifying their thermodynamic signatures? In this paper, we consider the minimization of unwanted conformational effects arising during the development of CD4/gp120 inhibitors, a new class of HIV-1 cell entry inhibitors. Competitive inhibitors of protein/protein interactions run the risk of triggering the very same signals that they are supposed to inhibit. Here, we show that for CD4/gp120 inhibitors the magnitude of those unwanted effects is related to the proportion in which the enthalpy and entropy changes contribute to the binding affinity. The thermodynamic optimization plot (TOP) previously proposed to optimize binding affinity can also be used to obtain appropriate enthalpy/entropy combinations for drug candidates.
PMCID: PMC3079564  PMID: 21288305
Binding Affinity; Enthalpy; Entropy; Thermodynamic Optimization; Isothermal Titration Calorimetry
17.  Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I, II, VII, XII, and XIII 
PLoS ONE  2014;9(12):e114106.
The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.
PMCID: PMC4262373  PMID: 25493428
18.  Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII 
BMC Biophysics  2012;5:12.
Human carbonic anhydrases (CAs) play crucial role in various physiological processes including carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions, and various pathological processes, especially tumor progression. Therefore, CAs are interesting targets for pharmaceutical research. The structure-activity relationships (SAR) of designed inhibitors require detailed thermodynamic and structural characterization of the binding reaction. Unfortunately, most publications list only the observed thermodynamic parameters that are significantly different from the intrinsic parameters. However, only intrinsic parameters could be used in the rational design and SAR of the novel compounds.
Intrinsic binding parameters for several inhibitors, including ethoxzolamide, trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIII isozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy, entropy, and the heat capacity. They were determined by titration calorimetry and thermal shift assay in a wide pH and temperature range to dissect all linked protonation reaction contributions.
Precise determination of the inhibitor binding thermodynamics enabled correct intrinsic affinity and enthalpy ranking of the compounds and provided the means for SAR analysis of other rationally designed CA inhibitors.
PMCID: PMC3534288  PMID: 22676044
19.  PHOENIX: A Scoring Function for Affinity Prediction Derived Using High-Resolution Crystal Structures and Calorimetry Measurements 
Binding affinity prediction is one of the most critical components to computer-aided structure-based drug design. Despite advances in first-principle methods for predicting binding affinity, empirical scoring functions that are fast and only relatively accurate are still widely used in structure-based drug design. With the increasing availability of X-ray crystallographic structures in the Protein Data Bank and continuing application of biophysical methods such as isothermal titration calorimetry to measure thermodynamic parameters contributing to binding free energy, sufficient experimental data exists that scoring functions can now be derived by separating enthalpic (ΔH) and entropic (TΔS) contributions to binding free energy (ΔG). PHOENIX, a scoring function to predict binding affinities of protein-ligand complexes, utilizes the increasing availability of experimental data to improve binding affinity predictions by the following: model training and testing using high-resolution crystallographic data to minimize structural noise, independent models of enthalpic and entropic contributions fitted to thermodynamic parameters assumed to be thermodynamically biased to calculate binding free energy, use of shape and volume descriptors to better capture entropic contributions. A set of 42 descriptors and 112 protein-ligand complexes were used to derive functions using partial least squares for change of enthalpy (ΔH) and change of entropy (TΔS) to calculate change of binding free energy (ΔG), resulting in a predictive r2 (r2pred) of 0.55 and a standard error (SE) of 1.34 kcal/mol. External validation using the 2009 version of the PDBbind “refined set” (n = 1612) resulted in a Pearson correlation coefficient (Rp) of 0.575 and a mean error (ME) of 1.41 pKd. Enthalpy and entropy predictions were of limited accuracy individually. However, their difference resulted in a relatively accurate binding free energy. While the development of an accurate and applicable scoring function was an objective of this study, the main focus was evaluation of the use of high-resolution X-ray crystal structures with high-quality thermodynamic parameters from isothermal titration calorimetry for scoring function development. With the increasing application of structure-based methods in molecular design, this study suggests that using high-resolution crystal structures, separating enthalpy and entropy contributions to binding free energy, and including descriptors to better capture entropic contributions may prove to be effective strategies towards rapid and accurate calculation of binding affinity.
PMCID: PMC3046228  PMID: 21214225
20.  Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work 
Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg2+, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.
PMCID: PMC3497075  PMID: 23148276
depletion attraction; electrostatic repulsion; entropy; grana; LHCII; van der Waals attraction
21.  Enthalpies of formation of Cd–Pr intermetallic compounds and thermodynamic assessment of the Cd–Pr system 
In the present study standard enthalpies of formation were measured by reaction and solution calorimetry at stoichiometric compositions of Cd2Pr, Cd3Pr, Cd58Pr13 and Cd6Pr. The corresponding values were determined to be −46.0, −38.8, −35.2 and −24.7 kJ/mol(at), respectively. These data together with thermodynamic data and phase diagram information from literature served as input data for a CALPHAD-type optimization of the Cd–Pr phase diagram. The complete composition range could be described precisely with the present models, both with respect to phase equilibria as well as to thermodynamic input data. The thermodynamic parameters of all intermetallic compounds were modelled following Neumann–Kopp rule. Temperature dependent contributions to the individual Gibbs energies were used for all compounds. Extended solid solubilities are well described for the low- and high-temperature modifications of Pr and also for the intermetallic compound CdPr. A quite good agreement with all viable data available from literature was found and is presented.
•ΔfH° was determined from Cd2Pr, Cd3Pr, Cd58Pr13 and Cd6Pr.•Thermodynamic assessment of the Cd–Pr phase diagram.•CALPHAD-type optimization of the complete Cd–Pr system is presented.•Temperature dependent contributions to the individual Gibbs energies were used.
PMCID: PMC4270480  PMID: 25540475
CALPHAD; Phase diagram Cd–Pr; Enthalpy of formation; Alloys
22.  Preparation and physicochemical characterization of 5 niclosamide solvates and 1 hemisolvate 
AAPS PharmSciTech  2004;5(1):86-95.
The purpose of the study was to characterize the physicochemical, structural, and spectral properties of the 1∶1 niclosamide and methanol, diethyl ether, dimethyl sulfoxide, N,N' dimethylformamide, and tetrahydrofuran solvates and the 2∶1 niclosamide and tetraethylene glycol hemisolvate prepared by recrystallization from these organic solvents. Structural, spectral, and thermal analysis results confirmed the presence of the solvents and differences in the structural properties of these solvates. In addition, differences in the activation energy of desolvation, batch solution calorimetry, and the aqueous solubility at 25°C, 24 hours, showed the stability of the solvates to be in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > methanol solvate > dimethyl sulfoxide solvate > N,N' dimethylformamide solvate. The intrinsic and powder dissolution rates of the solvates were in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > N,N' dimethylformamide solvate > methanol solvate > dimethyl sulfoxide solvate. Although these nonaqueous solvates had higher solubility and dissolution rates than the monohydrous forms, they were unstable in aqueous media and rapidly transformed to one of the monohydrous forms.
PMCID: PMC2784863  PMID: 15198533
niclosamide; solvates; solubility; physical stability
23.  What Drives Proteins into the Major or Minor Grooves of DNA? 
Journal of molecular biology  2006;365(1):1-9.
The energetic profiles of a significant number of protein-DNA systems at 20°C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves, namely that the water in the AT-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic.
PMCID: PMC1934558  PMID: 17055530
DNA binding; DNA grooves; hydration; thermodynamics; electrostatics
24.  Protein folding, protein collapse and Tanford’s transfer model: Lessons from single-molecule FRET 
The essential and non-trivial role of the denatured state of proteins in their folding reaction is being increasingly scrutinized in recent years. Single molecule FRET (smFRET) experiments show that the denatured state undergoes a continuous collapse (or coil-to-globule) transition as the concentration of a chemical denaturant is decreased, suggesting that conformational entropy of the denatured state is an important part of the free energy of folding. Such observations question the validity of the classical Tanford transfer model, which suggests that the folding free energy can be understood solely based on the difference in amino acid solvation between the folded state and a fixed unfolded state. An alternative to the transfer model is obtained here from a polymer theoretical analysis of a series of published smFRET data. The analysis shows that the free energy of denatured-state collapse has a linear dependence on denaturant concentration, an outcome of the interplay between enthalpic and entropic contributions. Surprisingly, the slope of the free energy of collapse agrees very well with the respective slope of the free energy of folding. This conformity of values obtained from two very different measurements shows that it is the collapse transition in the denatured state which mediates the effect of denaturants on folding. The energetics of folding are thus governed by the competition of solvation and conformational entropy in the denatured state.
PMCID: PMC2663972  PMID: 19239269
25.  Water-Membrane Partition Thermodynamics of an Amphiphilic Lipopeptide: An Enthalpy-Driven Hydrophobic Effect 
Biophysical Journal  2008;95(7):3269-3277.
To shed light on the driving force for the hydrophobic effect that partitions amphiphilic lipoproteins between water and membrane, we carried out an atomically detailed thermodynamic analysis of a triply lipid modified H-ras heptapeptide anchor (ANCH) in water and in a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Combining molecular mechanical and continuum solvent approaches with an improved technique for solute entropy calculation, we obtained an overall transfer free energy of ∼−13 kcal mol−1. This value is in qualitative agreement with free energy changes derived from a potential of mean force calculation and indirect experimental observations. Changes in free energies of solvation and ANCH conformational reorganization are unfavorable, whereas ANCH-DMPC interactions—especially van der Waals—favor insertion. These results are consistent with an enthalpy-driven hydrophobic effect, in accord with earlier calorimetric data on the membrane partition of other amphiphiles. Furthermore, structural and entropic analysis of molecular dynamics-generated ensembles suggests that conformational selection may play a hitherto unappreciated role in membrane insertion of lipid-modified peptides and proteins.
PMCID: PMC2547422  PMID: 18621822

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