Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53

A new strategy based on acid-alkali complexation for rapidly and accurately fishing phytochemicals in sennae folium / 中草药·英文版

Objective: There are some anthraquinones, anthraquinones and flavonones in Sennae Folium which exhibited significant acidity, such as sennoside A/B and sennoside C/D. The current strategies used in separating these components are mainly based on conventional column chromatography which is time consuming, laborious and costly. This study is aimed at exploring a method of precipitation extraction of acid components in Sennae Folium. Using alkaloid as a “hook”, it is reasonable to use the principle of “acid-alkali complexation” to "fish" the acidic components in Sennae Folium. Methods: Isothermal titration calorimeter (ITC) was used to measure the extraction efficiency of different alkaloids. Then, alkaloid determined by ITC was mixed with extracting solution of Sennae Folium to form complex. High performance liquid chromatography coupled with mass spectrometry (HPLC-MS2) was used to investigate the ingredients “fished” by berberine (Ber). The mechanism of “fishing” process was explained by ITC, optical activity, fluorescence spectrometry and scanning electron microscope. Results: The ITC results proved that the choice of “hook” was particularly important in the process of “fishing”. Among the hooks, the fishing efficiency of the isoquinoline alkaloids (Ber) was the highest, reaching 10.3%. Nine ingredients were detected and determined by HPLC-MS2, and the main components were sennoside A/B and sennoside C/D. Based on ITC test of Ber and sennoside A, the combination mechanism of the two ingredients was a chemical reaction with a nearly binding ratio (2:1). Fluorescence and optical properties of the active ingredients were changed after complexation. By scanning electron microscope, we found that two types of components had obviously self-assembled behavior during the formation process. Conclusion: Ber successfully “fished” the main acidic components, sennoside A/B and sennoside C/D, from Sennae Folium. Combined with different characterizations, the “fishing” process was determined as a chemical association reaction induced by electrostatic interaction or π-π stacking. Therefore, with special identification ability, the “fishing” process had the potential of practical application.

Thermodynamics of clay-drug complex dispersions: Isothermal titration calorimetry and high-performance liquid chromatography / 药物分析学报

An understanding of the thermodynamics of the complexation process utilized in sustaining drug release in clay matrices is of great importance. Several characterisation techniques as well as isothermal calo-rimetry were utilized in investigating the adsorption process of a model cationic drug (diltiazem hy-drochloride, DIL) onto a pharmaceutical clay system (magnesium aluminium silicate, MAS). X-ray powder diffraction (XRPD), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and optical microscopy confirmed the successful formation of the DIL-MAS complexes. Drug quantification from the complexes demonstrated variable behaviour in the differing media used with DIL degrading to desacetyl diltiazem hydrochloride (DC-DIL) in the 2 M HCl media. Here also, the authors report for the first time two binding processes that occurred for DIL and MAS. A competitor binding model was thus proposed and the thermodynamics obtained suggested their binding processes to be enthalpy driven and entropically unfavourable. This information is of great importance for a formulator as care and consideration should be given with appropriate media selection as well as the nature of binding in complexes.

Meridian tropism of Scutellaria baicalensis based on molecular thermodynamic characteristics / 药学学报

The property of medicine is the "identity card" of traditional Chinese medicine (TCM), and the key to crack the theory of property of TCM. Based on molecular thermodynamics, the effects of interaction between TCM and organs in vitro were studied from the perspective of micro-energy release and absorption in order to construct a new idea of characterizing meridian theory. Scutellaria baicalensis, for example, application of isothermal titration calorimetry (ITC) were used to determine the energy changes during the interaction of Scutellaria baicalensis and its main active ingredient baicalin with brain, heart, lung, spleen and kidney in vitro, comparison including the association constant (Ka) and disassociation constant (Kd), combined with thermodynamic parameters, such as stoichiometry ratio (n), enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy (ΔG), it is found that the interaction intensity between Scutellaria baicalensis and lung is significantly stronger than that of other organs, which is consistent with the theory of the return of Scutellaria baicalensis in ancient books. In addition, baicalin, the main active ingredient, showed the same action pattern as Scutellaria baicalensis. The thermodynamic parameters analysis showed that the action was a weak bond-induced spontaneous chemical binding reaction driven by both entropy and enthalpy. The results of specific curl measurement further proved the interaction between baicalin and lung, and were consistent with the results of ITC titration, indicating that ITC could be used to characterize the meridian tropism of TCM. Therefore, based on ITC, it is scientific and feasible to characterize the meridian of TCM by the energy change of the interaction between the decoction of TCM and its active components and the in vitro tissues respectively. This experiment provides a new idea for the discussion of meridian of TCM.

Application of isothermal titration calorimetry in drug research / 中草药

Isothermal titration calorimetry (ITC) is a real-time, quantitative, on-line, and dynamic thermal analysis technique for describing reaction processes. Some thermodynamic parameters, such as association constant, dissociation constant, stoichiometry ratio, enthalpy change, and entropy change of host and guest interaction, can be obtained in ITC analysis. This technique has the advantages of high calorimetric sensitivity and accurate measurement. In this paper, the principle and application characteristics of ITC technology are described, and the application of this technique in drug research are reviewed, including the interaction between drugs and proteins, the interaction between drugs and DNA molecules, clinical drug compatibility, the application in pharmaceutical preparations and the interaction between drugs and biomembrane. Lastly, the application of this technique in the nonspecific molecular interaction is prospected.

New qualities control strategy of Scutellaria baicalensis by isothermal titration calorimetry / 中国中药杂志

It has been focused on that there will be precipitates when decoction of Scutellariat Radix mixed with Coptidis Rhizoma. Precipitation was derived from interaction between acidic and basic compounds. This study was based on the interaction between active ingredients after compatibility, strived to explore whether it was feasible to judge the qualities of different Scutellariat Radix by isothermal titration calorimetry (ITC), build a new method established to characterize the qualities of traditional Chinese medicine by taking a series of active ingredients as index. We selected Scutellariat Radix (including three batches of different Scutellariat Radix bought from market and immature Scutellariat Radix which usually was used as adulterant) in different batches as the samples. First, we used ITC to determine the binding heat of the reactions between berberine and the decoctions of different Scutellariat Radix. The test showed that the binding heat of berberine titrated Scutellariat Radix was Scutellariat Radix A (－317.20 μJ), Scutellariat Radix B (－292.83 μJ), Scutellariat Radix C (－208.95 μJ) and immature Scutellariat Radix (－21.53 μJ), respectively. We chose deionized water titrated by berberine (2.51 μJ) as control. The heat change of berberine titrated immature Scutellariat Radix was much less than berberine titrated Scutellariat Radix. Then we determined the absorbance of different decoctions of Scutellariat Radix by UV Spectrophotometry on the maximum absorption wavelength, and the result is： Scutellariat Radix A (0.372), Scutellariat Radix B (0.333), Scutellariat Radix C (0.272), immature Scutellariat Radix (0.124). The absorbance of immature Scutellariat Radix was also less than Scutellariat Radix. The result of ITC assay was corresponded to UV spectrophotometry test. In conclusion, ITC could be used to characterize the quality of Scutellariat Radix. The new method to characterize the qualities of traditional Chinese medicine by taking a kind of active ingredients as index building by ITC was simple, scientific and feasible.

Formation mechanism of precipitation in Huanglian Jiedu Decoction based on molecular thermodynamic characteristics / 中草药

Objective To explore all sources of precipitation, parameters of interaction and binding abilities of different Chinese materia medica (CMM) combinations during the formation process of the precipitation in Huanglian Jiedu Decoction (HJD) using the thermodynamic method. The study was aim to reveal the mechanism of the formation of precipitation in CMM decoction. Methods First, the possible CMM combinations which had obvious precipitation phenomenon when combined were determined by the research on decomposed recipes. Then isothermal titration calorimetry (ITC) was used to determine the binding heat and thermodynamic parameters of binding reactions, thermodynamic parameters contents binding constant (Ka), dissociation constant (Kd), estimate of stoichiometric ratio (n), enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy change (ΔG), these data were used to explain the differences in binding abilities between different CMM combinations. Results The original decomposed recipes experiment indicated the CMM combinations that could produce obvious precipitate when they were mixed were Scutellariae Radix-Coptidis Rhizoma and Scutellariae Radix-Phellodendri Chinensis Cortex. Precipitation amount of Scutellariae Radix-Coptidis Rhizoma was the most. There was no precipitate in Scutellariae Radix-Gardeniae Fructus, Coptidis Rhizoma-Phellodendri Chinensis Cortex, Coptidis Rhizoma-Gardeniae Fructus or Phellodendri Chinensis Cortex-Gardeniae Fructus. In ITC, the thermodynamic parameters of the three experience groups were the same, ΔH 0, ΔG |-TΔS|, indicating the three reactions were all enthalpy-driving reactions. The Ka of Scutellariae Radix decoction titrated Coptidis Rhizoma decoction was 1.283 × 104, Scutellariae Radix decoction titrated Phellodendri Chinensis Cortex deccotion was 4.680 × 103, Scutellariae Radix decoction titrated Gardeniae Fructus decoction was 1.973 × 103. The value decreased gradually. The binding heat of Scutellariae Radix and Gardeniae Fructus was much smaller than the binding heats of Scutellariae Radix and Coptidis Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex. Conclusion During the formation process of the precipitation in HJD, the main source of acid-base complex-precipitation were Scutellariae Radix-Coptidis Rhizoma and Scutellariae Radix-Phellodendri Chinensis Cortex; Analysis of molecular thermodynamic indicates the binding energy of Scutellariae Radix and Coptidis Rhizoma was larger than that of Scutellariae Radix and Phellodendri Chinensis Cortex, compared with them, the binding energy of Scutellariae Radix and Gardeniae Fructus is tiny.

Characteristics and thermodynamics of the interaction of 6-shogaol with human serum albumin as studied by isothermal titration calorimetry

ABSTRACT The interaction between 6-shogaol, a pharmacologically active ginger constituent, and human serum albumin (HSA), the main in vivo drug transporter, was investigated using isothermal titration calorimetry (ITC). The value of the binding constant, Ka (5.02 ± 1.37 × 104 M−1) obtained for the 6-shogaol-HSA system suggested intermediate affinity. Analysis of the ITC data revealed feasibility of the binding reaction due to favorable enthalpy and entropy changes. The values of the thermodynamic parameters suggested involvement of van der Waals forces, hydrogen bonds and hydrophobic interactions in the 6-shogaol-HSA complex formation.

Calorimetric techniques applied to the thermodynamic study of interactions between proteins and polysaccharides / Técnicas calorimétricas aplicadas ao estudo termodinâmico das iterações entre proteínas e polissacarídeos

ABSTRACT: The interactions between biological macromolecules have been important for biotechnology, but further understanding is needed to maximize the utility of these interactions. Calorimetric techniques provide information regarding these interactions through the thermal energy that is produced or consumed during interactions. Notable techniques include differential scanning calorimetry, which generates a thermodynamic profile from temperature scanning, and isothermal titration calorimetry that provide the thermodynamic parameters directly related to the interaction. This review described how calorimetric techniques can be used to study interactions between proteins and polysaccharides, and provided valuable insight into the thermodynamics of their interaction.

RESUMO: As interações entre macromoléculas biológicas têm tido importante aplicação na biotecnologia, mas, para sua devida utilização, estudos mais detalhados são necessários. As técnicas calorimétricas permitem estudá-las ao serem capazes de fornecer informações referentes a essas interações através da energia térmica que é gerada ou absorvida durante o processo de interação. Dentre as técnicas que mais se destacam estão a Calorimetria Exploratória Diferencial, que é capaz de fornecer um perfil termodinâmico a partir de uma varredura de temperatura, e a Calorimetria de Titulação Isotérmica, que fornece parâmetros termodinâmicos diretamente relacionados ao processo de interação. Nesta revisão, descrevemos como essas técnicas calorimétricas podem ser efetivamente aplicadas no estudo das interações entre proteínas e polissacarídeos, com o propósito de obter informações valiosas sobre a termodinâmica da interação.

Application of isothermal titration calorimetry in molecular imprinting technique / 中国生化药物杂志

Molecular imprinting technique is a novel technology for preparing polymers with specific binding properties tosome specific molecules, and received widely attention in recent years.The article that was reviewed the applications of microcalorimetry in the molecular imprinting technique.The basic principle of microcalorimetry,characteristics and its advanced applications on evaluating imprinting effects of the molecularly imprinted polymers, screening of the monomers and the cross-linking agents, optimizing reaction conditions etc.were introduced.

Spectroscopy study on the interaction of γ-cyclodextrin and its two derivatives with rocuronium and vecuronium / 第二军医大学学报

Objective To study the interaction of γ-cyclodextrin (-CD) and its two derivatives with rocuronium and vecuronium. Methods The interactions of γ-CD and its two derivatives HS-7, Sugammadex with rocuronium and vecuronium were characterized by Isothermal Titration Calorimetry (ITC), and the thermodynamic constant and the binding constant was determined. The binding constant and inclusion ratio of γ-CD with rocuronium and vecuronium was observed by ultraviolet- visible spectrophotometry; the binding ability of three cyclodextrins with rocuronium and vecuronium was analyzed by nuclear magnetic resonance spectroscopy COSY and1HNMR. Results The results of ITC showed that HS-7, Sugammadex combined with rocuronium and vecuronium in a 1: 1 molar ratio, and γ-CDs combined with rocuronium in a 2: 1 molar ratio, with the binding constant of HS-7 and rocuronium being (3. 44 + 2. 18) X 107 L/mol, HS-7 and vecuronium being (5. 80 + 1. 83) X 106 L/mol, Sugammadex and rocuronium being (1. 04+0. 34) X 107 L/mol, Sugammadex and vecuronium being (2. 53 + 1. 07) X 106 L/mol, and γ-CD and rocuronium being (2. 84 + 3. 41) X 104 L/mol. The results of ultraviolet-visible spectrophotometry revealed that γ-CDs combined with rocuronium and vecuronium in a 2: 1 molar ratio, with the binding constant of y-CD and rocuronium being 6. 93 X 104 L/mol, and of γ-CD and vecuronium being 5. 17 X 104 L/mol. The results of nuclear magnetic resonance spectroscopy suggested that the binding ability of HS-7 with rocuronium and vecuronium was stronger than that of Sugammadex and γ -CD. Conclusion The binding ability of HS-7 with rocuronium and vecuronium is stronger than that of Sugammadex and γ -CD, and the three methods used in this study (ITC, ultraviolet-visible spectrophotometry and nuclear magnetic resonance spectroscopy) can characterize the inclusion complex from different perspectives.

Analysis on Interaction between Human Serum Albumin and Zinc Ions by Circular Dichroism and Isothermal Titration Calorimetry / 世界科学技术-中医药现代化

This article was aimed to study the mechanism of interaction between human serum albumin (HSA) and zinc ions, in order to provide the information on the secondary structure modification of HSA and the thermodynamics parameters using circular dichroism (CD) and isothermal titration calorimetry (ITC). CD and ITC were applied in the study. The concentration of HSA were 0.025 mmol·L-1, 0.05 mmol·L-1, 0.1 mmol·L-1, and 0.2 mmol·L-1, respectively. The concentration of zinc ion was 5 mmol·L-1. The CD analysis revealed that the secondary structure modification of HSA differed depending on the concentration of HAS. And the content ofα-helix was inversely proportional to the concentration of HSA. When the concentration of HSA was 0.2 mmol·L-1, the content ofα-helix was special. A series of thermodynamics parameters including association constants (Kb), stoichiometry (N), entropy (ΔS) and enthalpy (ΔH) were investigated by ITC analysis. And two types of binding sites were observed when ZnSO4(mmol·L-1)?HSA (mmol·L-1)= 5?0.2. The secondary structure modification of HSA interacting with zinc ions depended on the concentration of HSA, a dramatic reduction ofα-helix was detected when the concentration of HSA attained 0.2 mmol·L-1. And the protein hydrophobicity reduction and peptide chains expansion were equally observed at this concentration. The ITC analysis also revealed endothermic and exothermic binding sites in the ZnSO4-HSA interaction when ZnSO4 (mmol·L-1)?HSA (mmol·L-1)= 5?0.2, indicating that the endothermic sites were specific but not preferential for zinc ions interactions. These results provided theoretical supports for the application of Zn2+ to open the endothermic sites of HSA.

Caracterização físico-química de complexos de insulina: dimetil-beta-ciclodextrina e insulina: hidroxipropil-beta-ciclodextrina e avaliação da influência do tipo de complexo na produção de microesferas biodegradáveis / Physico-chemical caracterization of insulin: dimethyl-beta-cyclodextrin and insulin:hydroxypropil-beta-cyclodextrin complexes and evaluation of the kind of complexes on the biodegradable microspheres preparation

A etapa principal na ativação e ligação da insulina ao seu receptor é a dissociação dos hexâmeros do hormônio, normalmente presente nas preparações farmacêuticas, para a forma monomérica bioativa. A utilização de diferentes ciclodextrinas (CDs) como adjuvantes em formulações contendo insulina vem sendo explorada e os estudos realizados demonstram que estas substâncias podem aumentar a absorção da insulina principalmente por diminuírem sua capacidade de formar dímeros e hexâmeros em meio aquoso. No presente trabalho, complexos de insulina:hidroxipropil-beta-ciclodextrina (INS:HP-beta-CD) e insulina:dimetil-beta-ciclodextrina (INS:DM-beta-CD) foram caracterizados utilizando técnicas de titulação calorimétrica isotérmica e espalhamento dinâmico de luz. Por meio da titulação calorimétrica foram determinados os parâmetros termodinâmicos de interação entre a insulina e as CDs utilizadas, sugerindo que o mecanismo de complexação ocorre com aumento de entropia para ambos os sistemas. Os experimentos de espalhamento dinâmico de luz não indicaram diminuição do diâmetro hidrodinâmico das espécies moleculares de insulina após a complexação com as CDs. Os complexos INS:HP-beta-CD e INS:DM-beta-CD foram encapsulados em microesferas (MEs) de PLGA 50:50. A caracterização das MEs obtidas revelou aumento considerável na taxa de encapsulamento de insulina quando complexada com as CDs sem que ocorresse diferença significativa no diâmetro das partículas em função da complexação.

The main stage in the linking and activation of the specific receptors by the insulin is the dissociation of this peptide hexamers, normally present in pharmaceutical formulations, in the monomeric active form. Because of this, the use of different cyclodextrins as adjuvants in the formulations containing insulin has been explored and the realized studies have demonstrated that the cyclodextrins can increase the absorption of the insulin mainly by reducing the ability of insulin oligomerization in aqueous media. In this work, complexes of INS:HP-beta-CD and INS:DM-beta-CD have been characterized by the use of isothermal calorimetry titration (ICT) and dynamic scattering of light. By means of ICT, the thermodynamic parameters of interaction between insulin and the cyclodextrins have been determined, and it was observed that the complexation occurs with an increase of entropy for both systems. The experiments of dynamic scattering of light have not showed reduction in the size of insulin particles, which could indicate the dissociation of insulin hexamers after the complexation with cyclodextrins. Then, the INS: HP-beta-CD and INS:DM-beta-CD complexes were encapsulated in PLGA microspheres. These systems were characterized and it was not observed any significant difference in the microspheres diameter, but a considerable increase in the hormone loading after the complexation with HP-beta-CD and DM-beta-CD was shown.