Photo-stimulated leaving group isomerization of acyl donor esters in protease-catalyzed hydrolysis reactions

Abstract

Hydrolysis studies of photo-switchable N-maleyl-amino acid-(4-phenylazophenyl) esters of Ala, Gly, Met, Phe, and Pro were performed using the proteases trypsin and chymotrypsin. It has been found out that the cis-isomers were hydrolyzed faster than the trans-isomers. In dependence of the amino acid in the P1 position the velocity graduation is Phe?>?Met?>?Ala?>?Gly?>?Pro for trypsin and Phe???Met?>?Ala?>?Gly?>?Pro for chymotrypsin for both isomers.     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

Interaction of two flavonols with fat mass and obesity-associated protein investigated by fluorescence quenching and molecular docking

The binding of two flavonols with fat mass and obesity-associated protein (FTO) was studied using fluorescence spectroscopy, Stern-Volmer kinetics, UV-Vis absorption, and molecular docking. The quenching of FTO fluorescence was determined to be static with binding constants on the order of 10⁴ M^?1. The interaction was studied over three temperatures, and the binding was found to be exothermic with a positive change in entropy. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction and hydrogen bonding interaction are the main binding force in stabilizing the flavonol–FTO complex.     

Molecular characterization and expression analysis of fat mass and obesity-associated gene in rabbit

Fat mass and obesity-associated (FTO) gene codes for a nuclear protein of the AlkB related nonhaem iron and 2-oxoglutarate-dependent oxygenase superfamily, and is involved in animal fat deposition and human obesity. In this work, the molecular characterization and expression features of rabbit (Oryctolagus cuniculus) FTO cDNA were analysed. The rabbit FTO cDNA with a size of 2158 bp was cloned, including 1515 bp of the open reading frame that encoded a basic protein of 504 amino acids. Homologous comparison indicated that the rabbit FTO shared 36.36–91.88% identity with those from other species and phylogenetic analysis showed that the rabbit FTO is closely related to human, but more distantly related to zebrafish. The New Zealand rabbit FTO mRNA was detected in all tissues examined, with the highest levels found in the spleen and the lowest found in the kidney. However, no significant differences were seen in cerebellum, corpora quadrigemina, medulla oblongata and cerebral cortex of commercial adult rabbits. Moreover, mRNA levels of FTO in liver tissues were significantly increased in lactating New Zealand rabbits compared with 70-day-old, 90-day-old and gestating rabbits (P?<?0.05). In contrast, FTO mRNA levels were significantly lower in longissimus dorsi muscle of 90-day-old New Zealand rabbits than in 70-day-old rabbits (P?<?0.05). However, the expression levels of FTO in mammary gland and ovary of gestating and lactating rabbits were not significantly different (P?>?0.05).     

Forskolin-loaded human serum albumin nanoparticles and its biological importance

Abstract

In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340?nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24?h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26?±?0.1?×?10⁴ M^?1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be K_forskolin = 3.4?±?0.24?×?10⁴ M^?1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes.

Communicated by Ramaswamy H. Sarma     

Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach

Abstract

Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5?R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K_2-C-MU = 4?±?0.03?×?10⁴M^?1, K_5-CM-MU = 1.95?±?0.03?×?10⁴ M^?1 and K_5-P-MU =1.56?±?0.03?×?10⁴ M^?1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases.

Communicated by Ramaswamy H. Sarma     

Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic,cyclic voltammetric,and molecular docking technique

The interaction of fisetholz with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. The results revealed that there was a static quenching of BSA/HSA induced by fisetholz. The binding constants (K_a) and binding sites (n) were calculated at different temperatures (293, 303, and 311?K). The enthalpy change (ΔH) were calculated to be –17.20?kJ mol^?1 (BSA) and –18.28?kJ mol^?1 (HSA) and the entropy change (ΔS) were calculated to be 35.41?J mol^?1 (BSA) and 24.02?J mol^?1 (HSA), respectively, which indicated that the interaction between fisetholz and BSA/HSA was mainly by electrostatic attraction. Based on displacement experiments using site probes, indomethacin and ibuprofen, the binding site of fisetholz to BSA/HSA was identified as sub-domain IIIA, which was further confirmed by molecular docking method. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ on fisetholz-BSA or fisetholz-HSA complex. The spectra of synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) all showed that fisetholz binding to BSA/HSA leads to secondary structures change of the two serum albumins. According to the Förster non-radiation energy transfer theory, the binding distance between fisetholz and BSA/HSA was 2.94/4.68?nm. The cyclic voltammetry as a supporting tool also indicated that fisetholz interacted with protein.

Communicated by Ramaswamy H. Sarma     

Effect of (–)-epicatechin-3-gallate and (–)-epigallocatechin-3-gallate on the binding of tegafur to human serum albumin as determined by spectroscopy,isothermal titration calorimetry,and molecular docking

Green tea has attracted great interest as a cancer prevention agent. Interactions of tea polyphenols with serum albumin may influence the efficacy of drugs. The interactions of (–)-epigallocatechin-3-gallate (EGCG), (–)-epicatechin-3-gallate (ECG), and tegafur (TF) alone or in combination with human serum albumin (HSA) at pH 7.4 and different temperatures were investigated by spectroscopic methods, isothermal titration calorimetry (ITC), and molecular docking. The binding affinities to HSA were ranked in the order of EGCG?>?ECG?>?TF, and the interactions were spontaneous and exothermic. Ternary system studies showed that the presence of one component hindered the binding of another component to HSA. The secondary structures of HSA were slightly altered in the presence of the ligands. Site marking experiments and molecular docking showed that EGCG and ECG mainly bound to subdomain IIA and ΙΙΙA while TF bound to subdomain ΙΙA and ΙB. Results indicated that the existence of ECG and EGCG would influence the binding of TF to HSA and can increase the free concentration of TF. Obtained results would provide beneficial information about possible interference upon simultaneous co-administration of the tea components and drugs.

Communicated by Ramaswamy H. Sarma     

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum

Purpose

To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma.

Methods

Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C₁₈ column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients’ serum/plasma samples.

Results

Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient?>?0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range.

Conclusion

We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

     

Low-density lipoprotein-associated variables and the severity of coronary artery disease: an untreated Chinese cohort study

Abstract

Background and aims: Elevated low-density lipoprotein cholesterol (LDL-C) is causal risk for coronary artery disease (CAD) and LDL-associated variables including LDL-C, apolipoprotein B (apoB), non-high-density lipoprotein cholesterol (non-HDL-C), lipoprotein a [Lp(a)], small dense LDL (sd-LDL), and oxidized LDL (ox-LDL) have been widely used for predicting the risk of CAD. This study was aimed to compare the values of six LDL-related variables for predicting the severity of CAD using untreated patients undergoing coronary angiography (CAG).

Methods: A group of 1977 individuals were consecutively enrolled and divided into CAD (n?=?1151) and non-CAD groups (n?=?826) according to the results of CAG. LDL-C, apoB, non-HDL-C, Lp(a), sd-LDL and ox-LDL were measured, respectively. The numbers of stenotic arteries and Gensini Scores (GS) were used to calculate the severity of CAD and the associations of six variables with the severity of CAD and predicting value of these parameters were simultaneously examined.

Results: CAD patients had significantly higher concentrations of LDL-related variables than non-CAD ones (all p?<?0.05). Importantly, all variables rose with the increase in the severity of CAD. The predicting value of CAD manifested as sd-LDL?>?ox-LDL?>?apoB?>?non-HDL-C?>?LDL-C?>?Lp(a) [area under curve (AUC): sd-LDL 0.641; ox-LDL 0.640; apoB 0.611; non-HDL-C 0.587; LDL-C 0.583; Lp(a) 0.554; respectively]. In multivariate logistic analysis, all variables showed as independent risk factors for the severity of CAD [odds ratio (OR): ox-LDL?>?sd-LDL?>?apoB?>?non-HDL-C?>?LDL-C?>?Lp(a)].

Conclusions: All of LDL-related variables could be useful marker for predicting the severity of CAD but sd-LDL and ox-LDL appeared to litter better. Further study may be needed to validate our results.     

Influence of stearic acids on resveratrol-HSA interaction

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k _a ?=?(1.10?±?0.14)?×?10⁵?M^?1 and (1.09?±?0.02)?×?10⁵?M^?1, respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer <?(high [SA]/HSA)?<?HSA?<?(low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.     

Biomolecular interaction of a platelet aggregation inhibitor, 3,4-methylenedioxy-β-nitrostyrene with human serum albumin: multi-spectral and computational characterization

Abstract

Molecular interaction of the 3,4-methylenedioxy-β-nitrostyrene (MNS), an inhibitor of platelet aggregation with the main transport protein, albumin from human serum (HSA) was explored using absorption, fluorescence and circular dichroism (CD) spectroscopy in combination with in silico analyses. The MNS–HSA complexation was corroborated from the fluorescence and absorption spectral results. Implication of static quenching mechanism for MNS–HSA system was predicted from the Stern–Volmer constant, K_SV-temperature relationship as well as the bimolecular quenching rate constant, k_q values. Stabilization of the complex was affirmed by the value of the binding constant (K_a = 0.56-1.48?×?10⁴ M^?1). Thermodynamic data revealed that the MNS–HSA association was spontaneously driven mainly through hydrophobic interactions along with van der Waal’s interaction and H-bonds. These results were well supported by in silico interpretations. Far-UV and near-UV CD spectral results manifested small variations in the protein’s secondary and tertiary structures, respectively, while three-dimensional fluorescence spectra displayed microenvironmental fluctuations around protein’s fluorophores, upon MNS binding. Significant improvement in the protein’s thermostability was evident from the temperature-stability results of MNS-bound HSA. Binding locus of MNS, as identified by competitive drug displacement findings as well as in silico analysis, was found to be located in subdomain IIA (Sudlow’s site I) of the protein.

Communicated by Ramaswamy H. Sarma     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

Protective effect exerted by soil phosphorus on soybean subjected to arsenic and fluoride

Objetive: Arsenic (As) and fluoride (F) are found in groundwater and soils around the world, causing different problems to crops. Because these elements compete against phosphorus (P) in soils and plants, their relationship is complex. The aim of this work was to study the oxidative stress of soybean plants subjected to different concentrations of As and F, and the effect of P.

Methods: The following 10 treatments were carried out in each of two soils with different P content: three As levels (low 10?mg?As?kg^-1, medium 50?mg?As?kg^?1 and high 100?mg As kg^?1), three F levels (low 160?mg?F?kg^?1, medium 250?mg?F?kg^?1 and high 500?mg?F?kg^?1) and three As?+?F levels (same concentrations), and the control treatment (soil with the background As and F concentrations) Lipid peroxidation, chlorophyll, gluthatione contents and antioxidant enzymes activities were determination.

Results: Increased lipid peroxidation and alterations in glutathione content, catalase, superoxide dismutase and peroxidase activities as well as in chlorophyll content revealed that As causes higher oxidative stress in plants grown in soils with low P content.

Conclusion: Stress parameters in F treatments were less affected. Plants grown in soils enriched with P revealed a decrease in the toxic effects caused by As and F.     

Probing the Interaction of Human Serum Albumin With Bilirubin in the Presence of Aspirin by Multi-Spectroscopic,Molecular Modeling and Zeta Potential Techniques: Insight on Binary and Ternary Systems

Abstract

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K_a) and number of binding site (n) obtained were 1.6 × 10⁶ M^?1 and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10⁶ M^?1 and 0.84, respectively, in the presence of ASA (ternary complex) at δ_ex = 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C_CIAC) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N⁶-methyladenosine (m⁶A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m⁶A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m⁶A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.     

Effects of rs769217 and rs1001179 polymorphisms of catalase gene on blood catalase,carbohydrate and lipid biomarkers in diabetes mellitus

Abstract

Oxidative stress and deficiency of the enzyme catalase, which is the primary scavenger of the oxidant H₂O₂, may contribute to diabetes. The current study examined two polymorphisms in the catalase gene, ?262C>nT in the promoter and 111C>T in exon 9, and their effects on blood catalase activity as well as on concentrations of blood glucose, haemoglobin A1c, triglyceride, cholesterol, HDL, LDL, ApoA-I and ApoB. Subjects were type-1 and type-2 diabetics. We evaluated PCR-single strand conformational polymorphism for 111C>T and PCR-restriction fragment length polymorphism for ??262C>T. TT genotype frequency of 111C>T polymorphism was increased in type-1 diabetes. Type-2 diabetics with the CC or CT genotypes had decreased catalase and increased glucose, hemoglobinA1c and ApoB. Type-2 diabetics who have TT genotype in ?262C>T may have elevated risk for diabetes complications; these patients had the lowest mean catalase and HDL, as well as the highest glucose, haemoglobin A1c, cholesterol and ApoB.     

No association between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women: a one-year prospective study

Abstract

Introduction: The aim of the study was to explore the association between the vitamin D pathway gene variations and the bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women.

Methods: A total of 110 healthy postmenopausal Chinese women (61.51?±?6.93?years) were enrolled. The participants were supplemented with calcium (600?mg/d) and calcitriol (0.25?μg/d), for 1?year. Four biomarkers, serum levels of beta C-terminal cross-linked telopeptides of type I collagen (β-CTX), amino-terminal propeptide of type I collagen (P1NP), parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] were measured at baseline and 12-month follow-up. Multivariate regression models were established to explore the statistical association between the change rate of the four biomarkers and 15?key genes within the vitamin D metabolic pathway.

Results: This exclusion process left 98 participants for analysis. Serum levels of P1NP, β-CTX and PTH were significantly decreased at the 12-month follow-up (all p?<?0.05). Serum 25(OH)D level had no significant change (p?>?0.05). No association was found between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation.

Conclusions: Genetic background of postmenopausal Chinese women might not influence supplemental response of the biomarkers to calcium and low dose calcitriol.     

The role of chlorine atom on the binding between 2‐phenyl‐1H‐benzimidazole analogues and fat mass and obesity‐associated protein

In this work, nine 2‐phenyl‐1H‐benzimidazole structural analogues were screened for potential inhibitor of the fat mass and obesity‐associated protein (FTO) by isothermal titration calorimetry (ITC). The results show that the binding between 6‐chloro‐2‐phenyl‐1H‐benzimidazole (1d) and FTO was dominated by entropy. Results of enzymatic activity assays provided an IC₅₀ value of 24.65 μM for 1d. Our previous results and comparison of nine structural analogues indicated that the chlorine atom was crucial for the binding of small molecules with FTO. The identification of novel small molecules may provide information for the design of FTO inhibitors and the treatment of leukemia.