Androgen receptor binding affinity of pesticide "active" formulation ingredients. QSAR evaluation by COREPA method

The COREPA approach for identifying the COmmon REactivity PAttern of biologically similar chemicals was employed to upgrade the recently derived affinity pattern for high androgen receptor (AR) binding affinity. The training set consisted of 28 steroidal and nonsteroidal ligands whose AR binding affinity was determined in competitive binding assays (in terms of pKi). The interatomic distances between nucleophilic sites and their charges providing distinct and non-overlapping integral patterns for active and inactive chemicals were assumed that it was related with the endpoint, which was under study. These stereoelectronic characteristics were used to predict pKi values of pesticide "active" formulation ingredients in an attempt to identify chemicals with potential AR binding affinity.     

黄酮类醛糖还原酶抑制剂的抑制机理研究

为了研究黄酮类醛糖还原酶抑制剂的抑制机理, 选择了31个黄酮类化合物作为训练集, 使用Catalyst软件包构建了此类抑制剂的药效团模型. 并专门针对黄酮类化合物定制了氢键给体和受体模型, 效果优于使用Catalyst内预定义的模型. 最终的药效团模型由两个氢键给体和一个氢键受体组成, 对训练集具有较好预测能力(Correl=0.9013). 此外, 使用InsightII/Affinity对6个黄酮类化合物进行了分子对接研究. 综合药效团模型和分子对接研究的结果, 发现黄酮类化合物的抑制活性主要源于黄酮骨架上的C4’或C3’位的羟基与醛糖还原酶活性口袋中的TYR48、VAL47、GLN49和C7位的羟基与HIS110, TRP111所形成的两组氢键.     

3D-pharmacophores of flavonoid binding at the benzodiazepine GABA(A) receptor site using 4D-QSAR analysis

4D-QSAR analysis was applied to a training set of 38 flavonoids where affinity constants, Ki, to the GABA(A) benzodiazepine receptor site, BzR, were determined. It was found that the -logKi values of the compounds are highly dependent on the size and electrostatics character of the substituents at the R(3') and R(6) positions of the flavonoid scaffold. Polar negative groups correctly embedded in the R(3') and/or R(6) substituents are predicted to increase -logKi values. A planar conformation of the flavonoid scaffold was found not to be a requirement for the flavonoids to be active. A test set of four compounds was used to evaluate the predictivity of the 4D-QSAR models.     

Androgen receptor binding affinity of pesticide "active" formulation ingredients. QSAR evaluation by COREPA method

The COREPA approach for identifying the COmmon REactivity PAttern of biologically similar chemicals was employed to upgrade the recently derived affinity pattern for high androgen receptor (AR) binding affinity. The training set consisted of 28 steroidal and nonsteroidal ligands whose AR binding affinity was determined in competitive binding assays (in terms of p K i ). The interatomic distances between nucleophilic sites and their charges providing distinct and non-overlapping integral patterns for active and inactive chemicals were assumed that it was related with the endpoint, which was under study. These stereoelectronic characteristics were used to predict p K i values of pesticide "active" formulation ingredients in an attempt to identify chemicals with potential AR binding affinity.     

Synthesis,anticancer evaluation,and docking studies of some novel azo chromene derivatives

We have described a simple, convenient, and high-yielding one-pot synthesis of novel azo chromene derivatives via a three-component reaction of various azo aldehydes with dimedone and malononitrile using 10 mol% of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as catalyst and ethanol as solvent at reflux condition. All the synthesized compounds have been characterized using Fourier-transform infrared spectroscopy (FT-IR), ¹H NMR, ¹³C NMR, and HR-MS spectra and molecular docking was performed to explore new inhibitors of human placental aromatase cytochrome P450 and cyclooxygenase-2 enzymes. Of all the compounds docked, compound (E)-2-amino-4-(4,4-dimethyl-2,6-dioxocyclohexyl)-6-((3-methoxyphenyl)diazenyl-4H-chromene-3-carbonitrile ( 4o ) showed good binding affinity with the active site of human placental aromatase cytochrome P450 enzyme (PDB: 3EQM) with inhibition constant (Ki) 1.66 nM and compound 4o also showed good binding affinity with the active site of cyclooxygenase-2 enzyme (PDB: 6COX) with inhibition constant (Ki) 367.17 pM. In vitro anti-cancer activity studies against MCF-7 cells were also performed for compounds 4o , anastrozole and celecoxib. Compound 4o showed an effective cytotoxicity at 19.8 μg/ml compared to anastrozole and celecoxib (24.7 and 26.2 μg/ml).     

Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight

Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA–FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations.     

Computational design of protein–ligand binding: Modifying the specificity of asparaginyl‐tRNA synthetase

A method for computational design of protein–ligand interactions is implemented and tested on the asparaginyl‐ and aspartyl‐tRNA synthetase enzymes (AsnRS, AspRS). The substrate specificity of these enzymes is crucial for the accurate translation of the genetic code. The method relies on a molecular mechanics energy function and a simple, continuum electrostatic, implicit solvent model. As test calculations, we first compute AspRS‐substrate binding free energy changes due to nine point mutations, for which experimental data are available; we also perform large‐scale redesign of the entire active site of each enzyme (40 amino acids) and compare to experimental sequences. We then apply the method to engineer an increased binding of aspartyl‐adenylate (AspAMP) into AsnRS. Mutants are obtained using several directed evolution protocols, where four or five amino acid positions in the active site are randomized. Promising mutants are subjected to molecular dynamics simulations; Poisson‐Boltzmann calculations provide an estimate of the corresponding, AspAMP, binding free energy changes, relative to the native AsnRS. Several of the mutants are predicted to have an inverted binding specificity, preferring to bind AspAMP rather than the natural substrate, AsnAMP. The computed binding affinities are significantly weaker than the native, AsnRS:AsnAMP affinity, and in most cases, the active site structure is significantly changed, compared to the native complex. This almost certainly precludes catalytic activity. One of the designed sequences has a higher affinity and more native‐like structure and may represent a valid candidate for Asp activity. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.     

The Antagonist pGlu-βGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor

After we identified pGlu-βGlu-Pro-NH₂ as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH’s binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-βGlu-Pro-NH₂. The allosteric binding of this TRH antagonist is more robust than TRH’s binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.     

Identification of New Lead Molecules Against UBE2NL Enzyme for Cancer Therapy

Cancer is characterized by abnormal growth of cells. Targeting ubiquitin proteins in the discovery of new anticancer therapeutics is an attractive strategy. The present study uses the structure-based drug discovery methods to identify new lead structures, which are selective to the putative ubiquitin-conjugating enzyme E2N-like (UBE2NL). The 3D structure of the UBE2NL was evaluated using homology modeling techniques. The model was validated using standard in silico methods. The hydrophobic pocket of UBE2NL that aids in binding with its natural receptor ubiquitin-conjugating enzyme E2 variant (UBE2V) was identified through protein-protein docking study. The binding site region of the UBE2NL was identified using active site prediction tools. The binding site of UBE2NL which is responsible for cancer cell progression is considered for docking study. Virtual screening study with the small molecular structural database was carried out against the active site of UBE2NL. The ligand molecules that have shown affinity towards UBE2NL were considered for ADME prediction studies. The ligand molecules that obey the Lipinski’s rule of five and Jorgensen’s rule of three pharmacokinetic properties like human oral absorption etc. are prioritized. The resultant ligand molecules can be considered for the development of potent UBE2NL enzyme inhibitors for cancer therapy.     

Synthese und Antitumoraktivität voncis-Dichloroplatin(II)-Komplexen mit Östradiolderivaten

Summary Estrogen-receptor binding moieties were introduced into Pt(II) complexes in order to facilitate the selective transport into cancer cells. Estradiol esters of 2,3-diaminopropionic acid and estradiol ethers of 1,2-diamino-2-methyl-3-(p-hydroxyphenyl)propane were attached to Pt(II) complexes. The antitumor activity of the compounds was tested towards the human mammary carcinoma cell lines MDA-MB 231 and MCF-7, respectively, and the estrogen-receptor binding affinity of the Pt(II) complexes was determined. The steroidal Pt(II) complexes gave a maximum growth inhibition of 80% and a maximum estrogen-receptor binding affinity of 5.18.

     

Highlights on Steroidal Arylidene Derivatives as a Source of Pharmacologically Active Compounds: A Review

Steroids constitute a unique class of chemical compounds, playing an important role in physiopathological processes, and have high pharmacological interest. Additionally, steroids have been associated with a relatively low toxicity and high bioavailability. Nowadays, multiple steroidal derivatives are clinically available for the treatment of numerous diseases. Moreover, different structural modifications on their skeleton have been explored, aiming to develop compounds with new and improved pharmacological properties. Thus, steroidal arylidene derivatives emerged as a relevant example of these modifications. This family of compounds has been mainly described as 17β-hydroxysteroid dehydrogenase type 1 and aromatase inhibitors, as well as neuroprotective and anticancer agents. Besides, due to their straightforward preparation and intrinsic chemical reactivity, steroidal arylidene derivatives are important synthetic intermediates for the preparation of other compounds, particularly bearing heterocyclic systems. In fact, starting from arylidenesteroids, it was possible to develop bioactive steroidal pyrazolines, pyrazoles, pyrimidines, pyridines, spiro-pyrrolidines, amongst others. Most of these products have also been studied as anti-inflammatory and anticancer agents, as well as 5α-reductase and aromatase inhibitors. This work aims to provide a comprehensive overview of steroidal arylidene derivatives described in the literature, highlighting their bioactivities and importance as synthetic intermediates for other pharmacologically active compounds.     

Enzyme Selectivity Fine‐Tuned through Dynamic Control of a Loop

Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand‐selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations.     

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

Summary This study sheds new light on the role of acidic residues present in the active site cavity of human aromatase. Eight acidic residues (E129, D222, E245, E302, D309, E379, D380 and D476) lining the cavity are identified and studied using comparative modeling, docking, molecular dynamics as well as statistical techniques. The structural environment of these acidic residues is studied to assess the stability of the corresponding carboxylate anions. Results indicate that the environment of the residues E245, E302 and D222 is most suitable for carboxylate ion formation in the uncomplexed form. However, the stability of D309, D222 and D476 anions is seen to increase on complexation to steroidal substrates. In particular, the interaction between D309 and T310, which assists proton transfer, is found to be formed following androgen/nor-androgen complexation. The residue D309 is found to be clamped in the presence of substrate which is not observed in the case of the other residues although they exhibit changes in properties following substrate binding. Information entropic analysis indicates that the residues D309, D222 and D476 have more conformational flexibility compared to E302 and E245 prior to substrate binding. Interaction similar to that between D476 and D309, which is expected to assist androgen aromatization, is proposed between E302 and E245. The inhibition of aromatase activity by 4-hydroxy androstenedione (formestane) is attributed to a critical hydrogen bond formation between the hydroxy moiety and T310/D309 as well as the large distance from D476. The results corroborate well with earlier site directed mutagenesis studies.     

Determination of isoflavones in soy and selected foods containing soy by extraction, saponification, and liquid chromatography: collaborative study.

Isoflavones are biologically active compounds occurring naturally in a variety of plants, with relatively high levels found in soybeans. Twelve laboratories participated in a collaborative study to determine the aglycon isoflavone content of 8 test samples of soy and foods containing soy. The analytical method for the determination of isoflavones incorporates a mild saponification step that reduces the number of analytes measured and permits quantitation versus commercially available, stable reference standards. Test samples were extracted at 65 degrees C with methanol-water (80 + 20), saponified with dilute sodium hydroxide solution, and analyzed by reversed-phase liquid chromatography with UV detection at 260 nm. Isoflavone results were reported as microg/aglycon/g or microg aglycon equivalents/g. The 8 test samples included 2 blind duplicates and 4 single test samples with total isoflavone concentrations ranging from approximately 50 to 3000 microg/g. Test samples of soy ingredients and products made with soy were distributed to collaborators with appropriate reference standards. Collaborators were asked to analyze test samples in duplicate on 2 separate days. The data were analyzed for individual isoflavone components, subtotals of daidzin-daidzein, glycitin-glycitein, and genistin-genistein, and total isoflavones. The relative standard deviation (RSD) for repeatability was 1.8-7.1%, and the RSD for reproducibility was 3.2-16.1% for total isoflavone values of 47-3099 microg/g.     

Selective steroid recognition by a partially bridged resorcin[4]arene cavitand

The partially bridged resorcin[4]arene cavitand featuring a cleft-shaped recognition site formed by two anti-quinoxaline bridges and four convergent HO-groups was prepared in three steps and characterised by X-ray crystallography; cavitand was found to be a selective receptor for steroidal substrates in CDCl3, with the best binding observed for steroids with a flat A-ring and two H-bonding sites on rings A and C/D.     

A negative cooperativity mechanism of human CYP2E1 inferred from molecular dynamics simulations and free energy calculations

Human cytochrome P450 2E1 (CYP2E1) participates in the metabolism of over 2% of all the oral drugs. A hallmark peculiar feature of this enzyme is that it exhibits a pronounced negative cooperativity in substrate binding. However the mechanism by which the negative cooperativity occurs is unclear. Here, we performed molecular dynamics simulations and free energy calculations on human CYP2E1 to examine the structural differences between the substrate-free and the enzymes with one and two aniline molecules bound. Our results indicate that although the effector substrate does not bind in the active site cavity, it still can directly interact with the active site residues of human CYP2E1. The interaction of the effector substrate with the active site leads to a reorientation of active site residues, which thereby weakens the interactions of the active substrate with this site. We also identify a conserved residue T303 that plays a crucial role in the negative cooperative binding on the short-range effects. This residue is a key factor in the positioning of substrates and in proton delivery to the active site. Additionally, a long-range effect of the effector substrate is identified in which F478 is proposed to play a key role. As located in the interface between the active and effector sites, this residue structurally links the active and effector sites and is found to play a significant role in affecting substrate access and ligand positioning within the active site. In the negative cooperative binding, this residue can decrease the interactions of the active substrate with the active site by π-π stacking which then lowers the hydroxylation activity for the active substrate. These findings are in agreement with previous experimental observations and thus provide detailed atomistic insight into the poorly understood mechanism of the negative cooperativity in human CYP2E1.     

Site-selective metal binding by designed alpha-helical peptides

It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)4G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of 113Cd, 1H NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.