Protein–protein recognition: a computational mutagenesis study of the MDM2–P53 complex

Protein P53 is involved in more than 50% of the human cancers and the P53–MDM2 complex is a target for anticancer drug design. It is possible to engineer small P53 mimics that would be expected to disrupt the P53–MDM2 complex, and release P53 to initiate cell-cycle arrest or apoptosis. These small peptides should bind to the functional epitopes of the protein–protein interface, and prevent the interaction between P53 and MDM2. Here, we apply an improved computational alanine scanning mutagenesis method, which allows the determination of the hot spots present in both monomers, P53 and MDM2, of three protein complexes (the P53-binding domain of human MDM2, its analogue from Xenopus laevis, and the structure of human MDM2 in complex with an optimized P53 peptide). The importance of the hydrogen bonds formed by the protein backbone has been neglected due to the difficulty of measuring experimentally their contribution to the binding free energy. In this study we present a computational approach that allows the estimation of the contribution to the binding free energy of the C=O and N–H groups in the backbone of the P53 and MDM2 proteins. We have noticed that the hydrogen bond between the HE1 atom of the hot spot Trp23 and the O atom of the residue Leu54, as well as the NH-pi hydrogen bond between the Ile57 and Met58 were observed in the Molecular dynamics simulation, and their contribution to the binding free energy measured. This study not only shows the reliability of the computational mutagenesis method to detect hot spots but also demonstrates an excellent correlation between the quantitative calculated binding free energy contribution of the C=O and N–H backbone groups of the interfacial residues and the qualitative values expected for this kind of interaction. The study also increases our understanding of the P53–MDM2 interaction.     

Forces mediating protein–protein interactions: a computational study of p53 “approaching” MDM2

The protein MDM2 forms a complex with the tumor suppressing protein p53 and targets it for proteolysis in order to down-regulate p53 in normal cells. Inhibition of this interaction is of therapeutic importance. Molecular dynamics simulations of the association between p53 and MDM2 have revealed mutual modulation of the two surfaces. Analysis of the simulations of the two species approaching each other in solution shows how long range electrostatics steers these two proteins together. The net electrostatics is controlled largely by a few cationic residues that surround the MDM2 binding site. There is an overall separation in electrostatics of MDM2 and p53 that are mutually complementary and drive association. Upon close approach, there is significant energetic strain as the charges are occluded from water (desolvated). However, the complexation is driven by packing interactions that lead to highly favorable van der Waals interactions. Although the complementarity of the electrostatics of the two surfaces is essential for the two partners to form a complex, steric collisions of Y100 and short ranged van der Waals interactions of F19, W23, L26 of p53 determine the final steps of native complex formation. The electrostatics seem to be evolutionarily conserved, including variations in both partners.     

Transient structural disorder as a facilitator of protein-ligand binding: Native H/D exchange—Mass spectrometry study of cellular retinoic acid binding protein I

Binding of all-trans Retinoic Acid (RA) to Cellular Retinoic Acid Binding Protein I (CRABP I) does not result in significant changes of the protein tertiary structure, even though the binding site is inaccessible in a static apo-protein conformation. One of the proposed scenarios for the protein-ligand binding process invokes the notion of a flexible portal region adjacent to the binding site, while another model suggests that the requisite dynamic events are induced by dimerization of the apo-protein in solution. In this work, RA binding to CRABP I is studied in dilute solutions (low micro-molar range), where no dimer and/or oligomer formation occurs. Modulation of backbone dynamics within various segments of the protein by its ligand is assessed using a combination of hydrogen exchange, electrospray ionization mass spectrometry, and collision-induced dissociation of protein ions in the gas phase. Consistent with the portal model of ligand entry, several protein segments (most of them containing residues making hydrophobic contacts to RA in the holo-form of the protein) are flexible in the absence of the ligand. At the same time, the two segments containing arginine residues forming a salt bridge with RA form the least flexible region in the apo-form of the protein. Although the presence of RA in solution reduces flexibility of all protein segments, the largest effect is observed within four strands that form one of the two beta-sheets enveloping a cavity which houses the ligand-binding site. These results are consistent with a model in which ligand binding occurs through a partially unstructured state of the protein with unobstructed access to the ligand-binding site. This intermediate (whose core is formed by the two stable arginine-containing strands) corresponds to a relatively low-energy local minimum on the apo-protein energy surface and is frequently sampled under native conditions.     

Biomacromolecular quantitative structure–activity relationship (BioQSAR): a proof-of-concept study on the modeling, prediction and interpretation of protein–protein binding affinity

Quantitative structure–activity relationship (QSAR), a regression modeling methodology that establishes statistical correlation between structure feature and apparent behavior for a series of congeneric molecules quantitatively, has been widely used to evaluate the activity, toxicity and property of various small-molecule compounds such as drugs, toxicants and surfactants. However, it is surprising to see that such useful technique has only very limited applications to biomacromolecules, albeit the solved 3D atom-resolution structures of proteins, nucleic acids and their complexes have accumulated rapidly in past decades. Here, we present a proof-of-concept paradigm for the modeling, prediction and interpretation of the binding affinity of 144 sequence-nonredundant, structure-available and affinity-known protein complexes (Kastritis et al. Protein Sci 20:482–491, 2011) using a biomacromolecular QSAR (BioQSAR) scheme. We demonstrate that the modeling performance and predictive power of BioQSAR are comparable to or even better than that of traditional knowledge-based strategies, mechanism-type methods and empirical scoring algorithms, while BioQSAR possesses certain additional features compared to the traditional methods, such as adaptability, interpretability, deep-validation and high-efficiency. The BioQSAR scheme could be readily modified to infer the biological behavior and functions of other biomacromolecules, if their X-ray crystal structures, NMR conformation assemblies or computationally modeled structures are available.     

A validated LC–MS/MS method for the quantification of capivasertib in dog plasma: Application to its pharmacokinetics study

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated using acetonitrile. An Acquity BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) maintained at 40°C was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. Multiple reaction monitoring transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. Excellent linearity was achieved in the concentration range of 1–1,000 ng/ml with a correlation coefficient of >0.9981. The lower limit of quantification was 1 ng/ml. The extraction recovery of capivasertib from dog plasma was >85.81% and no significant matrix effect was found. The intra- and inter-day precision was <9.58% and the accuracy ranged from −10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.     

One-pot synthesis of Claisen–Schmidt reaction through (E)-chalcone derivatives: Spectral studies in human serum albumin protein binding and molecular docking investigation

An efficient and environmentally benign one-pot multicomponent synthesis of E-chalcones was developed using a mild and reusable new boron nitride-sulphonic acid catalyst. The catalyst was prepared by activating the boron nitride surface with nitric acid, followed by a simple reaction with 3-mercaptopropyl trimethoxysilane. The catalyst was characterized and morphological properties were studied by Fourier transform infrared, X-ray diffraction, transmission electron spectroscopy, scanning electron microscopy, Brunauer–Emmett–Teller theory, and Raman spectroscopy techniques. The solid acid catalyst was recycled five times in a Claisen–Schmidt reaction to synthesize new chalcone derivatives, and X-ray crystallography was used to elucidate the structure of (E)-1-(anthracen-9-yl)-3-(2-(4-methylpiperazin-1-yl)quinolin-3-yl)prop-2-en-1-one. A fluorescence quench titration method was used to assess its binding ability with human serum albumin (HSA), while molecular docking was also performed to get a more detailed insight into their interaction at the binding site of HSA.     

Selective quantification of lacosamide in human plasma using UPLC–MS/MS: Application to pharmacokinetic study in healthy subjects with different doses

A practical, sensitive, and robust UPLC–MS/MS method was developed and validated to quantify lacosamide in human plasma. A simple one-step protein precipitation was used to extract lacosamide and labeled lacosamide-13C, D3 as an internal standard (IS) from 150-μL plasma. The extracts were analyzed on an Eclipse Plus C18 column (50 × 2.1 mm, 1.8 μm) using 0.1% formic acid in water and methanol:acetonitrile (50:50, v/v) under gradient conditions. The extracts were quantified on LCMS-8040 using electrospray ionization source operated in positive ionization and multiple reaction monitoring modes. The method showed good linearity from 0.02 to 20 μg/mL, which was adequate to cover lacosamide concentration assayed in formulations with different strengths. The bioanalytical assay was fully validated as per current regulatory guidelines. The intra-batch and inter-batch precision values of lacosamide were less than 4.6%. Lacosamide was found to be stable at different storage conditions. The extraction recoveries and IS-normalized matrix factors for lacosamide ranged from 97.17 to 99.68% and from 0.973 to 1.012, respectively. The validated method was successfully applied to a pharmacokinetic study with three lacosamide formulations (50, 100, and 200 mg) in 36 healthy subjects. The assay reliability was determined by reanalysis of 81 subject samples.     

D3R Grand Challenge 2: blind prediction of protein–ligand poses,affinity rankings,and relative binding free energies

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank (http://www.pdb.org), and in affinity ranking and scoring of bound ligands.     

Raman study of the variation in anatase structure of TiO<Subscript>2</Subscript> nanopowders due to the changes of sol–gel synthesis conditions

TiO₂ nanopowders were produced by sol–gel technique under different synthesis conditions. XRD results have shown that obtained nanopowders are in anatase phase, with the presence of a small amount of highly disordered brookite phase, whereas nanocrystallite size and amount of brookite slightly depend on sol–gel synthesis conditions. Raman measurements confirm these results. The analyses of the shift and width of the most intensive anatase E _g Raman mode by phonon confinement model suggest that anatase crystallite size should be in the range between 11 and 15 nm, what is in excellent correlation with XRD results. Obtained results have shown that Raman spectroscopy is a highly sensitive method for the estimation of anatase crystallite size as well as brookite content in TiO₂ nanopowders synthesized by variable sol–gel synthesis conditions.     

Neutral C–H bond vs. electron pair of N(sp~2): A binding site effect study of macrocycle anion receptor

To evaluate the effect of neutral C–H bond or electron pair of nitrogen atom with sp2hybridization(N(sp2)) involving into the same chemical environment for anion binding, two analogous tetracationic imidazolium macrocycles, namely cyclo[2](2,6-bis-(1H-imidazol-1-yl)pyridine) [2](1,3-dimethylenebenzene)(14+), and cyclo[2](2,6-bis-(1H-imidazol-1-yl)pyridine)[2](2,6-di methylenepyridine)(24+)were studied in detail as small inorganic anion receptors. The guest anions with different shapes are Cl,N3, NO3, and H2PO4. The host–guest interactions were characterized via1 H NMR spectroscopy,electrospray ionization mass spectrometry(ESI-MS) and single crystal X-ray crystallography. The results implied that macrocyclic hosts with similar backbone but two distinct binding sites(14+with neutral C–H vs. 24+with N(sp2)) vary markedly in their response to anions, including the binding modes and association constants. The finding will serve to the construction of new anion receptors, even improve insights into the anion binding process in biology.     

A validated LC–MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study

A highly sensitive LC–MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid–liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25–500 ng/ml for SV (r² ≥ 0.9923) and 0.24–481.23 ng/ml for SVA (r² ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.     

A rapid UHPLC–MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study

A simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated for the determination of ARQ531, a Bruton’s tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C₁₈ column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5–1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from −13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1–10 mg/kg and good oral bioavailability (>50%).     

Adsorption/desorption of acid violet-7 onto magnetic MnO2 prior to its quantification by UV–visible spectroscopy: optimized by fractional factorial design

Research on Chemical Intermediates - A highly efficient magnetic adsorbent is presented to preconcentrate acid violet-7 (AV-7) prior quantifying by UV–visible spectroscopy...     

Insights into the binding mechanism of 2,5-substituted 4-pyrone derivatives as therapeutic agents for fused dimeric interactions: A computational study using QTAIM,dynamics and docking simulations of protein–ligand complexes

The study is focused on examining 2,5-Substituted 4-Pyrone based compounds through quantum chemical and topological analysis techniques, evaluating the properties of these compounds, including their geometrical structure, intermolecular interactions and assess their possible applications. Additionally, the molecular stability, charge delocalization and UV-Visible data was investigated and compared with the calculated energy and oscillator strength using the TD-DFT approach. The researchers observed that charge transfer occurred within the molecule, indicated by the HOMO and LUMO energies. It was also found that the compound exhibited planarity and higher chemical reactivity. The calculated Mulliken charges and molecular electrostatic potential were used to interpret the Fukui index data that help predict reactive sites and understand the reactivity patterns of specific atoms in a compound. The study is aimed to understand the role of NCI in the molecule under investigation using electron localization functions and localized orbit locator methods. Molecular docking and ADMET studies were conducting involving a detailed MD simulation of a protein-ligand complex using the OPLS3e force field and the SPC water model. These findings could prove to be beneficial in developing new therapeutic agents with various pharmacological effects and potential toxicities.     

General access to polyhydroxylated nortropane derivatives through hetero Diels–Alder cycloadditions. Part 3: Synthesis of natural (+)-calystegine B2

Cycloaddition of chiral nitroso derivatives with cyclohepta-1,3-diene gave one single stereoisomer with an excellent selectivity. The structures including absolute configurations have been assigned by spectroscopy and X-ray crystallography. These studies have been applied to the total synthesis of the naturally occurring calystegine B₂.     

A novel UPLC–MS/MS method for simultaneous quantification of trigonelline, 4-hydroxyisoleucine,and diosgenin from Trigonella foenum-graecum extract: Application to pharmacokinetic study in healthy and type 2 diabetic rats

Trigonelline (TR), 4-hydroxyisoleucine (4-HI), and diosgenin (DG) are the main bioactives of the purified standardized extract of the popular plant Trigonella foenum-graecum L. (TFG), and it has been proven effective for the treatment of various diseases. However, to the best of our knowledge, no study has investigated the pharmacokinetic parameters of purified standardized T. foenum-graecum extract in normal and diabetic Wistar rats. The present study has developed and validated a rapid, reliable, and sensitive simultaneous ultra-performance liquid chromatography MS method to estimate these bioactives. The chromatographic separation was achieved using methanol, acetonitrile, and 0.1% formic acid with the ideal gradient flow system on a BEH Shield RP 18 column. A positive electrospray ionization mode was selected to estimate m/z values of TR (138.14 > 94.63), 4-HI (148.19 > 74.08), and DG (415.54 > 271.33). The method was robust and reproducible over the linearity range of 60–5000, 6–5000, and 15–5000 ng/mL for TR, 4-HI, and DG, respectively. Using this novel validated method, we investigated the pharmacokinetic parameters of bioactives using Phoenix WinNonlin version 8.0 (Certera) in normal and diabetic rats. The assay was successfully applied for the estimation of pharmacokinetic parameters using noncompartmental analysis. This investigation shows that the absorption rate increased, whereas distribution and elimination processes slowed down in diabetic rats compared with normal rats.     

Theoretical study of the thermodynamic parameters of (CaO)n nanoclusters with n = 2–16 in the gas and solution phases: proton affinity,molecular basicity,and pKb values

Structural Chemistry - Thermodynamic quantities such as proton affinity (PA) and molecular basicity (GB) for (CaO)n nanoclusters with n?=?2–16 have been calculated using three...     

Multiple 3D- and 2D-quantitative structure–activity relationship models (QSAR), theoretical study and molecular modeling to identify structural requirements of imidazopyridine analogues as anti-infective agents against tuberculosis

Structural Chemistry - Tuberculosis (TB), an infectious remains a global health burden till date. Considering immense importance of theoretical tools in computer aided-drug designing, the current...