Conformational Profile Research Articles

Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).

Functionalized fullerene cations {R–C60}+ from a theoretical point of view

The first comprehensive theoretical investigation of a series of functionalized fullerene cations, {R-C(60)}(+), where R = H, CH(3), CH(2)Cl, CHCl(2), and CCl(3), is accomplished. A detailed consideration of conformational energetic profiles of these systems resulted in the structures of most stable conformers that fit all available experimental data. The trends in stability of {R-C(60)}(+) cations as well as transition barriers for migration of alkyl groups over the C(60)-surface are provided. A thorough study of energetics of {R-C(60)}(+) is augmented by in-depth investigation of their electronic structures and aromaticity. As the final part of this study, the detailed comparison of structures, energetics and properties of {R-C(60)}(+) with those of corannulene cations, {R-hub-C(20)H(10)}(+), is performed at the same level of theory. The similarities and differences between the surface functionalized ball- and bowl-shaped carbocations are illuminated.

Conformational flexibility and binding energy profile of c-Abl tyrosine kinase complexed with Imatinib: an insight from MD study

Structural biology of kinase and in particular of tyrosine kinase has given detailed insights into the intrinsic flexibility of the catalytic domain and has provided a rational basis for obtaining selective inhibitors. In this paper, we have studied the conformational flexibility of c-Abl tyrosine kinase complexed with Imatinib (STI), in the presence of TIP3P water in physiological conditions at neutral pH. The conformational studies suggest that the flexibility of activation loop is responsible to facilitate the nucleotide binding and release. Owing to the conformational adaptability, adenosine triphosphate (ATP) binds at a particular site in the loop region of the tyrosine kinase. The molecular mechanics Poisson–Boltzmann surface area methods are analysed, as is a free-energy pathways method, which shows the stable binding with free energy − 6.04 kcal/mol for STI. The binding energy calculated by the Sietraj method is approximately the same as the experimental binding energy of STI with c-Abl kinase. It is suggested that the conserved glutamic acid and lysine residues are necessary for the stability and optimum activity of inhibitor. This study may be helpful in rational drug designing of new kinase inhibitors.

Biophysical Characterization and Conformational Stability of Ebola and Marburg Virus-Like Particles

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.

4D-LQTA-QSAR and docking study on potent gram-negative specific LpxC inhibitors: a comparison to CoMFA modeling

A quasi 4D-QSAR has been carried out on a series of potent Gram-negative LpxC inhibitors. This approach makes use of the molecular dynamics (MD) trajectories and topology information retrieved from the GROMACS package. This new methodology is based on the generation of a conformational ensemble profile, CEP, for each compound instead of only one conformation, followed by the calculation intermolecular interaction energies at each grid point considering probes and all aligned conformations resulting from MD simulations. These interaction energies are independent variables employed in a QSAR analysis. The comparison of the proposed methodology to comparative molecular field analysis (CoMFA) formalism was performed. This methodology explores jointly the main features of CoMFA and 4D-QSAR models. Step-wise multiple linear regression was used for the selection of the most informative variables. After variable selection, multiple linear regression (MLR) and partial least squares (PLS) methods used for building the regression models. Leave-N-out cross-validation (LNO), and Y-randomization were performed in order to confirm the robustness of the model in addition to analysis of the independent test set. Best models provided the following statistics: [Formula in text] (PLS) and [Formula in text] (MLR). Docking study was applied to investigate the major interactions in protein-ligand complex with CDOCKER algorithm. Visualization of the descriptors of the best model helps us to interpret the model from the chemical point of view, supporting the applicability of this new approach in rational drug design.

Proposed Bioactive Conformations of Opiorphin, an Endogenous Dual APN/NEP Inhibitor.

The conformational profiles for the endogenous peptide Opiorphin and a set of seven analogues exhibiting different inhibitory activities toward human aminopeptidase N (hAPN) and human neprilysin (hNEP) were independently computed to deduce a bioactive conformation that Opiorphin may adopt when binding these two enzymes. The conformational space was thoroughly sampled using an iterative simulated annealing protocol, and a library of low-energy conformers was generated for each peptide. Bioactive Opiorphin conformations fitting our experimental structure-activity relationship data were identified for hAPN and hNEP using computational pairwise comparisons between each of the unique low-energy conformations of Opiorphin and its analogues. The obtained results provide a structural explanation for the dual hAPN and hNEP inhibitory activity of Opiorphin and show that the inborn flexibility of Opiorphin is essential for its analgesic activity.

FDTD modeling to enhance the performance of an organic solar cell embedded with gold nanoparticles

Optical enhancement is demonstrated in a bilayer P3HT-C60 solar cell by embedding gold nanoparticles directly into the P3HT layer of the photovoltaic device. FDTD simulations are used to model the observed performance gain. A qualitative agreement between the experimental and numerical results is achieved. This validates the numerical model and the simulation is subsequently extended to predict the performance gain of the bilayer device constructed with thinner P3HT layer. The numerical results reveal that the plasmonic structure has even larger effect on such thinner bilayer device. The enhancement is expected to be most significant when the p-n interface is allowed to assume the conformal hemispherical profile of the metal particles.

Free Energy Profiles along Consensus Normal Modes Provide Insight into HIV-1 Protease Flap Opening.

Describing biological macromolecular energetics from computer simulations can pose major challenges, and often necessitates enhanced conformational sampling. We describe the calculation of conformational free-energy profiles along carefully chosen collective coordinates: "consensus" normal modes, developed recently as robust alternatives to conventional normal modes. In an application to the HIV-1 protease, we obtain efficient sampling of significant flap opening movements governing inhibitor binding from relatively short simulations, in close correspondence with experimental results.

Chemodiversity and Molecular Plasticity: Recognition Processes as Explored by Property Spaces

In the last few years, a need to account for molecular flexibility in drug-design methodologies has emerged, even if the dynamic behavior of molecular properties is seldom made explicit. For a flexible molecule, it is indeed possible to compute different values for a given conformation-dependent property and the ensemble of such values defines a property space that can be used to describe its molecular variability; a most representative case is the lipophilicity space. In this review, a number of applications of lipophilicity space and other property spaces are presented, showing that this concept can be fruitfully exploited: to investigate the constraints exerted by media of different levels of structural organization, to examine processes of molecular recognition and binding at an atomic level, to derive informative descriptors to be included in quantitative structure--activity relationships and to analyze protein simulations extracting the relevant information. Much molecular information is neglected in the descriptors used by medicinal chemists, while the concept of property space can fill this gap by accounting for the often-disregarded dynamic behavior of both small ligands and biomacromolecules. Property space also introduces some innovative concepts such as molecular sensitivity and plasticity, which appear best suited to explore the ability of a molecule to adapt itself to the environment variously modulating its property and conformational profiles. Globally, such concepts can enhance our understanding of biological phenomena providing fruitful descriptors in drug-design and pharmaceutical sciences.

A computational study of Neuromedin B

A methodological study has been undertaken to assess the conformational profile of Neuromedin B (NMB) using classical molecular dynamics simulations (MD) and replica exchange molecular dynamics (REMD) simulations under generalized Born (GB) solvent conditions. Comparison of the results obtained from these simulations suggests that the peptide has a propensity to adopt both β-turns and α-helical conformations regardless of the simulation protocols used. However, the conformations adopted more helical character under REMD conditions and showed good agreement with the NMR supported structure reported in the literature.

Conformal profile theory for performance scaling of ultrabroadband optical parametric chirped pulse amplification

We developed a theory of optimization of amplifier bandwidth and conversion efficiency in ultrabroadband optical parametric chirped pulse amplification based on the principle of making the optimal conversion length uniform in time through pulse shaping. Conformal profiles, ideal combinations of pump intensity, seed intensity, and wave-vector mismatch temporal profiles can be found analytically by use of the exact Jacobi elliptic function solutions for signal gain and pump depletion in optical parametric amplification. We derive and investigate these gain equations for the general purpose of aiding the design of optical parametric amplifiers. Using these analytics, through an extensive analysis of conformal profile methods, we find conformal profile pulse shaping can significantly boost amplifier bandwidth as well as conversion efficiency, allowing in some amplifiers a severalfold increase in obtainable peak power. Pump pulse shaping is found to be particularly effective and flexible.

Extended and Clustered Conformers of Epothilone A

The conformational properties of epothilone A have been analyzed in detail using electronic structure calculations to better understand the effect of intramolecular hydrogen bonding on the conformational energies of this highly potent anticancer molecule. Single-point second-order Møller-Plesset calculations done in vacuo at the MP2/6-31+G(d,p)//B3LYP/6-31+G(d,p) level yielded data on the relative stability of conformers that were more distinct than data obtained from the standard DFT model, although the structural trends are in fair agreement. We studied torsional profiles of both hydroxyl groups and sampled energies of the side chain and thiazole moiety rotamers within the whole set of all experimentally accessible conformers. The aldol hydrogen bonds, though relatively weak, generally contribute to the conformational profile, while dipole-dipole interactions, ester group puckering, transannular repulsions between hydrogen atoms, steric effects, and syn-pentane effects have a limited influence. A salient result of our calculations is the determination that the energy of the clustered exo conformer P01 lays 9.3 kcal/mol below that of the extended, experimental conformer P11, apparently due to the unconstrained, near linear 3-OH hydrogen bond to thiazole. Another finding to be noted is the corroboration of the remarkable ability of 3-OH to form transannular hydrogen bonding with the epoxide, which releases the conformational strain of the macroring and thus leads to extra stabilization energy within the endoW subset. Finally, we found that the general trend of the conformer populations of epothilone A obtained from conformational energies resembles those derived from experiments and can be used to interpret values of NMR vicinal coupling constants. The calculated geometries and energies provide essential data for further discussion of the mechanism of biological activity of epothilone A and might be of importance in the explanation of its ADME properties.

Conformational profile and vibrational assignments of hippuric and 4-aminohippuric acids

The structural stability of hippuric acid and 4-aminohippuric acid was investigated for comparison purpose by the DFT-B3LYP and ab initio MP2 calculations with the 6-311G ** basis set. From the calculations at both levels of theory the near-planar Ctcnp structure was predicted to be the lowest energy minimum for the two acids. The stability of the corresponding planar structure ( Ctcp) was predicted to significantly decrease (6 kcal/mol) as going from the parent acid to the 4-amino derivative. The Tgcnp structure was predicted to be about 1 kcal/mol higher in energy than the most stable Ctcnp structure in both acids. The NH 2 inversion barrier was estimated from the planar Ctcp structure to be about 4 kcal/mol. The zwitterion structure was predicted to be about 45 and 50 kcal/mol higher in energy than the ground state conformation in the two acids, respectively, as a result of strong conjugation between the N atom of the NH moiety and the carbonyl C O of the benzoyl group. The vibrational frequencies of the lowest energy Ctcnp conformer of hippuric and 4-aminohippuric acids were computed at the B3LYP/6-311G ** level of theory. A comparison of the vibrational assignments for the two acids was provided on the basis of combined experimental and theoretical data.

Computational Study of the Free Energy Landscape of the Miniprotein CLN025 in Explicit and Implicit Solvent

The prediction capabilities of atomistic simulations of peptides are hampered by different difficulties, including the reliability of force fields, the treatment of the solvent or the adequate sampling of the conformational space. In this work, we have studied the conformational profile of the 10 residue miniprotein CLN025 known to exhibit a β-hairpin in its native state to understand the limitations of implicit methods to describe solvent effects and how these may be compensated by using different force fields. For this purpose, we carried out a thorough sampling of the conformational space of CLN025 in explicit solvent using the replica exchange molecular dynamics method as a sampling technique and compared the results with simulations of the system modeled using the analytical linearized Poisson-Boltzmann (ALPB) method with three different AMBER force fields: parm94, parm96, and parm99SB. The results show the peptide to exhibit a funnel-like free energy landscape with two minima in explicit solvent. In contrast, the higher minimum nearly disappears from the energy surface when the system is studied with an implicit representation of the solvent. Moreover, the different force fields used in combination with the ALPB method do not describe the system in the same manner. The results of this work suggest that the balance between intra- and intermolecular interactions is the cause of the differences between implicit and explicit solvent simulations in this system, stressing the role of the environment to define properly the conformational profile of a peptide in solution.

Atomistic modeling of peptides bound to a chemically active surface: conformational implications

This work presents a computational strategy to model flexible molecules tethered to a metallic rigid surface. The method is based on a previously developed procedure for inert surfaces, in which peptide-surface interactions were not considered. This procedure is able to generate uncorrelated relaxed microstructures at the atomistic level of systems containing relatively high densities of peptides tethered to the surface. The reliability of the strategy has been tested by simulating CREKA (Cys-Arg-Glu-Lys-Ala), a short linear pentapeptide that recognizes clotted plasma proteins and selectively homes to tumors, covalently tethered to a gold surface, results being compared with those obtained when the surface was represented as inert. The results indicate that the whole conformational profile of CREKA presents some correlation with the chemical activity of the surface, even though the bioactive conformation was found as the most favored in all cases. Specifically, simulations reflect that consideration of the peptide-surface interactions affect the geometrical orientation of the side chains, whereas the main chain conformation does not undergo significant modifications.

Exploring the energy landscape of a molecular engineered analog of a tumor-homing peptide.

Recently a new non-coded amino acid was designed as a replacement for Arg, to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This constrained Arg analog, denoted c(5)Arg, was engineered to also promote the stability of the CREKA bioactive conformation. The conformational profile of the CREKA analog obtained by replacing Arg by c(5)Arg has been extensively investigated in this work. Two molecular dynamics simulations-based strategies have been employed: a modified simulated annealing and replica exchange. Results obtained using both techniques show that the conformational features of the new analog fulfill the purpose of its design. The new CREKA analog not only preserves the main structural attributes found for the bioactive conformation of the parent peptide but also shows lower flexibility. Moreover, the conformational profile of the mutated peptide narrows towards the most stable structures previously observed for the parent CREKA peptide.

DFT Study of conformational flexibilities and interaction profiles of new class of nucleoside analogs having nucleic acid bases pairs (Pyrimidine analogues-Adenine) linked through a 1,2,3-triazole spacer

DFT calculations predict the existence of three stable conformations (one opened and two closed conformations) of the new class of nucleoside with various substituents (X = H, CH 3 , F, Cl, and Br). The calculations predict also that the two closed conformation are stabilized by intramolecular hydrogen bond. The N 17 –H 34 stretching is large red shift by as mush 380–455 cm -1 due to the intramolecular N 17 –H 34 …N 8 hydrogen bonding. The red shift is accompanied by a large enhancement of the N17–H34 absorption intensity. It shows that these intramolecular hydrogen bonds are classical H–bonds.

Conformational profile of bombesin assessed using different computational protocols

The present work involves the study of the conformational profile of bombesin using different computational procedures used to explore the configurational space based on molecular dynamics simulations. Specifically, the present study describes the effect of using Berendsen's versus Langevin's thermostat and on the other hand, the use of the multicanonical replica exchange molecular dynamics as compared to standard molecular dynamics. In these simulations the solvent was modeled using the Onufriev, Bashford and Case implementation of Generalized Born procedure. The detailed computational analysis agrees well with the aggregated information previously reported in the NMR study of the peptide in a mixture of trifluoroethanol/water. Present results show a clear preference for the peptide to attain a helical structure on the segment 6–14, with a tendency to adopt a α-helix at the C-terminus aligning the aromatic residues Trp 8 and His 12 together with Gln 7, known to be important for peptide mediated activation. Finally, the three methodologies used in the present work yield similar structural results, although a detailed analysis reveals biases that need to be considered when performing this kind of studies.

Conformational profile of a proline-arginine hybrid.

The intrinsic conformational preferences of a new nonproteinogenic amino acid have been explored by computational methods. This tailored molecule, named ((β)Pro)Arg, is conceived as a replacement for arginine in bioactive peptides when the stabilization of folded turn-like conformations is required. The new residue features a proline skeleton that bears the guanidilated side chain of arginine at the C(β) position of the five-membered pyrrolidine ring, in either a cis or a trans orientation with respect to the carboxylic acid. The conformational profiles of the N-acetyl-N'-methylamide derivatives of the cis and trans isomers of ((β)Pro)Arg have been examined in the gas phase and in solution by B3LYP/6-31+G(d,p) calculations and molecular dynamics simulations. The main conformational features of both isomers represent a balance between geometric restrictions imposed by the five-membered pyrrolidine ring and the ability of the guanidilated side chain to interact with the backbone through hydrogen bonds. Thus, both cis- and trans-((β)Pro)Arg exhibit a preference for the α(L) conformation as a consequence of the interactions established between the guanidinium moiety and the main-chain amide groups.

Conformational behavior and potential energy profile of gaseous histidine

Histidine as a natural amino acid is found to be biologically important and is known to function as a nucleophile or enzyme co-factor, or in proton transfer process. The properties of gaseous aromatic amino acid histidine depend on the structural forms it may take in gas-phase. Ab initio method has been used to characterize the gas-phase conformer/tautomers of histidine. Wide range of possible structures for histidine was surveyed at the MM level, and then the geometries of the unique conformers were refined at the B3LYP/6-311++G (d,p) levels. At this theoretical level, 25 conformers were located for both tautomers of histidine i.e., His [N πH] and His [N τH]. The MM level provides a poor description of the relative energies. Calculations at the B3LYP/6-311++G (d,p) level represent a significant improvement. Ab initio dipole moments are reported for all the conformers/tautomers. The results are compared to previous studies of amino acids and are analyzed in terms of intramolecular hydrogen-bonding interactions and Newman projections. In addition, we calculated the infrared frequencies and intensities of the most stable structures in order to assist in the assignment of hydrogen-bonding. Furthermore, Ramachandran backbone potential energy surfaces (PES) of 25 conformers of His [N πH] and His [N τH] tautomers of histidine were considered. The stationary points characterizing the potential energy profile of the various conformers/tautomers of histidine were investigated by the same density functional theory B3LYP/6-311++G (d,p). Minima and transition states characterizing the energetic paths for the interconversion of various structures of histidine were explored in detail.