Structure-based design and subsequent optimization of 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) inhibitors of p38 MAP kinase.

A computer-aided drug design strategy leads to the identification of a new class of p38 inhibitors based on the 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) scaffold. The tolyl triazole amides provided a potent platform amenable to optimization. Further exploration leads to compounds with greater than 100-fold improvement in binding affinity to p38. Derivatives prepared to alter the physicochemical properties produced inhibitors with IC(50)'s in human whole blood as low as 83 nM.

Pyrazinoindolone inhibitors of MAPKAP-K2.

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

Oltipraz attenuates the progression of heart failure in rats through inhibiting oxidative stress and inflammatory response.

OBJECTIVE: To evaluate the effect of oltipraz (OPZ) on isoproterenol-induced heart failure (HF) and heart function. We also explore the underlying molecular mechanism of OPZ. MATERIALS AND METHODS: The rats were randomly divided into four groups, including normal control group, isoproterenol (ISO) group, ISO +100 mg/kg OPZ group, and OPZ group. Hemodynamic parameters, such as left-ventricular systolic pressure, were statistically analyzed. Besides, plasma levels of brain natriuretic peptide (BNP), pro-inflammatory cytokines and antioxidant markers were assessed by using enzyme-linked immunosorbent assay (ELISA). Moreover, histopathological examination was applied to assess the degree of cardiac interstitial fibrosis. RESULTS: OPZ could statistically improve the hemodynamic parameters of the heart function, and could also obviously attenuate cardiac interstitial fibrosis in ISO-induced HF rats when compared with the ISO group. Besides, plasma level of BNP in ISO +100 mg/kg OPZ group dramatically decreased in comparison with that of ISO group. Moreover, compared with ISO group, OPZ treatment significantly reduced the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Moreover, OPZ treatment remarkably increased the levels of antioxidant markers such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in ISO-induced HF rats. CONCLUSIONS: OPZ administration may provide experimental evidence for the possible effect of OPZ on isoproterenol-induced heart failure in rats. Moreover, OPZ administration may have potential utility for the treatment of heart failure.

Designing potential energy functions for protein folding.

By following a consistent line of physical reasoning, some fundamental understanding about the foldability of proteins has been achieved. In recent years, this has led to the development of a number of successful algorithms for optimizing potential energy functions for folding protein models. The differences between the folding mechanisms of simple, contact-based lattice proteins and more traditional, realistic protein models, however, still call for further development of the potentials in addition to the optimization approaches.

Active transport of ions across membranes: energetic role of electrostatics and binding site asymmetry.

The active transport of ions across a membrane by an ATP-driven electrogenic ion pump is often described by an 'alternate access' model. The position of the binding site is assumed to be unchanged as the binding cavity opens alternatively to the uptake and discharge sides of the membrane. The ion binding affinity is higher on the uptake side of the membrane than on the discharge side. This difference in affinities is related to the maximum transport rate and to the efficiency with which ATP hydrolysis is coupled to active transport. Here we examine the electrostatic contribution to binding affinities, using a simple geometry for a model membrane-protein system, a continuum dielectric approximation, and a numerical method to calculate binding energy as a function of the binding site location. If the binding site is located asymmetrically, being further from the uptake side of the membrane than from the discharge side, there is a significant difference in binding free energy between the uptake and discharge states. This asymmetry can produce differences in affinities that are consistent with those measured for biological active transport systems. These results may account for the observed asymmetric location of the calcium binding site in the calcium ATPases from sarcoplasmic reticulum and from the plasma membrane. Electrostatic energy differences associated with binding site asymmetry may be a general feature of electrogenic transmembrane ion pumps.

Molecular mechanisms for cooperative folding of proteins.

The folding of single-domain globular proteins exhibits the character of first-order or two-state thermodynamics. The origin of such high cooperativity in relatively small polymer systems is still not well understood. Recently, the statistical mechanics of protein folding has been studied extensively with simple protein models such as short cubic-lattice chains with contact-based interactions. While many valuable insights about protein folding were gained with such models, some concerns have also arisen, viz. that they lack the character of protein backbones whose interactions would limit the folding patterns of proteins. Here, a comparative study of the conventional cubic-lattice chain model and a fine-grained more realistic lattice protein model with both backbone and side-chain interactions is carried out. It is found that, even though both types of models exhibit a cooperative two-state folding transition to the native structure with optimized force fields, the character and origin of cooperativity of the two models are different. In the simple contact-based model, the free-energy barrier occurs at the low end of the energy scale, and the cooperativity arises from a concerted formation of native contacts among many residues in a compact state. In the other more complicated model, the free-energy barrier occurs in the intermediate energy region, and the folding cooperativity arises from collective orientational arrangements of locally structured units in semi-open conformational states. On the basis of these results, two limiting molecular mechanisms for protein folding emerge, which can be used for analyzing the folding process of real proteins.

Molecular modeling and energy refinement of supercoiled DNA.

A method is presented for constructing the complete atomic structure of supercoiled DNA starting from a linear description of the double helical pathway. The folding pathway is defined by piecewise B-spline curves and the atoms are initially positioned with respect to the local Frenet trihedra determined by the equations of the curves. The resulting chemical structure is corrected and refined with an energy minimization procedure based on standard potential expressions. The refined molecular structure is then used to study the effects of supercoiling on the local secondary structure of DNA. The minimized structure is found to differ from an isotropic elastic rod model of the double helix, with the base pairs bending in an asymmetric fashion along the supercoiled trajectory. The starting trajectory is chosen so that the refined supercoiled structure is either underwound (10.37 base pairs per turn) or overwound (9.65 base pairs per turn) compared to the standard tenfold B-DNA fiber diffraction model. The underwound supercoil is also lower in energy than the overwound duplex. The variation of base pair sequence in poly(dA).poly(dT).poly(dAT).poly(dTA) and poly(dA5T5).poly(dT5A5) is additionally found to influence the secondary structural features along a given supercoiled pathway. Finally, the detailed features of the refined structures are found to be in agreement with known X-ray crystallographic structures of DNA oligomers.

How optimization of potential functions affects protein folding.

The relationship between the optimization of the potential function and the foldability of theoretical protein models is studied based on investigations of a 27-mer cubic-lattice protein model and a more realistic lattice model for the protein crambin. In both the simple and the more complicated systems, optimization of the energy parameters achieves significant improvements in the statistical-mechanical characteristics of the systems and leads to foldable protein models in simulation experiments. The foldability of the protein models is characterized by their statistical-mechanical properties--e.g., by the density of states and by Monte Carlo folding simulations of the models. With optimized energy parameters, a high level of consistency exists among different interactions in the native structures of the protein models, as revealed by a correlation function between the optimized energy parameters and the native structure of the model proteins. The results of this work are relevant to the design of a general potential function for folding proteins by theoretical simulations.

Analyzing the normal mode dynamics of macromolecules by the component synthesis method.

This paper presents a general method for studying the harmonic dynamics of large biomolecules and molecular complexes. The performance and accuracy of the method applied to a number of molecules are also reported. The basic approach of the method is to divide a macromolecule into a number of smaller components. The local normal modes of the components are first calculated by treating individual components and the interactions between nearest neighboring components. The physical displacements of all atoms are then represented in the local normal mode space, in which a selected range of high-frequency local modes is neglected. The equation of motion of the molecule in the local normal mode space will then have a smaller dimension, and consequently the normal modes of the whole structure, particularly for large molecules, can be solved much more easily. The normal modes of two polypeptides--(Ala)6 and (Ala)12--and a double-helical DNA--d(ATATA).d(TATAT)--are analyzed with this method. Reductions on the dimensions of harmonic dynamic equations for these molecules have been made, with the fraction of the deleted high-frequency modes ranging from 1/2 to 5/6. The calculated low-frequency normal modes are found to be very accurate as compared to the exact solutions by standard procedure. The major advantage of the present approach on macromolecule harmonic dynamics is that the reduction on the dimensionality of the eigenvalue problems can be varied according to the size of molecules, so the method can be easily applied to large macromolecules with controlled accuracy.

Modeling DNA supercoils and knots with B-spline functions.

A method is offered to model the complex trajectories of closed circular DNA supercoils and knots. The trajectories are approximated by polygons and analytical expressions of the curves are generated from the polygons with B-spline functions. The resulting curves are used to evaluate the writhe and elastic energy of a series of interrelated supercoils, and to generate detailed atomic models of the deformed double helix.

Effects of compact volume and chain stiffness on the conformations of native proteins.

An investigation of the statistical properties of the native conformations of proteins, observed from crystal structures, is reported. Protein conformations were analyzed in terms of a bond vector correlation function and molecular volume. It was observed that, while the volume of a protein structure varies nearly linearly with the number of residues, the bond vector correlation function exhibits a universal feature for all sizes of proteins. To interpret the nature of the bond vector correlation function of native protein structures quantitatively, Monte Carlo simulations of realistic polypeptide chains of specific but arbitrary amino acid sequence were carried out. The molecule was constrained in an ellipsoidal volume determined by its chain length, and conformations with unacceptable nonbonded contacts between different amino acid residues were excluded. The interactions within a terminally blocked single residue, which correlate two nearest-neighbor peptide groups in a chain, were taken into account by an energetically biased sampling of its phi-psi space. The simulated chain correlation functions were found to be in good agreement with those of the crystal structures of beta-sheet-type and mixed-type (alpha+beta) proteins of similar length. On the basis of these calculations, it is concluded that the observed conformations of these native proteins may arise from two basic factors: the compactness of structures under hydrophobic interactions and the intrinsic stiffness of polypeptide chains due to the interactions within each terminally blocked residue.

Unfolding and refolding of the native structure of bovine pancreatic trypsin inhibitor studied by computer simulations.

A new procedure for studying the folding and unfolding of proteins, with an application to bovine pancreatic trypsin inhibitor (BPTI), is reported. The unfolding and refolding of the native structure of the protein are characterized by the dimensions of the protein, expressed in terms of the three principal radii of the structure considered as an ellipsoid. A dynamic equation, describing the variations of the principal radii on the unfolding path, and a numerical procedure to solve this equation are proposed. Expanded and distorted conformations are refolded to the native structure by a dimensional-constraint energy minimization procedure. A unique and reproducible unfolding pathway for an intermediate of BPTI lacking the [30,51] disulfide bond is obtained. The resulting unfolded conformations are extended; they contain near-native local structure, but their longest principal radii are more than 2.5 times greater than that of the native structure. The most interesting finding is that the majority of expanded conformations, generated under various conditions, can be refolded closely to the native structure, as measured by the correct overall chain fold, by the rms deviations from the native structure of only 1.9-3.1 A, and by the energy differences of about 10 kcal/mol from the native structure. Introduction of the [30,51] disulfide bond at this stage, followed by minimization, improves the closeness of the refolded structures to the native structure, reducing the rms deviations to 0.9-2.0 A. The unique refolding of these expanded structures over such a large conformational space implies that the folding is strongly dictated by the interactions in the amino acid sequence of BPTI. The simulations indicate that, under conditions that favor a compact structure as mimicked by the volume constraints in our algorithm, the expanded conformations have a strong tendency to move toward the native structure; therefore, they probably would be favorable folding intermediates. The results presented here support a general model for protein folding, i.e., progressive formation of partially folded structural units, followed by collapse to the compact native structure. The general applicability of the procedure is also discussed.