Denise D. Beusen

Systematic search in conformational analysis

Abstract The coupling of conformation to activity and reactivity is a widely accepted concept, and as such has driven the development of tools which execute conformational searches in rapid and robust fashion [T.F. Havel, Prog. Biophys. Molec. Biol., 56 (1991) 43–78; A.R. Leach, In Rev. Comput. Chem.; K.B. Lipkowitz and D.B. Boyd, Ed.; VCH Publishers, Inc.: New York, N.Y., 1991, Vol. II, pp. 1–55]. Among the aims of these methods are the determination of a complete set of local minima from which the global energy minimum can be identified, or the generation of conformations consistent with constraints derived from SAR or structural studies. Most methods fall into two broad categories: those which are random or stochastic, and those which are systematic. Yet another group consists of those which are based on heuristics and artificial intelligence [A.R. Leach, K. Prout, D.P. Dolata, J. Comput. Chem. 11 (1990) 680–693]. The first category is typified by molecular dynamics [W.F. van Gunsteren and H.J.C. Berendsen, Angew. Chem. Int. Ed. Eng., 29 (1990) 992–1023], Monte Carlo [M.P. Alien and D.J. Tildesley, Computer Simulation of Liquids, Oxford Science Publications, 1989], distance geometry [J.M. Blaney and J.S. Dixon, in K.B. Lipkowitz and D.B. Boyd (Eds.), Reviews in Computational Chemistry, VCH, New York, Vol. 5, pp. 299–335, 1994], and other approaches [M. Saunders, J. Comput. Chem., 10 (1989) 203–208] in which the path by which conformational space is examined is ideally completely random, but bounded by the geometries of covalent bond lengths and angles. In traditional systematic searches, the variable to be examined, e.g. torsion angles, is divided into a regular grid. Each and every grid point is evaluated in a systematic fashion to determine its validity. The path through the grid points is regular and defined. In principle, systematic search can, within the resolution of the grid, identify all sterically allowed conformations of a molecule. Consequently, systematic search is an ideal tool for conformational analysis because it is not path dependent and cannot become entrapped in local minima. In this article we review some of the basics of systematic search, algorithmic improvements that have enhanced its speed, and new developments that have increased its accuracy by moving away from the limitations of a fixed torsional grid.

Anodic amide oxidations: Conformationally restricted peptide building blocks from the direct oxidation of dipeptides

Abstract A pair of bicyclic lactam based conformationally restricted peptide mimetics have been synthesized in good yield by the direct anodic oxidation of dipeptides. This work highlights the simplicity of using electrochemistry to construct peptide mimetics and serves to further define the nature of the substituents that are compatible with an electrochemical procedure for annulating rings onto amino acid derivatives.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31P in S3P and Glp, biosynthetically 13C-labeled S3P, specifically 13C and 15N labeled Glp, and a variety of protein-15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

Metabolism of 19-methyl substituted steroids and a proposal for the third aromatase monooxygenation.

The article summarizes the results of recent studies on the metabolism of 10-ethylestr-4-ene-3,17-dione, 10-[(1R)-1-hydroxyethyl]-, and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione, in placenta. These compounds are the 19-methyl analogs of androstenedione, 19-hydroxyandrostenedione, and 19-oxoandrostenedione, respectively. No conversion of 10-ethylestr-4-ene-3,17-dione to either estrogens or oxygenated metabolites was detected. Both 10-[(1R)-1-hydroxyethyl]- and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione were oxygenated to 10-(1,1-dihydroxyethyl)estr-4-ene-3,17-dione and isolated following in situ dehydration as 10-acetylestr-4-ene-3,17-dione. Evidence for the involvement of aromatase in these conversions is discussed. No conversion of 10-acetylestr-4-ene-3,17-dione to either estrogens or other oxygenated products was detected. These results lead us to propose a new mechanism for the third aromatase monooxygenation. We propose that the third oxygenation is initiated by 1 beta-hydrogen abstraction at C1 of 19,19-dihydroxyandrostenedione, followed by homolytic cleavage of the C10-C19 bond with concurrent formation of a delta 1(10),4-3-ketosteroid and a C19 carbon radical, and terminated by oxygen rebound at C19.

Molecular Modeling in Drug Design

This chapter examines the approaches to molecular modeling and drug design and emphasizes their limitations. Useful information to guide the design and synthesis of potential novel therapeutics can be developed from an analysis of structure-activity data in the three-dimensional framework provided by current molecular modeling techniques. Although most of the techniques and approaches described have broader application than shown, the examples chosen should be sufficient to illustrate their use. Keywords: drug design; molecular modeling; molecular mechanics; quantum mechanics; receptors; ligands; affinity; protein structure

Study of the role of schiff base formation in the aromatization of 3-[18O]testosterone and 3,17-DI-[18O]androstenedione by human placental aromatase

Testosterone, prepared with 18O at the 3 position, and androstenedione, prepared with 18O at both the 3 and 17 positions, were incubated with human placental microsomes. The resulting estrogen metabolites (estradiol and estrone, respectively) as well as unconverted starting material were isolated and analyzed for their 18O content by gas chromatography-mass spectrometry. In each case, greater than 90% of the 18O present in the original substrate was retained in the products. This result argues against a role for Schiff base formation in the aromatase reaction.

5 – Peptide Conformation: Stability and Dynamics

This chapter focuses on the computational and experimental tools used to elucidate the conformational stability and dynamics of peptides. Peptides are unique in having both a large number of degrees of freedom and a broad spectrum of functional groups. The importance of peptides in regulating biological processes and their seemingly infinite conformational possibilities define both the impetus and the impediment to understanding their mechanism of action. As mediators in precisely regulated, complex biological systems, peptides have a combination of flexibility and functionality that gives them the countervailing properties of adaptability and specificity. The innate properties of peptides that allow them to be multipotent with regard to encoded 3D message complement the expression of receptor subtypes having specific biological functions. It is generally accepted that the biological activity of a peptide is coupled to its conformation. Because of the linkage between conformation and activity, the field of conformationally directed peptide design has arisen in order to understand, elicit, or inhibit a given biological activity. There are two basic elements of this process: identifying the relevant conformation and developing means to stabilize that conformation. Any evaluation of peptide conformational stability and dynamics requires a description of the statistical distribution between equilibrium states of a peptide as well as the energetic barriers separating them. The role of dynamics in the bioactivity of a peptide is not certain. This chapter also discusses specific cases in which the conformational stability of peptides has been studied and/or rationally perturbed.