Robert S. McDowell

Structural and functional aspects of RGD-containing protein antagonists of glycoprotein IIb-IIIa

In the past few years, knowledge of the structure and function of RGD-containing protein antagonists of the platelet fibrinogen receptor glycoprotein IIb-IIIa has advanced rapidly. The RGD sequence is found at the apex of extended, solvent accessible, and conformationally flexible loops in proteins of vastly different structural frameworks. Conformation of the RGD epitope and the immediate surrounding sequence are critical factors affecting potency and selectivity as integrin antagonists. The RGD sequence has recently been introduced into a number of protein and peptide scaffolds.

Benzodiazepine peptidomimetic inhibitors of farnesyltransferase.

A structural survey of protein Zn2+ binding geometries was instigated based upon the functional requirement of Ras farnesyltransferase for Zn2+. The Cys-X-X-Cys motif found in Zn(2+)-binding proteins such as aspartate transcarbamylase was used as a template to devise a bidentate-coordination model for Cys-A1-A2-X peptide inhibitors. Accordingly, replacement of the central dipeptide with the hydrophobic scaffold 3-amino-1-carboxymethyl-2,3-dihydro-5- phenyl-1H-1,4-benzodiazepin-2-one (BZA) yielded a peptidomimetic inhibitor, Cys(BZA)Met, of moderate potency (IC50 = 400 nM). N-Methylation of the cysteine amide improved potency almost 100-fold (IC50 = 0.3-1 nM). The increased affinity presumably correlates with a preferred conformation of the inhibitor which maximizes a hydrophobic interaction between the scaffold and the enzyme, and the proper presentation of cysteine and methionine to allow bidentate coordination at Zn2+. These non-peptide inhibitors have been shown to block farnesylation of the Ras protein in intact cells and provide lead compounds for the development of new cancer therapeutic agents.

Model Peptide Studies Demonstrate That Amphipathic Secondary Structures Can Be Recognized by the Chaperonin GroEL (cpn60)

The molecular chaperone cpn60 binds many unfolded proteins and facilitates their proper folding. Synthetic peptides have been used to probe the question of how cpn60 might recognize such a diverse set of unfolded proteins. Three hybrid peptides were synthesized encompassing portions of the bee venom peptide, apamin, and the sequence KWLAESVRAGK from an amphipathic helix in the NH2-terminal region of bovine rhodanese. Two disulfides connecting cysteine residues hold the peptides in stable helical conformations with unobstructed faces oriented away from the disulfides. Peptides were designed to present either a hydrophobic or hydrophilic face of the amphipathic helix that is similar to the one near the amino terminus of rhodanese. Aggregation of these peptides was detected by measuring 1,1′-bis(4-anilino)napthalene-5,5′-disulfonic acid (bisANS) fluorescence at increasing peptide concentrations, and aggregation was not apparent below 2 μM. Thus, all experiments with the peptides were performed at a concentration of 1 μM. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a Kd = ∼106 μM for binding to cpn60, whereas there was no detectable binding of the reduced form. The peptide with the exposed hydrophilic face did not bind to cpn60 in either the oxidized or reduced states. Fluorescence experiments utilizing bisANS as a probe showed that binding of the helical hydrophobic peptide could induce the exposure of hydrophobic surfaces on cpn60, whereas the same peptide in its random coil form had no effect. Thus, binding to cpn60 is favored by a secondary structure that organizes and exposes a hydrophobic surface, a feature found in amphipathic helices. Further, the binding of a hydrophobic surface to cpn60 can induce further exposure of complementary surfaces on cpn60 complexes, thus amplifying interactions available for target proteins.

N-Benzoyl amino acids as LFA-1/ICAM inhibitors 1: amino acid structure-activity relationship.

The association of ICAM-1 with LFA-1 plays a critical role in several autoimmune diseases. N-2-Bromobenzoyl L-tryptophan, compound 1, was identified as an inhibitor to the formation of the LFA-1/ICAM complex. The SAR of the amino acid indicates that the carboxylic acid is required for inhibition and that L-histidine is the most favored amino acid.

Solution conformation of an atrial natriuretic peptide variant selective for the type A receptor.

Two-dimensional NMR spectroscopy has been used to characterize the solution conformation of an atrial natriuretic peptide (ANP) variant which is selective for the human natriuretic peptide receptor A (NPR-A) relative to receptor C (NPR-C). The ANP mutant, containing six substitutions, has reduced flexibility in aqueous solution relative to wild-type ANP and allows the observation of sufficient NOE connectivities for structure determination by distance geometry and restrained molecular dynamics calculations. The solution conformation is reasonably well defined, having an average backbone atom rms deviation from the average coordinates of approximately 1.1 A for residues 7-27. The structure is consistent with available functional data and shows a spatial separation between known receptor binding determinants and residues found to be outside the hormone-receptor interface.

Chapter 28. Structure-Based Design from Flexible Ligands

Publisher Summary This chapter discusses some strategies that have been used to obtain structural information from the well flexible molecules. While these strategies may not yield a model that has the atomic level of detail afforded by X-ray crystallography, examples illustrates that even approximate structural models can provide a useful springboard for the discovery of novel lead series. Although the chapter discusses the peptides, it is clear that similar strategies can be applied to other compounds that can be assembled in a modular fashion, including hits obtained from combinatorial synthesis. It is clear from the examples mentioned in the chapter that many powerful tools exist for deducing putative bioactive conformations of flexible molecules. The biological importance of the Phe-Trp-Lys-Thr turn in somatostatin is underscored by the many potent, structured peptide analogs that have been developed. Successive rigidification of the arginine-glycine-aspartic acid (RGD) epitope in cyclic peptides has led in two cases to models that directed the design of novel nonpeptide lead compounds. Angiotensin II and bradykinin illustrate how local conformational constraints can be combined with cyclization to the sequentially develop high-confidence structural models. Excepting the GPIIb/IIIa antagonists, however, few compelling examples exist, in which these types of models have been used successfully to design fundamentally the new chemical entities.

Hydroxyl and Water Molecule Orientations in Trypsin

A comparison is presented of experimentally observed hydroxyl and water hydrogens in trypsin determined from neutron density maps with the results of a 140ps molecular dynamics (MD) simulation. Experimental determination of hydrogen and deuterium atom positions in molecules as large as proteins is a unique capability of neutron diffraction. The comparison addresses the degree to which a standard force-field approach can adequately describe the local electrostatic and van der Waals forces that determine the orientations of these hydrogens. The molecular dynamics simulation, based on the all-atom AMBER force-field, allowed free rotation of all hydroxyl groups and movement of water molecules making up a bath surrounding the protein. The neutron densities, derived from 2.1A D2O-H2O difference Fourier maps, provide a database of 27 well-ordered hydroxyl hydrogens. Virtually all of the simulated hydroxyl orientations are within a standard deviation of the experimentally-observed positions, including several examples in which both the simulation and the neutron density indicate that a hydroxyl group is shifted from a ‘standard’ rotamer. For the most highly ordered water molecules, the hydrogen distributions calculated from the trajectory were in good agreement with neutron density; simulated water molecules that displayed multiple hydrogen bonding networks had correspondingly broadened neutron density profiles. This comparison was facilitated by development of a method to construct a pseudo 2A density map based on the hydrogen atom distributions from the simulation. The degree of disorder of internal water molecules is shown to result primarily from the electrostatic environment surrounding that water molecule as opposed to the cavity size available to the molecule. A method is presented for comparing the discrete observations sampled in a dynamics trajectory with the time-averaged data obtained from X-ray or neutron diffraction studies. This method is particularly useful for statically-disordered water molecules, in which the average location assigned from a trajectory may represent a site of relatively low occupancy.