Pharmacophore refinement of gpIIb/IIIa antagonists based on comparative studies of antiadhesive cyclic and acyclic RGD peptides

Abstract

Structurally guided design approaches to low-molecular-weight platelet aggregation antagonists addressing the platelet-associated heterodimeric cell surface receptor gpIIb/IIIa rely on comparative studies of an ensemble of conformationally and biologically characterized compounds, since no high-resolution structure of the receptor system is available. We report a classical indirect and comparative pharmacophore refinement approach based on a series of small cyclic Arg-Gly-Asp (RGD) peptides as gpIIb/IIIa-fibrinogen interaction antagonists. These peptides have previously been investigated as potent and selective tumor cell adhesion inhibitors. The definition of geometrical descriptors classifying the RGD peptide conformations and their subsequent analysis over selected RGD- and RXD-containing protein structures allows for a correlation of distinct structural features for platelet aggregation inhibition. An almost parallel alignment of the Arg and Asp side chains was identified by a vector analysis as being present in all active cyclic hexa- and pentapeptides. This orientation is induced mainly by the constraint of backbone cyclization and is not of any covalent tripeptide-inherent origin, which was rationalized by a 500 ps high-energy MD simulation of a sequentially related linear model peptide. The incorporation of the recognition tripeptide Arg-Gly-Asp into the cyclic peptide templates acted as a filter mechanism, restricting the otherwise free torsional relation of both side chains to a parallel orientation. Based on the derived results, several detailed features of the receptor binding site could be deduced in terms of receptor complementarity. These findings should govern the design of next-generation compounds with enhanced activities. Furthermore, the complementary stereochemical characteristics of the substrate can be used as boundary conditions for pseudoreceptor modelling studies that are capable of reconstructing a hypothetical binding pocket, qualitatively resembling the steric and electronic demands of gpIIb/IIIa. It is interesting to note that these features provide clear differentiation to requirements for inhibition of alpha v beta 3 substrate binding. This can account for the extremely high selectivity and activity of some of our constrained peptides for either the alpha IIb beta 3 or the alpha v beta 3 receptor.

Analysis of the RGD sequence in protein structures: comparison to the conformations of the RGDW and D RGDW peptides determined by molecular dynamics simulations

“…A similar approach was taken in the study by MuÈ ller et al [27], where the structure of 18 cyclic RGD peptides were determined by two-dimensional NMR spectroscopy and restrained molecular dynamics simulations; their activities were characterized by IC 50 values and were compared to the linear peptide GRGDS and to ®brinogen, vitronectin and ®bronectin.…”

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

“…In matched cases, cyclization yields superactivity and higher receptor specificity. For our RGD peptides we used cyclic penta-and hexapeptides as template structures [4][5][6][7]. Within these relatively small cyclic peptides, the preferred backbone conformation is mainly determined by the number and position of D and L amino acids (glycine can act as a D or an L amino acid) [8].…”

“…In case of a single D amino acid and four L amino acids, the homodetic cyclic pentapeptide prefers a ~II'/y conformation with the D amino acid in the i+ 1 position of the [3II'-turn [8]. Using the sequence -RGDFV-, we synthesized five cyclic pentapeptides in which one of the five amino acids is in D-configuration [4][5][6][7]. This procedure results in a shift of the functional groups around the skeleton of the [3II'/T-backbone ('spatial screening').…”

The Constrained Amino Acid β‐Acc Confers Potency and Selectivity to Integrin Ligands

“…[10] As d-amino acids prefer the i + 1 position of bII' turns, the RGD sequence of cyclic pentapeptides like cyclo-(-Arg-Gly-Asp-d-Phe-Val-) adopts a nonstretched conformation with glycine in the central position of a g turn, resulting in highly active integrin a V b 3 ligands with a distance of 668 pm between C b atoms of Arg and Asp. [4,20] In another previously investigated peptide, cyclo-(-Arg-Gly-Asp-Phe-d-Val-), aspartic acid is found in the central position of a g turn with an Arg to Asp C b atom distance of 905 pm. [20] Therefore, the RGD sequence is more stretched, which accounts for the peptide being a somewhat less-active and less-specific ligand of integrin a V b 3 .…”

“…[4,20] In another previously investigated peptide, cyclo-(-Arg-Gly-Asp-Phe-d-Val-), aspartic acid is found in the central position of a g turn with an Arg to Asp C b atom distance of 905 pm. [20] Therefore, the RGD sequence is more stretched, which accounts for the peptide being a somewhat less-active and less-specific ligand of integrin a V b 3 . Reference peptide 2 is also characterized by an elongated conformation of the RGD sequence with a distance of 930 pm between C b atoms of Arg and Asp, which correlates well with the lower affinity toward integrin a V b 3 .…”