Discovery of the first non-peptide antagonist of the motilin receptor.

A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.

NOE-derived conformation of GRGDSP cell adhesion recognition site in the presence of SDS micelles and integrin receptor GPIIB/IIIA.

The tripeptide RGD is well known for its role in integrin receptor-mediated cell-cell surface adhesion. Here, NMR and transferred NOE studies have been done with the fibrinogen/fibronectin-derived hexapeptide GRGDSP in the presence of sodium dodecyl sulfate (SDS) and purified platelet glycoprotein integrin receptor GPIIb/IIIa. In the presence of SDS and absence of receptor, GRGDSP gives NOE-based distance geometry-generated structures characteristic of two "nested' beta-turns centered at RG and GD. In the presence of integrin GPIIb/IIIa, GRGDSP resonances are chemically shifted and broadened consistent with a dynamic equilibrium between free and receptor "bound' peptide. NOEs characteristic of the nested beta-turns are either absent or weaker indicating a significant conformational change in GRGDSP in the receptor bound state. GRGDSP appears to bind the receptor in a more extended backbone conformation which positions aspartic acid and arginine residues spatially close for potential electrostatic interactions.

RGD induces conformational transition in purified platelet integrin GPIIb/IIIa-SDS system yielding multiple binding states for fibrinogen gamma-chain C-terminal peptide.

Fibrinogen gamma-chain C-terminal peptide HHLG-GAKQAGDV (gamma 12) and alpha-chain peptide GRGDSP are known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet integrin receptor GPIIb/IIIa. NMR studies of gamma 12 in the presence of purified GPIIb/IIIa in SDS/water solution have demonstrated the presence of two gamma 12 binding states, one of which is eliminated by GRGDSP (RGD) up to a RGD: gamma 12 ratio of 2:1. RGD: gamma 12 ratios greater than 2:1 produce multiple sets of gamma 12 NMR signals in TOCSY spectra. At a ratio of 4:1, two to four such resonance sets can be resolved for A405, Q407, A408, G409, D410 and V411 spin systems. The number of multiple resonances remains unchanged at ratios of 6:1 and 8:1. Addition of gamma 12 to reverse the ratio to 8:8 (1:1) has no apparent effect on the RGD-induced distribution. Results suggest that RGD irreversibly induces a conformational transition(s) in GPIIb/IIIa to produce multiple gamma 12 binding sites on the receptor.

Design and evaluation of nonpeptide fibrinogen gamma-chain based GPIIb/IIIa antagonists.

Two series of nonpeptide turn mimetics were designed by analysis of the solution NMR structure of the 385-411 sequence of the gamma-chain of fibrinogen. These compounds, based on the KQAGD (Lys-Gln-Ala-Gly-Asp, 406-410) sequence, were synthesized and studied in vitro. The most interesting compound from our study, RWJ 50042 (25), exhibits potent inhibition of fibrinogen binding to GPIIb/IIIa (IC50 = 0.009 microM), as well as thrombin- or collagen-induced platelet aggregation (IC50 = 0.76, 0.14 microM). Since the 400-411 sequence is required for gamma-chain bioactivity and is a unique recognition sequence among ligands for integrins, vis-a-vis other RGD (Arg-Gly-Asp)-presenting proteins, these turn mimetics may represent a new, selective approach to antagonism of the fibrinogen receptor.