Susanne Trumpp-Kallmeyer

Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-A deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.