Current Medicinal Chemistry - Volume 7, Issue 9, 2000

Current strategies in pharmaceutical research comprise two methodologically different but complementary approaches for lead finding purposes, namely the random screening of compound libraries and the structure-based effort, commonly termed rational drug design. The structure-based approach is aimed to exploit 3D structure data of the molecular compo-nents involved in the molecular recognition event that underlies the attempt to therapeutically modulate the biological function of a macromolecular target with proven pathophysiological relevance for a disease state.In this context, G protein-coupled receptors (GPCRs) constitute the most prominent family of validated drug targets within biomedical research, since approximately 60 percent of approved drugs elicit their therapeutic effects by selectively addressing members of that target family. From a 3D structure point of view, these transmembrane signal transduction systems represent the most challenging task for structure determination, which is due to the heterogeneous and fine-balanced environment conditions that are necessary for structural and functional integrity of the receptor protein.This contribution will address the different concepts to derive structurally relevant information on the transmemebrane seven-helix protein (7TM) domain of GPCRs with special emphasis laid on the multidisciplinarity of the applied methodologies. The current status of electron-cryo-microscopy on 2D crystals and even high-resolution x-ray crystallography on 7TM proteins will be introduced highlighting the transferability of the emerging structural principles onto the GPCR superfamily. Special techniques from bioinformatics and homology-related molecular modeling in combination with tailor-made protein simulation methodologies complement the experimentally derived data, in that they facilitate the 3D structure generation and structure validation process.This contribution summarises the most recent results of GPCR structure studies with the aim to underline the impact of structure data not only for the purpose of rationalising structure-activity data on low-molecular weight antagonists within the context of a protein binding pocket, but also for a better understanding of e.g. mutagenesis experiments, thus qualifying GPCR structure models as valid communication platforms establishing a functional link between molecular biology, biophysics, bioinformatics and organic chemistry in a highly efficient manner. This contribution summarises the most recent results of GPCR structure studies with the aim to underline the impact of structure data not only for the purpose of rationalising structure-activity data on low-molecular weight antagonists within the context of a protein binding pocket, but also for a better understanding of e.g. mutagenesis experiments, thus qualifying GPCR structure models as valid communication platforms establishing a functional link between molecular biology, biophysics, bioinformatics and organic chemistry in a highly efficient manner.

During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in vivo manipulation of levels of GPCRs using transgenic and gene knock out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology.Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.

G-protein-coupled receptors constitute a superfamily of integral membrane proteins encompassing hundreds of receptors for all types of chemical messengers, as well as, for example, the key molecules of our light and smell sensory systems, bioactive amines, peptide hormones, neurotrans-mitters and even proteins. Because of their complicated organisation with the characteristic seven transmembrane segments (7 TM) it has yet been impossible to structurally characterise any G-protein coupled receptor by crystallography or magnetic resonance. However, a number of indirect methods to study the structure and ligand binding of these proteins have been developed. Various studies have shown that antibodies produced against G-protein-coupled receptors are valuable tools. In this review we focus on the use of anti-receptor antibodies for the characterisation of membranes, cells and tissue, for mapping of the binding site, for purification by immunoaffinity chromatography and for biochemical studies of G-protein-coupled receptors. As an example we describe the characterisation of the G-protein-coupled neuropeptide Y receptor subtypes.

G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44 p42 mitogen activated protein kinases (MAPK) and frequently transactivation of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.

The design of peptidomimetic ligands with agonist biological activities in vitro and in vivo has been challenging. Lofty goals have been set for this research including high potency, high receptor type selectivity, high stability in vitro and in vivo, and high efficacy in vitro and in vivo for agonists. A systematic stepwise strategy has been developed to accomplish these goals. These include determining the primary amino acid side chain residues required for molecular recognition and, in the case of agonist activity, those required for information transduction. In addition to determining the preferred backbone conformation which can serve as a template for the bioactive conformation (an a-helix, b-turn, b-sheet, etc.), a strategy has been developed to examine and determine the preferred side chain conformations in chi space (chi 1 , chi 2 , etc.). These include specific covalent and non-covalent constraints which can place the constrained side chains at highly preferred gauche (minus), or gauche (plus), or trans conformations. Examples are provided that illustrate this methodology and provide insight into the topographical requirements for ligand receptor interactions. Often, at this juncture one can obtain a quite precise 3D pharmacophore for the ligand, as well as high stability to agonist biodegradation and good bioavailability including the ability to cross membrane barriers.If a non-peptide ligand is desired, efforts are in progress to develop templates, and aspects of conformational design that permit assembling of all components necessary for molecular recognition and transduction. Here the proper choice of template that can place the key side chain residue in 3D space is still difficult, and thus only partial success has been achieved in terms of potent and selective ligands. A few of these approaches are presented and discussed in some detail.

Receptor targeting with radiolabeled peptides has become very important in nuclear oncology in the past few years. The most frequently used peptides in the clinic are analogs of somatostatin (SRIF), e.g. OctreoScan, which contain chelators for the radioisotopes 111 In, 86Y, 90 Y, 67Ga, 68Ga and 64Cu or for 99m Tc and 188Re. and were labelled with the halogens 123I and 18F. Radiolabeled analogs of a-melanocyte-stimulating hormone (a-MSH), neurotensin, vasoactive intestinal peptide (VIP), bombesin (BN), substance P (SP) and gastrin/cholecystokinin (CCK) are also being developed, evaluated in vitro and in vivo and tested for clinical application. This review focuses on the expression in tumors and the regulation of receptors for these neuropeptides as well as the development of novel chelator-peptide conjugates suitable for in vivo scintigraphy or internal radiotherapy. The state of the art of radiopeptide pharmaceuticals is illustrated with four SRIF analogs, modified with the macrocyclic chelator 1, 4, 7, 10-tetraazacyclododecane- 1, 4, 7, 10-tetraacetic acid (DOTA) (D-Phe 1 )-octreotide (DOTAOC), [D-Phe 1 , Tyr 3 )-octreotide (DOTATOC), vapreotide (DOTAVAP) and lanreotide (DOTALAN). DOTA is almost a universal chelator capable of strongly encapsulating hard metals such as 111 In and 67 Ga for Single Photon Emission Tomography (SPET), 68 Ga, 86 Y and 64 Cu for Positron Emission Tomography (PET) as well as 90 Y for receptor-mediated radionuclide therapy and radiolanthanides which exhibit different interesting decay schemes. From biodistribution studies in experimental animals and from clinical data it is concluded that DOTATOC is currently the most suitable SRIF radiopeptide with the best potential in the clinic.