Abstracts of papers Medicinal chemistry meeting

s of papers Medicinal Chemistry Meeting Organized by the Division of Medicinal Chemistry of the Royal Dutch Chemical Society (Sectie Farmacochemie van de Koninklijke Nederlandse Chemische Vereniging) Weesp (the Netherlands), 15 December 1989 Organizing committee: Prof. Dr. H. Timmerman (Amsterdam) Dr. J. Wilting (Utrecht) Dr. M.T.M. Tulp (Weesp) Dr. J. Kelder (Oss) 11 -1989 Pharmaceutisch Weekblad Scientific edition M1 HOW THE KNOWLEDGE OF DNA SEQUENCE CAN HELP US TO UNDERSTAND NEURORECEPTOR STRUCTURE AND FUNCTION M.G. Darlison r A.N. Bateson R.J. Harvey, W. Wisdenf E.A. Barnard. The application of recombinant DNA technology to the study of neuroreceptors has yielded important information on their primary structures, membrane topologies, structure/function relationships and evolution. Such investigations have, to date, revealed the existence of three superfamilieslof receptors. The ligand-gated ion channel superfamily includes GABA a receptors, glycine receptors and the muscle and neuroHal types o~ nicotinic . acetylcholine receptors. Members of this class are multi-subunit; each subunit contains four membranespadning domains that form the integral ion channel, a long extracellular domain that contains agonist and antagonist binding sites and an intracellular loop that may be subject to phosphorylation/dephosphorylation. 2 In contrast, the G-protein-coupled receptor superfamily includes the adrenergic, muscarinic and serotonin receptor subtypes and at least some neuropeptide receptors (e.g. the substance K receptor}. Receptors of this class have a single polypeptide chain which contains seven membrane-spanning domains; here the ligand-binding site is formed by amino-acid side-chains in the transmembrane segments and is, therefore, in or close to the plane of the membrane. Recently, the cloning of complementary DNAs for atrial natriuretic peptide receptors has revealed the existence of an additional class of neuroreceptor. These also comprise a single polypeptide, but they have one membrane-spanning domain and integral guanylate cyclase activity. Molecular biological studies on the vertebrate GABA receptor in this and other laboratories have shown ~hat this receptor/ion channel comprises at least a, 8 and subunit types and that these must associate in different neuronal cell populations to form many receptor subtypes. Such complexity had not previously been realised from pharmacological and biochemical studies. 1 E.A. Barnard et al. Trends Neurosci., i0 (1987) 502. 2 M.G. Caron. Current Opinion in Cell Biol., 1 (1989) 159. 3 M.-S. Chang et al. Nature 341 (1989} 68. MRC Molecular Neurobiology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. MUSCARINIC A C E T Y L C H O L I N E RECEPTORS: STRUCTURE AND FUNCTION Chris J. van Konncn * and Nell M. Nathanson 9 Muscarinic acetylcholine receptors (mAChR) arc the predominant cholinergic receptors in the mammalian cenmal nervous syslem and play a major role in regulating the funcdons of the target organs of the parasympathedc nervous system. Acdvafion of mAChRs by acctylcholinr can result in a wide variety of cellular responses including changes in incracellalar levels of cAMP and cGMP, activation of phospholipase C, and the opening or closing of ion channels. Genomic and eDNA clones encoding at least five different receptor subtypes have been idendfiud from a variety of mammalian species. Muscarinic receptors belong m a family of receptors which ate ingolved in the acdvadon of various signal mmsducdon pathways through their interaction with guanine nucleodde-binding proteins (G-proteins). This "superfamily" includes the five subtypes of mammalian mAChR, the serotonJn receptors, the ft. and [}-admnergic receptors,the dopamine D2 receptor, the substance K and P receptors, the intropin-choriogonadom~pin receptor, and a yeast pheromone receptor. These receptors all sham a predicted seven mansmembrane domain structure with highly conserved amino acid sequences, especially within certain u'ansmembrane regions. The least homology is found in the proposed third cytoplasmic loop, the cytoplasmic carboxyl tcmdnai tail and the exmacellular amino terminal loop. Among the well conserved amino acids am the asparaginc residues in the putative extracellular loop (N-glycosylafion), the cysteine residues in the second and third exa'acellular loop ( disulfide bond), a cysteine residue in the cytoplasmic carboxyl tail and praline residues in the mansmembrane regions 4, 5, 6 and 7. The determinants of receptor function (ligand binding and G-protein coupling) and reguladon (receptor processing and desensidzadon) am thought to reside in both the regions of high homology and in those of variable amino acid idandty. To understand the reladonship of mAChR structure and function, we have employed site-directed mulagencsis to define domains of functional importance, i.r regions involved in G-prolein coupling and receptor processing. Dam on this work will he pmsemed. * Ch. J. van Koppen is a recipient of an American Heart Association, Washington Affiliate postdocorai fellowship. Dept. of Pharmacology, University of Washington. Seattle WA 98195 USA THE PROGESTERONE RECEPTOR: A MEMBER OF A SUPERFAMILY OF GENE TRANSCRIPTION REGULATORY FACTORS. H. LOosFelt, M. At~er, A. Oulochon-Mantel~ M. Mlsrahi, M. Perrot-Applanat, M.T. Vu Hal, F. Lorenzo, g. Mlldrom. Rabble I and human 2 progesterone receptor eDNA's were cloned and sequenced allowing to deduce the primary structure Of the corresponding proteins. The human progesterone receptor was mapped to chromosome l lq22-q233. The promoter and 5' Flanking region oF the gene was Isolated and analyzed 4. In vitro mutagenesis experiments allowed to analyze the role OF the hormone and antlhormane in receptor Funotlon 5 and the mechanisms OF its nuclear localization 6. Over 50 monoclonal antibodies were prepared against the rabbit and over 30 against the human progesterone receptor. A new method was devised and used to map the corresponding epitopes7, 8. The antibodies were used to study the receptor by Immunocytochemistry in breast cancer g and endometrlum I0. I. Loos fe l t R. e t a l . Proc. Nat l . Acad. So t . , 83 (1986) 9045. 2. Miarahi M. et al. Biochem. Biophys. Res. Commun., I~3 (1987) 740. 3. Rousseau-Merck et al. Human Oenet., 77 (1987) 280. 4. Mlsrahl M. et al. Nucleic Acids Res., 16 (1988) 5459. 5. Dulochon-Mentel A. et al. Nature, 336 (1988) 695. 6. Guiochon-Mantel A. et al. Cell, 5? (1g~9) 1147. 7. Lorenzo et al. gut. J. glochem., 176 (1988) 53. 8. Vu Hal M.T. et al Bioehem J., 260 (7989) 371. 9. Perrot-Applanat M. et al. Cancer Rea., 47 (1987) 2652. 10.Oarcia E. et al. J. Clin. Endocrinol. Me~ab., 67 (1988) 80. "Hormones et Reproduction", INSERM U. 135 , Facultd de M4declne Parls-Sud, 94275 be Kremllo-Bic~tre Cedex, France. HERBICIDE BINDING KINETICS AND PARTIAL SEQUENCE OF THE psbA GENE, WHICH CODES FOR THE HERBICIDE-BINDING DI PROTEIN J.D. Naber and J.J.S. van Rensen The DI protein of the reaction center of photosystem II in chloroplast membranes is the receptor protein of many different classes of chemical weed control agents: herbicides. This protein is encoded by the chloroplast psbA @erie. Several weeds have become resistant to trlazine-type herbicides. We investigated the klnetics of the binding of several herbxcides to the DI protein and sequenced part of the chloroplast psbA gene from plants, either resistant or susceptible for triazine-type herbzcides. The exchange parameters of the herbicide binding kinetics were determined using a kinetic model, as described by Naber (1989). In this model, association and dissociation parameters can be discriminated. DNA and RNA were isolated simultaneously from fresh leaf material. Sequence analysis were performed with total DNAand RNA-preparations, using a synthetic primer complementary to a highly conserved region of the psbA gene (Naber, 1989). For the herblcldes atrazlne, diuron, ioxynil, and pairs of S and R stereo-zsomers of cyanoacrylates it is found that differences in activlty can be ascribed to different release rates, rather tha~ to differences in binding velocities. This indxcates that the approach of the inhibitor to its binding niche is determined by physical properties of the inhibitor, like e.g. hydrophobicity. A stationary bxnding, resulting in a significant inhibitzon of photosynthetic electron transport, requires a strict molecular shape. Triazine resxstance is not related to a decreased bindlng rate, but to an approximately hundred-fold increased value of the release parameters of the triazine herbicide. The resistance to triazine herbicides is caused by an alteration in the psbA-codon 264: adenine is exchanged for guanine. This results in a substitution of serine in the susceptible plants for glycine in the resistant plants of Chenopodium album and Brassica naDus. J.J.S. van Rensen and J.F.H. Snel, Photosynthesis Res., 6 (1985) 231. J.J.S. van Rensen and L.E.E.M. Spatsens, Z. Naturforsch., 42c (1987) 794. J.D. Naber, Ph.D. Thesls, Agricultural University Wageningen, t989. Lab. of Plant Physzological Research, Agricultural University Wageningen, Gen. Foulkesweg 72, 6703 BW Wageningen, The Netherlands. 11-1989 Pharmaceutisch Weekblad Scientific edition M3 M O D E L I N G T H E A C T I V E SITE O F C Y T O C H R O M E P450-c A. Baarslae. L. Kovma'ns. G.M. Donnd-On den Kelder and N.P.E. Vcrmeulen* Cytochromr P450 isozymes arc important in the metabolism of many endogcnic and xenobiotic compounds. In metabolizing these compounds cytochromc P450 isozymes also give rise to the formation of very toxic chemicals. Since the discovery of multiple forms it is known that the isozym9 cytochrome P450..c activates polycyclic hydrocarbons. Based on these data Jerina ct al. (1985) constructed a geometrical model for the bindingsitc of polycyolic hydrocarbons. This bindingsite catalyzes highly stercospccific epoxidat ions result ing in primarily (R,R) dihydrodiols. Limitation of this