Cloning and characterization of a novel human histamine receptor.

Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular [Ca(2+)] in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of [(3)H]histamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both [(35)S]GTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.

Fatty acid sulfonyl fluorides inhibit anandamide metabolism and bind to the cannabinoid receptor.

Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (palmitylsulfonyl fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50 values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylfluoride had an IC50 of 18 nM and C20 sulfonylfluoride had an IC50 of 78 microM. The C14, C16, and C18 sulfonyl fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.

A novel electrophilic high affinity irreversible probe for the cannabinoid receptor.

In order to explore the structural requirements for cannabinoid activity we have been involved in the design and synthesis of stereochemically defined high affinity probes for the cannabinoid receptor. This effort has involved the development of irreversible ligands which will allow us to obtain detailed information on the cannabinoid receptor active site(s). The irreversible ligands, which incorporate highly reactive functional groups in a strategic position of the ligand, may form covalent bonds with amino acid residues at the receptor active site or in the neighborhood of this site. We shall discuss the biochemical properties of one of these probes, which incorporates the electrophilic isothiocyanate group into the structure of the highly potent cannabinoid agonist (-)-1',1'-dimethylheptyl-delta 8-THC. This ligand, (-)-7'-isothiocyanato-1',1'-dimethylheptyl-delta 8-THC (7'-NCS-DMH-delta 8-THC), was evaluated for its affinity for cannabinoid binding sites using rat forebrain membrane preparations and found to have an apparent IC50 value of 660 pM. Incubation of the membrane preparation with a ligand concentration of five times the apparent IC50 resulted in the irreversible occupation of nearly all of the receptor specific binding sites.