Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.

Structure-activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor.

Potent and selective antagonists of the human EP3 receptor have been identified. The structure-activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K(i) values at the EP3 receptor and micromolar activities at the EP1, EP2 and EP4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP3 receptor was also investigated.

Maintenance of caspase-3 proenzyme dormancy by an intrinsic "safety catch" regulatory tripeptide.

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp "safety-catch" regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this "safety catch" results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.

Structure-activity relationship on the human EP3 prostanoid receptor by use of solid-support chemistry.

Potent and selective EP3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24-95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis.

Cysteine proteases related to mammalian interleukin-1 beta converting enzyme (ICE) and to its Caenorhabditis elegans homologue, CED-3, play a critical role in the biochemical events that culminate in apoptosis. We have determined the three-dimensional structure of a complex of the human CED-3 homologue CPP32/apopain with a potent tetrapeptide-aldehyde inhibitor. The protein resembles ICE in overall structure, but its S4 subsite is strikingly different in size and chemical composition. These differences account for the variation in specificity between the ICE- and CED-3-related proteases and enable the design of specific inhibitors that can probe the physiological functions of the proteins and disease states with which they are associated.

Activation of the human peripheral cannabinoid receptor results in inhibition of adenylyl cyclase.

The human peripheral cannabinoid (CB2) receptor has been cloned by reverse transcription-polymerase chain reaction from human spleen RNA and expressed, to study both ligand binding characteristics and signal transduction pathways. Receptor binding assays used the aminoalkylindole [3H]Win 55212-2 and membranes from transiently transfected COS-M6 cells. Saturation analysis showed that [3H]Win 55212-2 specific binding to the CB2 receptor was of high affinity, with a Kd of 2.1 +/- 0.2 nM (four experiments), and a high level of expression was attained, with a maximal number of saturable binding sites of 24.1 +/- 4.4 pmol/mg of protein (four experiments). The rates of association and dissociation for [3H]Win 55212-2 specific binding were both rapid when measured at 30 degrees. [3H]Win 55212-2 specific binding to the CB2 receptor was moderately enhanced by divalent and monovalent cations but was only slightly inhibited by guanosine-5'-O-(3-thio)-triphosphate. Competition for [3H]Win 55212-2 specific binding to the CB2 receptor was stereoselective, with the following rank order of potency for the more active stereoisomers: HU-210 > (-)-CP-55940 approximately Win 55212-2 >> (-)delta 9-THC > anandamide. The signaling pathway of the human CB2 receptor was investigated in a CB2-CHO-K1 stable cell line. CB2 receptor activation by cannabinoid agonists inhibited forskolin-induced cAMP production in a concentration-dependent and stereoselective manner but did not increase either cAMP production or Ca2+ mobilization in fura-2/acetoxymethyl ester-loaded CB2-CHO-K1 cells. The CB2 receptor-mediated inhibition of forskolin-induced cAMP production was abolished by pretreatment of the cells with 10 ng/ml pertussis toxin. These results demonstrate that the CB2 receptor is functionally coupled to inhibition of adenylyl cyclase activity via a pertussis toxin-sensitive G protein.

Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.

Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung.

Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.