Discovery of novel selective norepinephrine inhibitors: 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides (WYE-114152).

Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound 10b (WYE-114152) had low nanomolar hNET potency (IC(50) = 15 nM) and good selectivity for hNET over hSERT (>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated efficacy in rat models of acute, inflammatory, and neuropathic pain.

Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231).

Structural modification of a virtual screening hit led to the identification of a new series of 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols which are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound S-17b (WYE-103231) had low nanomolar hNET potency (IC(50) = 1.2 nM) and excellent selectivity for hNET over hSERT (>1600-fold) and hDAT (>600-fold). S-17b additionally had a good pharmacokinetic profile and demonstrated oral efficacy in rat models of ovariectomized-induced thermoregulatory dysfunction and morphine dependent flush as well as the hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain.

Pharmacological characterization of the muscarinic agonist (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983) in in vitro and in vivo models of chronic pain.

Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4-29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M(1) (IC(50) = 6.6 nM; E(max) = 65% of 10 muM carbachol-stimulation) over the M(3) (IC(50) = 23 nM; E(max) = 41%) and M(5) receptors (IC(50) = 300 nM; E(max) = 18%). WAY-132983 also activated the M(4) receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC(50) = 10.5 nM); at the M(2) receptor its potency was reduced by 5-fold (IC(50) = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M(4) receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.

The role of transmembrane helix 5 in agonist binding to the human H3 receptor.

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.