The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors; however, mutations at SMO have been found to abolish their anti-tumor effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6–2.8Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D4736.55 elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules.
Several 2-alkyl-5-methoxytryptamine analogues were designed and prepared as potential 5-HT(6) serotonin agonists. It was found that 5-HT(6) receptors accommodate small alkyl substituents at the indole 2-position and that the resulting compounds can bind with affinities comparable to that of serotonin. In particular, 2-ethyl-5-methoxy-N, N-dimethyltryptamine (8) binds with high affinity at human 5-HT(6) receptors (K(i) = 16 nM) relative to 5-HT (K(i) = 75 nM) and was a full agonist, at least as potent (8: K(act) = 3.6 nM) as serotonin (K(act) = 5.0 nM), in activating adenylate cyclase. Compound 8 displays modest affinity for several other populations of 5-HT receptors, notably h5-HT(1A) (K(i) = 170 nM), h5-HT(1D) (K(i) = 290 nM), and h5-HT(7) (K(i) = 300 nM) receptors, but is otherwise quite selective. Compound 8 represents the first and most selective 5-HT(6) agonist reported to date. Replacing the 2-ethyl substituent with a phenyl group results in a compound that retains 5-HT(6) receptor affinity (i.e., 10: K(i) = 20 nM) but lacks agonist character. 2-Substituted tryptamines, then, might allow entry to a novel class of 5-HT(6) agonists and antagonists.
Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.
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