Receptors have well-conserved regions that are recognized and activated by hormones and neurotransmitters. Most drugs bind to these sites and mimic or block the action of the native ligands. Using a high-throughput functional screen, we identified a potent and selective M 1 muscarinic receptor agonist from a novel structural class. Using a series of chimeric receptors, we demonstrated that this ligand activates the receptor through a region that is not conserved among receptor subtypes, explaining its unprecedented selectivity. This region of the receptor is distinct from the conserved region that is recognized by traditional ligands. The finding that receptors for small-molecule transmitters can have multiple, structurally distinct activation sites has broad implications for the study of receptor structure/function and the potential for the discovery of novel ligands with high selectivity.G-protein-coupled receptors that bind monoamine ligands (e.g., serotonin, adrenaline, dopamine, histamine, and acetylcholine) comprise the most intensively studied and exploited receptor family for the development of therapeutic agents by the pharmaceutical industry. The natural ligands for monoamine receptors are believed to bind a highly conserved pocket located deep within the transmembrane (TM)-spanning regions and to mediate receptor activation primarily through TM3, TM5, TM6, and TM7 (Spalding et al., 1994;Baldwin et al., 1997;Gether, 2000;Lu et al., 2001). Of the amino acids in these regions, 74% are identical in all five muscarinic receptor subtypes (Bonner et al., 1988). Potent small-molecule agonists are also believed to bind monoamine receptors through the same highly conserved regions (Strader et al., 1989(Strader et al., , 1991Wess et al., 1991;Page et al., 1995;Spalding et al., 1998;Ward et al., 1999;Allman et al., 2000).The muscarinic M 1 receptor has been targeted for the discovery of therapeutics for Alzheimer's Disease, and several companies have developed M 1 -selective agonists (e.g., Tecle et al., 1998;Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). Many potent compounds came out of these programs, and several were shown to improve cognition in animals (WAY-132983 and CI-1017; Bartolomeo et al., 2000;Weiss et al., 2000) and people (Xanomeline, Bodick et al., 1997). However, many of the compounds also produced classic muscarinic side effects such as sweating, nausea and diarrhea (Bodick et al., 1997, Bartolomeo et al., 2000Thal et al., 2000). In vitro assays have shown that these compounds activate the M 1 , M 3 , M 4 , and M 5 muscarinic receptor subtypes at similar concentrations (Table 1 and Tecle Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). This may be a direct result of the ligands activating the receptors through regions where the amino acid sequence is almost identical. Since drug interactions with nontarget receptor subtypes are often responsible for the unwanted side effects of commercial pharmaceuticals, there is strong motivation to design more selec...

Opioid receptor agonists are known to alter the activity of membrane ionic conductances and receptor-activated channels in CNS neurons and, via these mechanisms, to modulate neuronal excitability and synaptic transmission. In neuronal-like cell lines opioids also have been reported to induce intracellular Ca(2+) signals and to alter Ca(2+) signals evoked by membrane depolarization; these effects on intracellular Ca(2+) may provide an additional mechanism through which opioids modulate neuronal activity. However, opioid effects on resting or stimulated intracellular Ca(2+) levels have not been demonstrated in native CNS neurons. Thus, we investigated opioid effects on intracellular Ca(2+) in cultured rat hippocampal neurons by using fura-2-based microscopic Ca(2+) imaging. The opioid receptor agonist D-Ala(2)-N-Me-Phe(4),Gly-ol(5)-enkephalin (DAMGO; 1 microM) dramatically increased the amplitude of spontaneous intracellular Ca(2+) oscillations in the hippocampal neurons, with synchronization of the Ca(2+) oscillations across neurons in a given field. The effects of DAMGO were blocked by the opioid receptor antagonist naloxone (1 microM) and were dependent on functional NMDA receptors and L-type Ca(2+) channels. In parallel whole-cell recordings, DAMGO enhanced spontaneous, synaptically driven NMDA receptor-mediated burst events, depolarizing responses to exogenous NMDA and current-evoked Ca(2+) spikes. These results show that the activation of opioid receptors can augment several components of neuronal Ca(2+) signaling pathways significantly and, as a consequence, enhance intracellular Ca(2+) signals. These results provide evidence of a novel neuronal mechanism of opioid action on CNS neuronal networks that may contribute to both short- and long-term effects of opioids.

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Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.

Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.