Author Correction: Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs.

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Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs.

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F6.52. Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for drug discovery.

The 5-hydroxytryptamine4 receptor agonists prucalopride and PRX-03140 increase acetylcholine and histamine levels in the rat prefrontal cortex and the power of stimulated hippocampal θ oscillations.

5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.

Essential role of CCR6 in directing activated T cells to the skin during contact hypersensitivity.

CCR6 is expressed in a number of dermatological inflammatory diseases. Here, we report that mice sensitized with the hapten oxazolone had increased numbers of CCR6+ T cells in the draining lymph nodes. Using CCR6-/- mice, we assessed the role of CCR6 on the development of contact hypersensitivity. After hapten sensitization and re-challenge, ear swelling in CCR6-/- animals was reduced 80% as compared with wild-type (WT) control mice. This decreased level of inflammation was not related to an inhibition in T-cell activation, because CCR6-/- lymph node cells from sensitized mice produced threefold higher levels of IFN-gamma in culture than cells from sensitized WT mice and, when these cells were directly injected into the site of hapten challenge, induced a robust inflammatory response. However, intravenous injection of CCR6-/- lymph node cells from sensitized mice were unable to prime naive mice to re-challenge whereas cells from primed WT mice were able to sensitize animals. These results suggest that CCR6 plays an important role in directing the trafficking of activated T cells into the skin and suggests that a CCR6 antagonist could be useful to treat skin-mediated inflammatory reactions.

The human specific CCR1 antagonist CP-481,715 inhibits cell infiltration and inflammatory responses in human CCR1 transgenic mice.

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-gamma and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.