By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.

The dopamine D2/D3 receptor (D2R/D3R) agonists are used as therapeutics for Parkinson’s disease (PD) and other motor disorders. Selective targeting of D3R over D2R is attractive because of D3R’s restricted tissue distribution with potentially fewer side-effects and its putative neuroprotective effect. However, the high sequence homology between the D2R and D3R poses a challenge in the development of D3R selective agonists. To address the ligand selectivity, bitopic ligands were designed and synthesized previously based on a potent D3R-preferential agonist PF592,379 as the primary pharmacophore (PP). This PP was attached to various secondary pharmacophores (SPs) using chemically different linkers. Here, we characterize some of these novel bitopic ligands at both D3R and D2R using BRET-based functional assays. The bitopic ligands showed varying differences in potencies and efficacies. In addition, the chirality of the PP was key to conferring improved D3R potency, selectivity, and G protein signaling bias. In particular, compound AB04-88 exhibited significant D3R over D2R selectivity, and G protein bias at D3R. This bias was consistently observed at various time-points ranging from 8 to 46 min. Together, the structure-activity relationships derived from these functional studies reveal unique pharmacology at D3R and support further evaluation of functionally biased D3R agonists for their therapeutic potential.

The nonmedical (i.e., recreational) misuse of synthetic cannabinoids (SCs) is a worldwide public health problem. When compared to cannabis, the misuse of SCs is associated with a higher incidence of serious adverse effects, suggesting the possible involvement of noncannabinoid sites of action. Here, we find that, unlike the phytocannabinoid Δ 9tetrahydrocannabinol, the indole-moiety containing SCs, AM2201 and JWH-018, act as positive allosteric modulators (PAMs) at the 5-HT 1A receptor (5-HT 1A R). This suggests that some biological effects of SCs might involve allosteric interactions with 5-HT 1A Rs. To test this hypothesis, we examined effects of AM2201 on 5-HT 1A R agonist-activated G protein-coupled inwardly rectifying potassium channel currents in neurons in vitro and on the hypothermic response to 5-HT 1A R stimulation in mice lacking the cannabinoid receptor 1. We found that both 5-HT 1A R effects were potentiated by AM2201, suggesting that PAM activity at 5-HT 1A R may represent a novel noncannabinoid receptor mechanism underlying the complex profile of effects for certain SCs.

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

No abstract

The dopamine 2 and 3 receptors (D2R and D3R) are well‐characterized targets for neuropsychiatric disorders. D2R/D3R agonists are used as add‐on therapies for Parkinson’s disease (PD) and have been studied for other motor‐associated disorders. Selectively targeting D3R over D2R is attractive for two reasons: i) restricted tissue distribution of D3R compared to D2R exhibits potential for lesser side‐effects; ii) there is evidence for dominant role of D3R over D2R for neuroprotective and neurorestorative actions of dopaminergic agonists used for the treatment of PD. However, lack of highly selective D3R agonists has made it difficult to separate the D3R vs. D2R physiological effects. Indeed, the development of highly selective D3R agonists has remained a challenge due to their 78% sequence identity in the transmembrane domain. To improve the outlook of D3R agonist‐based medication development, based on a potent D3R‐selective agonist PF592,379, we have designed, synthesized, and pharmacologically characterized bitopic ligands with a secondary pharmacophore tethered to the PF592,379 primary pharmacophore by a linker. Using BRET based functional assays that measure G‐protein activation and β‐arrestin recruitment, we characterized these bitopic ligands at both D3R and D2R. We found that the addition of secondary pharmacophore to the PF592,379 scaffold improved potency at D3R in G‐protein activation with maximal efficacy similar or higher than that of the parent compound. In contrast, the potencies at D3R in recruitment of β‐arrestin remained the same. Some of these compounds also showed modest D3R over D2R selectivity. Interestingly, certain combinations of secondary pharmacophores and linkers resulted in G‐protein signaling bias at either D3R or D2R but not both, resulting in receptor‐selective bias. We also found that given the same ligand structure, chirality can confer subtype selectivity and promote G‐protein bias. Together, our novel bitopic D3R agonists based on PF592,379 scaffold provide novel tools to further probe unique pharmacology at D3R and evaluate the therapeutic potential of targeting D3R vs. D2R for PD. Support or Funding Information National Institute on Drug Abuse – Intramural Research Program, National Institutes of Health

, to study the effect of post harvest treatments on quality and shelf life of sweet orange (Citrus sinensis) cultivar Local. It was carried out in Completely Randomized Design (CRD) with five treatments of different types of post harvest treatments viz. T1 = control (distilled water), T2 = bavistin (0.1%), T3 = calcium chloride (1%), T4 = Jeevatu (5%) and T5 = cinnamon oil (2%) replicated four times. Among these post harvest treatments, T1 showed highest percentage of weight loss (15.83%), lowest firmness (2.22 kg/cm 2) and highest TSS (10.70˚ Brix), lowest TA (0.395%) at final day of storage as compared to other treatments. Bavistin was found as the most effective in reducing the physiological loss in weight (10.80%), retained maximum firmness (3.13 kg/cm 2), highest tritrable acidity (0.76%), highest pH (5.08). The minimum total soluble solid (8.75˚Brix) was retained by cinnamon oil. This study revealed that sweet orange treated with bavistin recorded lowest physiological loss in weight (31.77%) and retains more firmness (24.73kg/cm 2) than that of control .Thus, present findings indicate that sweet oranges treated with bavistin increase the shelf life where as cinnamon oil also found to be promising treatment for retaining the quality of the sweet oranges stored up to 28 th days under laboratory condition.