Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ('iDrugSnFRs') for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Multipotent Stromal Cells from Subcutaneous Adipose Tissue of Normal Weight and Obese Subjects: Modulation of Their Adipogenic Differentiation by Adenosine A1 Receptor Ligands

Adenosine A1 receptor (A1R) activation, stimulating lipogenesis and decreasing insulin resistance, could be useful for metabolic syndrome management in obese subjects. Since full A1R agonists induce harmful side-effects, while partial agonists show a better pharmacological profile, we investigated the influence of two derivatives of the full A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA), C1 and C2 behaving as A1R partial agonists in animal models, on the adipogenic differentiation of stromal/stem cells (ASCs) from human subcutaneous adipose tissue, which mainly contribute to increase fat mass in obesity. The ASCs from normal-weight subjects showed increased proliferation and A1R expression but reduced adipogenic differentiation compared to obese individual-derived ASCs. Cell exposure to CCPA, C1, C2 or DPCPX, an A1R antagonist, did not affect ASC proliferation, while mainly C2 and DPCPX significantly decreased adipogenic differentiation of both ASC types, reducing the activity of glycerol-3-phosphate dehydrogenase and the expression of PPARγ and FABP-4, all adipogenic markers, and phosphorylation of Akt in the phosphatidylinositol-3-kinase pathway, which plays a key-role in adipogenesis. While requiring confirmation in in vivo models, our results suggest that A1R partial agonists or antagonists, by limiting ASC differentiation into adipocytes and, thereby, fat mass expansion, could favor development/worsening of metabolic syndrome in obese subjects without a dietary control.

Partial Agonists and Dual Disorders: Focus on Dual Schizophrenia

Dual disorder is a term applied to patients with an addictive disorder and other mental disorder. Epidemiological studies have established that dual disorders are an expectation rather than an exception. They are difficult to diagnose and treat and constitute a huge burden for both patients and their relatives and society. Current treatments are a combination of those needed to treat the addictive disorder with those focused on the co-occurring psychiatric disorder. Focusing specifically on schizophrenia, growing scientific evidence supports the existence of a shared vulnerability for substance use in these patients and those at risk. Various antipsychotics have been found to be useful in the treatment of psychotic symptoms and disorders; however, few effective treatments have been identified until now for substance use disorders in patients with dual schizophrenia. Partial agonism stands as a new pharmacological option available in recent years. Molecules with this kind of action may act as functional agonists or as antagonists, depending on the surrounding levels of the neurotransmitter. Studies have found their efficacy in schizophrenia, addiction, anxiety and depression. Certain partial agonist antipsychotics seem to have a role in the treatment of dual schizophrenia. That could be the case with cariprazine. Because of its higher affinity for dopaminergic D3 receptors compared to D2, a potential to prevent relapse to addiction, added to its antipsychotic efficacy, has been suggested. Here we briefly review current advances and future directions and introduce some personal insights into the role of partial agonists in co-occurring schizophrenia and substance use.

Reducing Addiction in Bipolar Disorder via Hacking the Dopaminergic System

The dopaminergic system plays a central and decisive role in substance use disorder (SUD), bipolar disorder (BD), and possibly in a subgroup of patients with refractory depression. Common genetic markers and underlying cellular processes, such as kindling, support the close link between these disorders, which is also expressed by the high rate of comorbidity. Although partial dopamine agonists/antagonists acting on D2 and D3 receptors have an established role in treating BD, their usefulness in SUD is less clear. However, dopamine D3 receptors were shown to play a central role in SUD and BD, making D2/D3 partial agonists/antagonists a potential target for both disorders. This narrative review examines whether these substances bear the promise of a future therapeutic approach especially in patients with comorbid BD and SUD.

Fluorescence Activation Mechanism and Imaging of Drug Permeation with New Sensors for Smoking-Cessation Ligands

ABSTRACTNicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters (“iDrugSnFRs”) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives – 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs, in combination with previously described nicotine and varenicline sensors, provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Predicting the Effects of Per- and Polyfluoroalkyl Substance Mixtures on Peroxisome Proliferator-Activated Receptor Alpha Activity in Vitro

Human exposure to per- and polyfluoroalkyl substances (PFAS) is ubiquitous, with mixtures of PFAS detected in drinking water, food, household dust, and other exposure sources. Animal toxicity studies and human epidemiology indicate that PFAS may act through shared mechanisms including activation of peroxisome proliferator activated receptor α (PPARα). However, the effect of PFAS mixtures on human relevant molecular initiating events remains an important data gap in the PFAS literature. Here, we tested the ability of modeling approaches to predict the effect of diverse PPARα ligands on receptor activity using Cos7 cells transiently transfected with a full length human PPARα (hPPARα) expression construct and a peroxisome proliferator response element-driven luciferase reporter. Cells were treated for 24 hours with two full hPPARα agonists (pemafibrate and GW7647), a full and a partial hPPARα agonist (pemafibrate and mono(2-ethylhexyl) phthalate), or a full hPPARα agonist and a competitive antagonist (pemafibrate and GW6471). Receptor activity was modeled with three additive approaches: effect summation, relative potency factors (RPF), and generalized concentration addition (GCA). While RPF and GCA accurately predicted activity for mixtures of full hPPARα agonists, only GCA predicted activity for full and partial hPPARα agonists and a full agonist and antagonist. We then generated concentration response curves for seven PFAS, which were well-fit with three-parameter Hill functions. The four perfluorinated carboxylic acids (PFCA) tended to act as full hPPARα agonists while the three perfluorinated sulfonic acids (PFSA) tended to act as partial agonists that varied in efficacy between 28-67% of the full agonist, positive control level. GCA and RPF performed equally well at predicting the effects of mixtures with three PFCAs, but only GCA predicted experimental activity with mixtures of PFSAs and a mixture of PFCAs and PFSAs at ratios found in the general population. We conclude that of the three approaches, GCA most accurately models the effect of PFAS mixtures on hPPARα activity in vitro.HighlightsPerfluorinated carboxylic acids are full human PPARα agonistsPerfluorinated sulfonic acids are partial human PPARα agonistsGCA predicts human PPARα activity for mixtures of full and partial agonistsGCA predicts human PPARα activity for mixtures of agonists and competitive antagonistsGCA accurately predicts human PPARα activity in response to PFAS mixtures