Ultrapotent chemogenetics for research and potential clinical applications

Magnus, Christopher; Lee, Peter H.; Bonaventura, Jordi; Zemla, Roland; Gómez, Juan L.; Ramirez, Melissa H.; Hu, Xing; Galván, Adriana; Basu, Jayeeta; Michaelides, Michael; Sternson, Scott M.

doi:10.1126/science.aav5282

Cited by 145 publications

(150 citation statements)

References 75 publications

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“…Only iDianiSnFR, which lacks His68, binds varenicline with EC 50 > 10 μM. Even tighter binding has been achieved with varenicline derivatives at mutated ligand-gated channels ( Magnus et al, 2019 ). On the one hand, the cellular experiments described here cannot use iDrugSnFR pairs with dissociation rate constants less than ~0.1 s –1 , corresponding to an EC 50 of less than ~100 nM.…”

Section: Discussionmentioning

confidence: 99%

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

Nichols

Blumenfeld

Fan

et al. 2022

eLife

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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.

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Section: Discussionmentioning

confidence: 99%

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

Nichols

Blumenfeld

Fan

et al. 2022

eLife

View full text Add to dashboard Cite

show abstract

“…Only iDianiSnFR, which lacks His68, binds varenicline with EC50 > 10 μM. Even tighter binding has been achieved with varenicline derivatives at mutated ligand-gated channels (Magnus, et al, 2019). On the one hand, the cellular experiments described here cannot use iDrugSnFR pairs with dissociation rate constants less than ~ 0.1 s -1 , corresponding to an EC50 of less than ~ 100 nM.…”

Section: The Idrugsnfr Paradigmmentioning

confidence: 86%

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

Nichols

Blumenfeld

Fan

et al. 2021

Preprint

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Nicotinic partial agonists provide a partial 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. Development of the series was aided and explained by the first atomic-scale structural studies on liganded periplasmic binding protein-based biosensors. Members of the series detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and primary 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.

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“…Genetic tools have made a dramatic contribution to a better understanding of the nervous system. Thus human genetic studies have identified causative mutations for a variety of disorders 29 , whilst gene and cell silencing 30 and in particular activity-based reporter systems such as CANE have proved extraordinarily informative 31 .…”

Section: Discussionmentioning

confidence: 99%

Tools for analysis and conditional deletion of subsets of sensory neurons

et al. 2021

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Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.

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Ultrapotent chemogenetics for research and potential clinical applications

Cited by 145 publications

References 75 publications

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

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

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

Tools for analysis and conditional deletion of subsets of sensory neurons

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