Shivange, A. V. | Search bioRxiv

bioRxiv2021

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.

pharmacology and toxicology↗

bioRxiv2020

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

neuroscience↗

bioRxiv2019

Structural features underlying the activity of benzimidazole derivatives that target phosphopeptide recognition by the tandem BRCT domain of the BRCA1 protein

The tandem BRCT (tBRCT) domains of BRCA1 engage pSer-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, a benzimidazole that represents a first selective small molecule inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domains, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features that underlie the biochemical and cellular activities of Bractoppin. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing single residue substitutions that alter K1702, a key residue mediating phosphopeptide recognition (K1702A), or alter hydrophobic residues (F1662R or L1701K) that adjoin the pSer-recognition site. However, mutation of BRCA1 tBRCT residue M1775R, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding. Collectively, these findings confirm a binding mode for Bractoppin that blocks the phosphopeptide-binding site via structural features distinct from the substrate phosphopeptide. We explored these structural features through structure-guided chemical elaboration of Bractoppin, synthesizing analogs bearing modifications on the left and right hand side (LHS/RHS) of Bractoppins benzimidazole ring. Characterization of these analogs in biochemical assay reveal structural features underlying potency. Analogs where the LHS phenyl is replaced by cyanomethyl (2091) and 4-methoxyphenoxypropyl (2113) conceptualized from structure-guided strategies like GIST and dimer interface analysis expose the role of phenyl and isopropyl as critical hydrophobic anchors. Two Bractoppin analogs, 2088 and 2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the biochemical and cellular activity of a novel benzimidazole inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target the protein-protein interactions of this previously undrugged family of protein domains.

cancer biology↗