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A series of benzyloxy and phenoxy derivatives of the adenosine receptor agonists N6-cyclopentyl adenosine (CPA) and N6-cyclopentyl 5'-N-ethylcarboxamidoadenosine (CP-NECA) were synthesized, and their potency and selectivity were assessed. We observed that the most potent were the compounds with a halogen in the meta position on the aromatic ring of the benzyloxy- or phenoxycyclopentyl substituent. In general, the NECA-based compounds displayed greater A1R selectivity than the adenosine-based compounds, with N6-2-(3-bromobenzyloxy)cyclopentyl-NECA and N6-2-(3-methoxyphenoxy)cyclopentyl-NECA showing ∼1500-fold improved A1R selectivity compared to NECA. In addition, we quantified the compounds' affinity and kinetics of binding at both human and rat A1R using a NanoBRET binding assay and found that the halogen substituent in the benzyloxy- or phenoxycyclopentyl moiety seems to confer high affinity for the A1R. Molecular modeling studies suggested a hydrophobic subpocket as contributing to the A1R selectivity displayed. We believe that the identified selective potent A1R agonists are valuable tool compounds for adenosine receptor research.

Antibodies are immensely useful tools for biochemical research and have found application in numerous protein detection and purification methods. Moreover, monoclonal antibodies are increasingly utilised as therapeutics or, conjugated to active pharmaceutical ingredients, in targeted chemotherapy. Several reagents and protocols are reported to synthesise fluorescent antibodies for protein target detection and immunofluorescence applications. However, most of these protocols lead to non-selective conjugation, over-labelling or in the worst case antigen binding site modification. Here, we have used the antibody disulphide cleavage and re-bridging strategy to introduce bright fluorescent dyes without loss of the antibody function. The resulting fluorescent IgG1 type antibodies were shown to be effective imaging tools in western blot and direct immunofluorescence experiments.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.

One third of all medicines act at G protein-coupled receptors (GPCRs). However, the identification of new therapeutic GPCR targets is limited by a fundamental property of GPCRs: their propensity to couple to different G protein alpha subunits (Gα) leading to multiple downstream cellular effects. This is especially the case for adenosine A 1 receptors (A 1 Rs), the activation of which results in unwanted cardiorespiratory effects, severely limiting their clinical potential. We have discovered that the novel A 1 R agonist, BnOCPA, unlike typical A 1 R agonists, has a unique and highly selective Gα activation preference. Moreover, we found that BnOCPA is a potent and powerful analgesic without causing bradycardia, hypotension or respiratory depression. BnOCPA thus demonstrates a hitherto unknown Gα-selective activation of the native A 1 R, sheds new light on the fundamentals of GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of biased agonism. Abbreviated summary We describe the selective activation of an adenosine A 1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of biased agonism.

Epibatidine is an alkaloid toxin that binds with high affinity to nicotinic and muscarinic acetylcholine receptors, and has been extensively used as a research tool. To examine binding interactions at the nicotinic receptor, it has been co-crystallised with the structural homologue acetylcholine binding protein (AChBP; PDB ID 2BYQ), and with an AChBP chimaera (3SQ6) that shares 64% sequence identity with the α7 nACh receptor. However, the binding orientations revealed by AChBP co-crystal structures may not precisely represent their receptor homologues and experimental evidence is needed to verify the ligand poses. Here we identify potential binding site interactions between epibatidine and AChBP residues, and substitute equivalent positions in the α7 nACh receptor. The effects of these are probed by [3H]epibatidine binding following the expression α7 nACh receptor cysteine mutants in HEK 293 cells. Of the sixteen mutants created, the affinity of epibatidine was unaffected by the substitutions Q55C, L106C, L116C, T146C, D160C and S162C, reduced by C186A and C187A, increased by Q114C and S144C, and abolished by W53C, Y91C, N104C, W145C, Y184C and Y191C. These results are consistent with the predicted orientations in AChBP and suggest that epibatidine is likely to occupy a similar location at α7 nACh receptors. We speculate that steric constraints placed upon the C-5 position of the pyridine ring in 3SQ6 may account for the relatively poor affinities of epibatidine derivatives that are substituted at this position., Highlights • The binding orientation of epibatidine is known from AChBP crystal structures. • In silico docking shows this orientation is conserved across AChBP proteins. • By mutagenesis we confirm a similar orientation in α7 nACh receptors. • This suggests a basis for designing epibatidine-based probes.

BACKGROUND AND PURPOSE: TRPM4 is a calcium-activated nonselective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small-molecule inhibitor by combining in silico methods and cell based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors. EXPERIMENTAL APPROACH: Here, we developed a HTS compatible assay to record TRPM4-mediated Na+ influx in cells using a Na+ -sensitive dye and used this assay to screen a small set of compounds selected by ligand-based virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using western blots and electrophysiology experiments. KEY RESULTS: A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also show for the first time that a naturally occurring variant of TRPM4, which display loss-of-expression and function, is rescued by the most promising compound 5 identified in this study. CONCLUSIONS AND IMPLICATIONS: The discovery of compound 5, the most potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.

Crystal structures can identify ligand-receptor interactions and assist the development of novel therapeutics, but experimental challenges sometimes necessitate the use of homologous proteins. Tropisetron is an orthosteric ligand at both 5-HT$_{3}$ and α7 nACh receptors and its binding orientation has been determined in the structural homologue AChBP (pdbid: 2WNC). Co-crystallisation with a structurally-related ligand, granisetron, reveals an almost identical orientation (pdbid; 2YME). However, there is a >1000-fold difference in the affinity of tropisetron at 5-HT$_{3}$ versus α7 nACh receptors, and α7 nACh receptors do not bind granisetron. These striking pharmacological differences prompt questions about which receptor the crystal structures most closely represent and whether the ligand orientations are correct. Here we probe the binding orientation of tropisetron and granisetron at 5-HT$_{3}$ receptors by $\textit{in silico}$ modelling and docking, radioligand binding on cysteine-substituted 5-HT$_{3}$ receptor mutants transiently expressed in HEK 293 cells, and synthetic modification of the ligands. For 15 of the 23 cysteine substitutions, the effects on tropisetron and granisetron were different. Structure-activity relationships on synthesised derivatives of both ligands were also consistent with different orientations, revealing that contrary to the crystallographic evidence from AChBP, the two ligands adopt different orientations in the 5-HT3 receptor binding site. Our results show that even quite structurally similar molecules can adopt different orientations in the same binding site, and that caution may be needed when using homologous proteins to predict ligand binding.

Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [3H]granisetron (Ki = 6.76 µM) and G-FL (Ki = 4.90 µM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 µM, and competed with G-FL with a Ki of 7.94 µM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 µM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects.

The multiple object tracking task (Pylyshyn and Storm, 1988) has long been a standard tool for use in understanding how we attend to multiple moving points in the visual field. In the current experiments, it is first demonstrated that this classical task can be adapted for use in a simulated driving environment, where it is commonly thought to apply. Standard requirements of driving (steering, maintaining headway) are shown to reduce tracking ability. Subsequent experiments (2a, 2b, 2c) investigate the way in which participants respond to events at target and distractor locations, and have bearing on Pylyshyn’s (1989) “indexing” hypothesis. The final experiment investigates the effect of the colour-composition of the tracking set on performance, and may have implications for our theoretical understanding of how tracking is performed. AUTO21, NSERC, CANDrive

Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [3H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects., Graphical abstract, Highlights • Muscarinic ligands scopolamine and atropine also have micromolar affinity at 5-HT3 receptors. • The 5-HT3 receptor ligand granisetron also has micromolar affinity at muscarinic receptors. • Scopolamine has a tetrahedral carbon linker that is responsible for its lower affinity at 5-HT3 receptors. • Scopolamine is used as a preclinical model for inducing cognitive dysfunction. • Use of high concentrations may inhibit 5-HT3 receptors and complicate analysis.

SummaryThe development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective Gα agonism.

Serotonin gated ionotropic 5 HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5 HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective high affinity antagonists such as granisetron (Kytril®) and palonosetron (Aloxi®) belong to a family of drugs (the ‘setrons’) that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo we report the synthesis of 8 fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1 (methyl 11C) N granisetron ([11C]2) through N alkylation with [11C]CH3I respectively. Both compounds [18F]15 (chemical and radiochemical purity >95 specific activity 41 GBq/µmol) and [11C]2 (chemical and radiochemical purity =99 specific activity 170 GBq/µmol) were evaluated for their utility as PET probes. Using mouse and rat brain slices in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5 HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5 HT3 receptors in vivo.