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SummaryWe have previously reported that in rat hippocampal area CA1, the A1R-selective agonist, BnOCPA, potently inhibited excitatory synaptic transmission but did not cause membrane hyperpolarisation in CA1 pyramidal neurons, as would be expected of A1R agonists. This functional discrimination by BnOCPA may arise from its ability, in cAMP inhibition assays, to selectively activate only Gob out of the six Gαi/o subtypes. This may explain why BnOCPA is a potent analgesic that does not cause sedation or cardiorespiratory depression in the rat. Since many preclinical studies are performed using mice, we have here investigated whether BnOCPA’s functional discrimination extends to the mouse. While the potency of BnOCPA against the inhibition of hippocampal synaptic transmission was comparable between rats and mice, we discovered that low concentrations of BnOCPA hyperpolarised mouse CA1 neurons and reduced both their input resistance and firing rate in an A1R-dependent manner. In interleaved experiments we confirmed our previous observations in the rat that concentrations of BnOCPA equivalent to those tested in the mouse had little or no effect on membrane potential or input resistance. Using NanoBRET binding we established that BnOCPA had similar affinity at the mouse and rat A1Rs, and displayed little difference in G protein coupling, as determined using the TRUPATH assay. Thus, although the mechanism for the loss of BnOCPA functional selectivity between pre- and postsynaptic receptors in the mouse hippocampus is currently unclear, it may stem from differences in expression of the individual G proteins subunits or the coupling to murine K+channels.Short summaryWe describe the differential actions of the selective A1R agonist BnOCPA in mouse vs rat hippocampus. In mice, BnOCPA does not show a selectivity between pre and postsynaptic A1Rs, unlike in rats. This may stem from differences in the G protein coupling to K+channels.

A series of benzyloxy and phenoxy derivatives of the adenosine receptor agonists

The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions and can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and therefore, cellular defense mechanisms for superoxide neutralization have evolved. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561(CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potential of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in context of described cytochrome b561 proteins and potential physiological roles of CybB.

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.

BackgroundThe Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all afore-mentioned compounds were screened using assays expressing human TRPM4, whereas the efficacy on mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy.AimIn this study, we performed comparative electro-physiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells.Methods and ResultsWe identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, it increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials.ConclusionAltogether, these results suggest that pharmacological compounds screened using “humanised assays” should be extensively characterised before application in in vivo mouse models.

[Image: see text] Despite being among the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A(1)R) was determined in the active, G(i) protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A(1)R. Our results identify new residues involved in the association and dissociation pathway, they suggest the binding mode of N(6)-cyclopentyladenosine (CPA) related ligands, and they highlight the dramatic effect that chemical modifications can have on the overall binding mechanism, paving the way for the rational development of a structure-kinetics relationship of A(1)R agonists.

Bile salts, such as cholate, glycocholate, taurocholate, and glycochenodeoxycholate, are taken up from the portal blood into hepatocytes via transporters, such as the Na+-taurocholate-cotransporting polypeptide (NTCP) and organic anion-transporting polypeptides (OATPs). These bile salts are later secreted into bile across the canalicular membrane, which is facilitated by the bile salt export pump (BSEP). Apart from bile salt transport, some of these proteins (e.g., OATPs) are also key transporters for drug uptake into hepatocytes. In vivo studies of transporter function in patients by using tracer compounds have emerged as an important diagnostic tool to complement classic liver parameter measurements by determining dynamic liver function both for diagnosis and monitoring progression or improvement of liver diseases. Such approaches include use of radioactively labeled bile salts (e.g., for positron emission tomography) and fluorescent bile salt derivatives or dyes (e.g., indocyanine green). To expand the list of liver function markers, we synthesized fluorescent derivatives of cholic and chenodeoxycholic acid by conjugating small organic dyes to the bile acid side chain. These novel fluorescent probes were able to block substrate transport in a concentration-dependent manner of NTCP, OATP1B1, OATP1B3, OATP2B1, BSEP, and intestinal apical sodium-dependent bile salt transporter (ASBT). Whereas the fluorescent bile acid derivatives themselves were transported across the membrane by OATP1B1, OATP1B3, and OATP2B1, they were not transport substrates for NTCP, ASBT, BSEP, and multidrug resistance-related protein 2. Accordingly, these novel fluorescent bile acid probes can potentially be used as imaging agents to monitor the function of OATPs. SIGNIFICANCE STATEMENT: Synthetic modification of common bile acids by attachment of small organic fluorescent dyes to the bile acid side chain resulted in bright, fluorescent probes that interact with hepatic and intestinal organic anion [organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1], bile salt uptake (Na+-taurocholate-cotransporting polypeptide, apical sodium-dependent bile salt transporter), and bile salt efflux (bile salt export pump, multidrug resistance-related protein 2) transporters. Although the fluorescent bile salt derivatives are taken up into cells via the OATPs, the efflux transporters do not transport any of them but one.

The 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 A

Store-operated heteromeric Orai1/Orai3 channels have been discussed in the context of aging, cancer, and immune cell differentiation. In contrast to homomeric Orai1 channels, they exhibit a different pharmacology upon application of reactive oxygen species (ROS) or 2-aminoethoxydiphenyl borate (2-APB) in various cell types. In endogenous cells, subunit composition and arrangement may vary and cannot be defined precisely. In this study, we used patch-clamp electrophysiology to investigate the 2-APB profile of store-operated and store-independent homomeric Orai1 and heteromeric Orai1/Orai3 concatenated channels with defined subunit compositions. As has been shown previous, one or more Orai3 subunit(s) within the channel result(s) in decreased Ca2+ release activated Ca2+ current (ICRAC). Upon application of 50 &micro, M 2-APB, channels with two or more Orai3 subunits exhibit large outward currents and can be activated by 2-APB independent from storedepletion and/or the presence of STIM1. The number and position of Orai3 subunits within the heteromeric store-operated channel change ion conductivity of 2-APB-activated outward current. Compared to homomeric Orai1 channels, one Orai3 subunit within the channel does not alter 2-APB pharmacology. None of the concatenated channel constructs were able to exactly simulate the complex 2-APB pharmacology observed in prostate cancer cells. However, 2-APB profiles of prostate cancer cells are similar to those of concatenated channels with Orai3 subunit(s). Considering the presented and previous results, this indicates that distinct subtypes of heteromeric SOCE channels may be selectively activated or blocked. In the future, targeting distinct heteromeric SOCE channel subtypes may be the key to tailored SOCE-based therapies.

The serotonin-gated 5-HT3 receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT3 receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [3H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT3 receptor. Detailed analysis by protein-MS/M...

Background and purpose TRPM4 is a calcium-activated non-selective 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 inhibitors 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 high throughput screening 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 showed for the first time that a naturally occurring variant of TRPM4, which displays 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, a 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.

Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC50 being 295 μM. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC50 of 100 μM, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins so called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O Benzylated L threo ß hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB TBOA ((2S3S) 2 amino 3 ((3 (4 (trifluoromethyl) benzamido)benzyl)oxy)succinic acid) standing out as low nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB TBOA in less than a fifth of synthetic steps than the published route. For the first time the inhibitory activity and isoform selectivity of these TFB TBOA enantio and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore we synthesized potent photoaffinity probes based on TFB TBOA using our novel synthetic strategy. KEYWORDS: Glutamate transporters asymmetric synthesis inhibitors aspartate derivatives isoform selectivity photoaffinity probes

The serotonin-gated 5-HT

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.

The introduction of next-generation technologies to the maritime shipping industry, including Portable Pilotage Units, Remote Pilotage, advanced situation awareness aids, and Autonomous Shipping, creates an urgent need to understand operator workload during Bridge Team operations, and co-operations with shore based personnel. In this paper we analyse mental workload of maritime Captains, Pilots and Tug Masters during standard and emergency scenarios, using traditional measures (SWAT, ISA), communications analysis, and the collection of simultaneous electro-dermal activity (EDA) of team members. Results indicate that the EDA measure overcomes some of the problems with paper-based techniques, and has excellent temporal resolution for emergency events. Implications for testing of novel technologies are discussed.

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

TRPM4 is a calcium-activated non-selective 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 inhibitors by combining in silico methods and cell-based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors.Here, we developed a high throughput screening compatible assay to record TRPM4-mediated NaA series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss-of-expression and function, is rescued by the most promising compound 5 identified in this study.The discovery of compound 5, a 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.

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [(3)H]granisetron. Kinetic measurements (kon = 4.0 × 10(4) M(-1) s(-1) ; koff = 0.23 s(-1)) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

Many contend that driving an automobile involves multiple-object tracking. At this point, no one has tested this idea, and it is unclear how multiple-object tracking would coordinate with the other activities involved in driving. To address some of the initial and most basic questions about multiple-object tracking while driving, we modified the tracking task for use in a driving simulator, creating the multiple-vehicle tracking task. In Experiment 1, we employed a dual-task methodology to determine whether there was interference between tracking and driving. Findings suggest that although it is possible to track multiple vehicles while driving, driving reduces tracking performance, and tracking compromises headway and lane position maintenance while driving. Modified change-detection paradigms were used to assess whether there were change localization advantages for tracked targets in multiple-vehicle tracking. When changes occurred during a blanking interval, drivers were more accurate (Experiment 2a) and ~250 ms faster (Experiment 2b) at locating the vehicle that changed when it was a target rather than a distractor in tracking. In a more realistic driving task where drivers had to brake in response to the sudden onset of brake lights in one of the lead vehicles, drivers were more accurate at localizing the vehicle that braked if it was a tracking target, although there was no advantage in terms of braking response time. Overall, results suggest that multiple-object tracking is possible while driving and perhaps even advantageous in some situations, but further research is required to determine whether multiple-object tracking is actually used in day-to-day driving.

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-β-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

Serotonin-gated ionotropic 5-HT

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-HT3 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-HT3 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-HT3 receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT3 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., Highlights • The drugs granisetron and tropisetron are structurally similar. • Crystals of them bound to AChBP suggest they have similar binding orientations. • At 5-HT3R, the effects of mutagenesis indicate that their orientations differ. • SAR on both of these drugs also supports different orientations.

Graphical abstract, The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT3 receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT3AR.

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

A series of N(6)-bicyclic and N(6)-(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N(6)-adamantyl substitution in combination with 5'-N-ethylcarboxamido or 5'-hydroxymethyl groups. In addition, we determined that 5'-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compounds might have therapeutic potential.

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors.

The crystal structure of the resting state of cytochrome P450cam (CYP101), a heme thiolate protein, shows a cluster of six water molecules in the substrate binding pocket, one of which is coordinating to iron(III) as sixth ligand. The resting state is low-spin and changes to high-spin when substrate camphor binds and H2O is removed. In contrast to the protein, previously synthesised enzyme models such as H2O[BOND]FeIII(porph)(ArS−) were shown to be purely high-spin. Iron(S−)porphyrins with different distal sites mimicking proposed remote effects have been prepared and studied by cw-EPR. The results indicate that the low-spin of the resting state of P450cam is due to the fact that the water molecule coordinating to iron has an OH−-like character because of hydrogen bonding and polarisation of the water cluster, respectively.