Equilibrium Binding Studies Research Articles

Dual allosteric and orthosteric pharmacology of synthetic analog cannabidiol-dimethylheptyl, but not cannabidiol, on the cannabinoid CB2 receptor

Cannabinoid CB2 receptor (CB2R) is a class A G protein-coupled receptor (GPCR) involved in a broad spectrum of physiological processes and pathological conditions. For that reason, targeting CB2R might provide therapeutic opportunities in neurodegenerative disorders, neuropathic pain, inflammatory diseases, and cancer. The main components from Cannabis sativa, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been therapeutically exploited and synthetically-derived analogs have been generated. One example is cannabidiol-dimethylheptyl (CBD-DMH), which exhibits anti-inflammatory effects. Nevertheless, its pharmacological mechanism of action is not yet fully understood and is hypothesized for multiple targets, including CB2R. The aim of this study was to further investigate the molecular pharmacology of CBD-DMH on CB2R while CBD was taken along as control. These compounds were screened in equilibrium and kinetic radioligand binding studies and various functional assays, including G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. In dissociation studies, CBD-DMH allosterically modulated the radioligand binding. Furthermore, CBD-DMH negatively modulated the G protein activation of reference agonists CP55,940, AEA and 2-AG, but not the agonist-induced ß-arrestin-2 recruitment. Nevertheless, CBD-DMH also displayed competitive binding to CB2R and partial agonism on G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. CBD did not exhibit such allosteric behavior and only very weakly bound CB2R without activation. This study shows a dual binding mode of CBD-DMH, but not CBD, to CB2R with the suggestion of two different binding sites. Altogether, it encourages further research into this dual mechanism which might provide a new class of molecules targeting CB2R.

Sterol-activated amyloid beta fibril formation

The metabolic processes that link Alzheimer’s disease (AD) to elevated cholesterol levels in the brain are not fully defined. Amyloid beta (Aβ) plaque accumulation is believed to begin decades prior to symptoms and to contribute significantly to the disease. Cholesterol and its metabolites accelerate plaque formation through as-yet undefined mechanisms. Here, the mechanism of cholesterol (CH) and cholesterol 3-sulfate (CS) induced acceleration of Aβ fibril formation is explored in quantitative ligand binding, Aβ fibril polymerization, and molecular dynamics studies. Equilibrium and presteady-state binding studies reveal that monomeric Aβ42•ligand complexes form and dissociate rapidly relative to oligomerization, that the ligand/peptide stoichiometry is 1-to-1 and that the peptide is likely saturated in vivo. Analysis of Aβ42 polymerization progress curves demonstrates that ligands accelerate polymer synthesis by catalyzing conversion of peptide monomers into dimers that nucleate the polymerization reaction. Nucleation is accelerated ∼49-fold by CH, and ∼13,000-fold by CS — a minor CH metabolite. Polymerization kinetic models predict that at presumed disease-relevant CS and CH concentrations, approximately half of polymerization nuclei will contain CS, small oligomers of neurotoxic dimensions (∼12-mers) will contain substantial CS, and fibril-formation lag times will decrease 13-fold relative to unliganded Aβ42. Molecular dynamics models, which quantitatively predict all experimental findings, indicate that the acceleration mechanism is rooted in ligand-induced stabilization of the peptide in non-helical conformations that readily form polymerization nuclei.

The Impact of Solution Ionic Strength, Hardness, and pH on the Sorption Efficiency of Polychlorinated Biphenyls in Magnetic Nanocomposite Microparticle (MNM) Gels

Environmental conditions of groundwater and surface water greatly vary as a function of location. Factors such as ionic strength, water hardness, and solution pH can change the physical and chemical properties of the nanocomposites used in remediation and the pollutants of interest. In this work, magnetic nanocomposite microparticle (MNM) gels are used as sorbents for remediation of PCB 126 as model organic contaminant. Three MNM systems are used: curcumin multiacrylate MNMs (CMA MNMs), quercetin multiacrylate MNMs (QMA MNMs), and polyethylene glycol-400-dimethacrylate MNMs (PEG MNMs). The effect of ionic strength, water hardness, and pH were studied on the sorption efficiency of the MNMs for PCB 126 by performing equilibrium binding studies. It is seen that the ionic strength and water hardness have a minimal effect on the MNM gel system sorption of PCB 126. However, a decrease in binding was observed when the pH increased from 6.5 to 8.5, attributed to anion-π interactions between the buffer ions in solution and the PCB molecules as well as with the aromatic rings of the MNM gel systems. Overall, the results indicate that the developed MNM gels can be used as magnetic sorbents for polychlorinated biphenyls in groundwater and surface water remediation, provided that the solution pH is controlled.

Curvature dependence of BAR protein membrane association and dissociation kinetics

BAR (Bin/Amphiphysin/Rvs) domain containing proteins function as lipid bilayer benders and curvature sensors, and they contribute to membrane shaping involved in cell signaling and metabolism. The mechanism for their membrane shape sensing has been investigated by both equilibrium binding and kinetic studies. In prior research, stopped-flow spectroscopy has been used to deduce a positive dependence on membrane curvature for the binding rate constant, kon, of a BAR protein called endophilin. However, the impact of bulk diffusion of endophilin, on the kinetic binding parameters has not been thoroughly considered. Employing similar methods, and using lipid vesicles of multiple sizes, we obtained a linear dependence of kon on vesicle curvature. However, we found that the observed relation can be explained without considering the local curvature sensing ability of endophilin in the membrane association process. In contrast, the diffusion-independent unbinding rate constant (koff) obtained from stopped-flow measurements shows a negative dependence on membrane curvature, which is controlled/mediated by endophilin-membrane interactions. This latter dependency, in addition to protein–protein interactions on the membrane, explains the selective binding of BAR proteins to highly curved membranes in equilibrium binding experiments.

De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G

Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10−5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83–93% and yields of 11–89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.

Chemisch‐biologischer Werkzeugkasten für die intrazelluläre Bindungsstelle von CCR9: Fluoreszierende Liganden, neue Leitstrukturen und PROTACs

AbstractEine konservierte intrazelluläre allosterische Bindungsstelle (IABS) wurde kürzlich an mehreren G‐Protein‐gekoppelten Rezeptoren (GPCRs) identifiziert. Ausgehend von Vercirnon, einem intrazellulären Antagonisten am C−C‐Chemokin‐Rezeptor‐Typ‐9 (CCR9), entwickelten wir molekulare Werkzeuge, die an der IABS von CCR9 angreifen. Zunächst synthetisierten wir fluoreszierende CCR9‐Liganden, die Bindungsstudien via NanoBRET sowie Fluoreszenzmikroskopie ermöglichten. Deren Anwendung in zellfreien sowie zellulären Testsystemen erlaubte die Identifizierung eines 4‐Aminopyrimidin‐Analogons als neuen intrazellulären CCR9‐Antagonisten mit verbesserter Affinität. Um den proteasomalen Abbau von CCR9 einzuleiten, entwickelten wir das erste PROTAC, das die IABS von GPCRs als Angriffsort nutzt. In einer Machbarkeitsstudie zeigen wir, dass unser CCR9‐PROTAC in der Lage ist, die CCR9‐Spiegel zu reduzieren, was einen konzeptionell neuartigen Ansatz zur Modulation der GPCR‐Aktivität darstellt.

A Chemical Biology Toolbox Targeting the Intracellular Binding Site of CCR9: Fluorescent Ligands, New Drug Leads and PROTACs.

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein‐coupled receptors (GPCRs). Starting from vercirnon, an intracellular C−C chemokine receptor type 9 (CCR9) antagonist and previous phase III clinical candidate for the treatment of Crohn's disease, we developed a chemical biology toolbox targeting the IABS of CCR9. We first synthesized a fluorescent ligand enabling equilibrium and kinetic binding studies via NanoBRET as well as fluorescence microscopy. Applying this molecular tool in a membrane‐based setup and in living cells, we discovered a 4‐aminopyrimidine analogue as a new intracellular CCR9 antagonist with improved affinity. To chemically induce CCR9 degradation, we then developed the first PROTAC targeting the IABS of GPCRs. In a proof‐of‐principle study, we succeeded in showing that our CCR9‐PROTAC is able to reduce CCR9 levels, thereby offering an unprecedented approach to modulate GPCR activity.

Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-β-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.

Interactions of a Signal Transduction Protein Investigated by Fluorescence Stopped-Flow Kinetics.

To understand cellular processes such as biochemical pathways and signaling networks, we need to understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational modifications, or other mechanisms. To do so, we break these systems down into their elementary steps, which are almost invariably either unimolecular or bimolecular reactions that frequently occur on sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing in less than 2ms, are generally suitable for the study of such reactions. The application of these techniques to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis, 1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the pre-steady state) under single turnover conditions.The individual steps in complex biochemical reaction schemes determine how fast systems can respond to incoming signals and adapt to changed conditions[1, 2]. This chapter is concerned with in vitro techniques that have been developed to study fast reactions in solution, and we present the study of various interactions of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

The selective 5-HT2A receptor agonist 25CN-NBOH: Structure-activity relationship, in vivo pharmacology, and in vitro and ex vivo binding characteristics of [3H]25CN-NBOH

The remarkable effects exhibited by classical psychedelics in recent clinical trials have spawned considerable interest in 5-HT2A receptor (5-HT2AR) activation as a treatment strategy for several psychiatric/cognitive disorders. In this study we have continued our development of 25CN-NBOH, one of the most 5-HT2AR-selective agonists reported to date, as a pharmacological tool for exploration of 5-HT2AR expression and functions. The importance of the 2′ and 3′ positions in 25CN-NBOH as structural hotspots for its 5-HT2AR activity was investigated by synthesis and pharmacological characterization of six novel analogs at 5-HT2AR and 5-HT2CR in binding and functional assays. While the 5-HT2AR activity of 25CN-NBOH was retained in 3′-methyl, 2′,3′-chroman, 2′,3′-dihydrofuran and 2′,3′-furan analogs, the 3′-methoxy and 3′-ethyl analogs displayed substantially lower binding affinities and agonist potencies than 25CN-NBOH. Interestingly, the 2′,3′-substitution pattern was also a key determinant of agonist efficacy, as all six analogs exhibited low-efficacy partial agonism or de facto antagonism at the 5-HT2AR in the functional assays. Systemic administration of 25CN-NBOH and its close structural analog 25CN-NBMD induced robust head-twitch response in mice, a well-established behavioural effect of 5-HT2AR activation in vivo, and 25CN-NBOH mediated robust reductions in the activity of mice in an anxiety-related marble burying assay, which supports the proposed beneficial effects of 5-HT2AR activation on disorders characterized by cognitive rigidity. Finally, tritiated 25CN-NBOH exhibited high 5-HT2AR binding affinity (KD ~1 nM) and selectivity against 5-HT2BR and 5-HT2CR in equilibrium and kinetic binding studies of the recombinant receptors, and in concordance [3H]25CN-NBOH displayed substantial specific, ketanserin-sensitive binding to cortex and small levels of binding to choroid plexus in rat brain slices in autoradiography studies. In conclusion, this work delineates the subtle molecular determinants of the 5-HT2AR activity in 25CN-NBOH, substantiates the potential in this compound and its analogs as tools for in vivo studies of the 5-HT2AR, and introduces a novel selective agonist radioligand as another potentially valuable tool for future explorations of this receptor.

Preparation and evaluation of phosphate binding capacity of micronized lanthanum carbonate formulation

The aim of this study was to prepare lanthanum carbonate formulations and to investigate the effects of particle size reduction of lanthanum carbonate to the submicron/micron (SM/M) range by ball milling process. It is a common way to enhance the dissolution rate and therefore the bioactivity of poorly water soluble or insoluble active drug substances by using nanotechnological methods. For this purpose, one of the methods is to prepare drug microcrystals/nanocrystals by decreasing the particle size and thus, increasing the surface area for enhancement of dissolution rate and bioactivity. For this purpose, assay analysis, in-vitro dissolution rates and bioactivity of the formulations were investigated. Assay and in-vitro dissolution rate were analysed by a validated complexometric titration method. Phosphate binding and equilibrium binding studies within the scope of bioactivity studies were carried out by a validated ion chromatography. As a result of these experiments, the phosphate binding capacity and dissolution rate of SM/M lanthanum formulation were found to be higher than both lanthanum formulation (prepared by untreated lanthanum carbonate) and the reference product-Fosrenol® 1000 mg Sachet.

Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study.

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore “resting-like” under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD’s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

Biophysical Characterization of Aptenodytes forsteri Cytochrome P450 19 (Aromatase)

Cytochrome P450 19 (aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. As the terminal enzyme in estrogen biosynthesis, it is critical for the maintenance of myriad tissues and has emerged as a successful target for the treatment of estrogen‐dependent breast cancer. Aromatase is a phylogenetically‐ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming of neural circuits controlling song. Aromatase also plays a role in sex determination of fish, reptiles, and amphibians. Biophysical characterization of the human enzyme has been limited by low yields from recombinant expression systems. Accordingly, we sought to identify an aromatase homolog with enhanced expression properties in the absence of stabilizing ligands. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin) aromatase (afCYP19A1). Using human cytochrome P450 reductase as a redox partner, the enzyme kinetic parameters for turnover of androstenedione were like the human enzyme and mass spectrometry analysis conformed that afCYP19A1 likewise catalyzes the transformation through 19‐hydroxy‐ and 19‐oxoandrostenedione intermediates. Equilibrium binding studies revealed that androstenedione and anastrozole had the highest affinity for the enzyme and were closely followed by 19‐hydroxy‐androstenedione and testosterone. The affinity of 19‐oxoandrostenedione was an order‐of‐magnitude lower. Ligand‐binding kinetics were determined by stopped‐flow UV‐visible spectroscopy. Both androstenedione and testosterone where determined to bind to the enzyme in a step‐wise fashion with a spectroscopically‐detectable intermediate. In summary, these studies describe the first biophysical characterization of a non‐human aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme despite bearing 75% sequence identity. In addition, they provide access to an alternative model to study the catalytic mechanism of the enzyme using methods requiring substantial amounts of ligand‐free enzyme.Support or Funding InformationNational Institutes of Health National Institute of General Medical Sciences [Grant R01GM114168]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro Bioequivalence Studies in Tablet Formulation Containing 625 mg of Colesevelam Hydrochlorides

Colesevelam hydrochloride is a second generation bile acid sequestrant that is used principally for treatment of elevated LDL cholesterol. Additionally, it is indicated to improve glycemic control in adults with type 2 diabetes mellitus. Colesevelam hydrochloride is an insoluble, non-absorbed polymer that binds bile acids in the intestine, impeding their reabsorption. Because the drug is not absorbed into the systemic circulation, pharmacokinetic information is not available. The present abstract focuses on the study of in vitro BE in tablet formulation containing 625 mg of Colesevelam hydrochloride. Bile acid salts of Glycocholic Acid (GC), Glycochenodeoxycholic Acid (GCDC) and Taurodeoxycholic Acid (TDC) were used in vitro BE studies. The binding capacity HPLC method was developed and validated for these bile acid salts. The equilibrium binding study that is the pivotal BE study and in vitro kinetic binding study that is the support the pivotal equilibrium binding study was repeated twelve times. In the in vitro equilibrium binding studies, the Langmuir binding constants k1 (affinity) and k2 (capacity) were calculated for the three salts, individually and combined (GC+GCDC+TDC) using both nonlinear Langmuir equation and linear Langmuir equation for the test and reference products. The calculated capacity constant (k2), the more important parameter, were obtained very similar between test and reference products in the 90% confidence interval and acceptance criteria of 80% to 120%. The test/reference ratio for capacity constant, k2, was obtained 0.918 and 0.922 using equation 1 and 2 for total bile acid salts from the without acid pre-treatment equilibrium binding study and 0.948, 0.953 using equation 1 and 2 for total bile acid salts from the with acid pre-treatment equilibrium binding study.

Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.

Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has ‘post-recruitment’ roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from −4 to +1 in the open complex with all three proteins and from −4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.

Enhanced potency of bivalent small molecule gp41 inhibitors

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40–60times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors

The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT3ARs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT3ARs were heterologously expressed in HEK-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT3ARs reversibly and dose-dependently with inhibitory potencies of 87 μM and 112 μM, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect.

Isozyme Specific Allosteric Regulation of Human Sulfotransferase 1A1.

The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates the activities of hundreds, perhaps thousands, of signaling small molecules via regiospecific transfer of the sulfuryl moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyls and amines of acceptors. Signaling molecules regulated by sulfonation include numerous steroid and thyroid hormones, epinephrine, serotonin, and dopamine. SULT1A1, a major phase II metabolism SULT isoform, is found at a high concentration in liver and has recently been show to harbor two allosteric binding sites, each of which binds a separate and complex class of compounds: the catechins (naturally occurring polyphenols) and nonsteroidal anti-inflammatory drugs. Among catechins, epigallocatechin gallate (EGCG) displays high affinity and specificity for SULT1A1. The allosteric network associated with either site has yet to be defined. Here, using equilibrium binding and pre-steady state studies, the network is shown to involve 14 distinct complexes. ECGG binds both the allosteric site and, relatively weakly, the active site of SULT1A1. It is not a SULT1A1 substrate but is sulfonated by SULT2A1. EGCG binds 17-fold more tightly when the active-site cap of the enzyme is closed by the binding of the nucleotide. When nucleotide is saturating, EGCG binds in two phases. In the first, it binds to the cap-open conformer; in the second, it traps the cap in the closed configuration. Cap closure encapsulates the nucleotide, preventing its release; hence, the EGCG-induced cap stabilization slows nucleotide release, inhibiting turnover. Finally, a comprehensive quantitative model of the network is presented.

Kinetic mechanism for binding and flipping of damaged bases by DNA repair dealkylases

DNA is susceptible to alkylation by endogenous metabolites and by exogenous reactive species and multiple pathways have evolved to detect and repair the sites of alkylation damage. The best characterized pathway employs DNA repair glycosylases to remove alkylated bases and initiate the multi‐step base excision repair pathway. In contrast, the AlkB family of direct repair enzymes use oxygen, iron(II) and 2‐oxoglutarate to catalyze the oxidative removal of alkyl groups from nucleobases and restore them to their natural structures in a single step. Structural work shows that base flipping is used to bring the alkylated base into proximity of the catalytic iron, but kinetic studies have been difficult and crucial information is lacking regarding the timing of the searching, flipping and chemical steps. We have used equilibrium binding and transient kinetic studies to define the minimal kinetic mechanism for AlkB enzymes. We compare this mechanism to that which is employed by DNA repair glycosylases. Interesting parallels between these different pathways have been uncovered that provide insight into the fundamental problem of how aberrant methyl groups can be detected in duplex DNA. However, there are also several differences that are relevant to pathway choice in cells where both pathways are operational.