A new, simple and robust radioligand binding method used to determine kinetic off-rate constants for unlabeled ligands. Application at α2A- and α2C-adrenoceptors.

Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form. Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing α2A- and α2C-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the koff constants for various unlabeled ligands. The results show that the α2C-selectivity of MK912 depends mainly on a very slow off-rate at the α2C-adrenoceptor subtype. Regarding the markedly α2C- over α2A-selective compound spiroxatrine, its much faster on-rate at α2C- than α2A-adrenoceptors explains much of its exceptional α2C-selectivity. Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining koff constants of unlabeled drugs.

The C-terminal half of the α2C-adrenoceptor determines the receptor's membrane expression level and drug selectivity.

In tissues as well as in transfected cells, α2C-adrenoceptors show poorer expression levels compared to α2A-adrenoceptors. In order to characterize which regions of the α2C-adrenoceptor are involved in regulating the expression of binding-competent receptors at the plasma membrane, six chimeric α2A-/α2C-adrenoceptors were constructed. The wild-type α2A- and α2C-adrenoceptors and the six chimeric α2A-/α2C-adrenoceptors were transiently transfected into human embryonic kidney 293 (HEK293) cells, and the expression levels were investigated by radioligand binding. The results show that the C-terminal half of the α2C-adrenoceptor, ranging from the second extracellular loop to the C-terminus, is the main determinant of the low expression level of binding-competent α2C-adrenoceptors in HEK293 cell membranes. The so-called retention signal in the N-terminus of the α2C-adrenoceptor had a less profound effect on the expression levels of the chimeric receptors. For seven drugs competing for [(3)H]-RX821002 binding, the K i values were determined at the wild-type α2A- and α2C-adrenoceptors and at four of the chimeric α2A-/α2C-adrenoceptors. The results show that the α2C- over α2A-selectivity of spiroxatrine, spiperone, clozapine, MK912, and chlorpromazine, as well as the α2A- over α2C-selectivity of BRL44408, resides mainly in the C-terminal half of the receptors. To some extent, the region comprising the N-terminal half of the receptors contributed to the α2C-selectivity of spiperone, clozapine, and chlorpromazine.

The predicted N-terminal signal sequence of the human α2C-adrenoceptor does not act as a functional cleavable signal peptide.

The N-terminal region of the human α(2C)-adrenoceptor has a 22 amino acid sequence MASPALAAALAVAAAAGPNASG. This stretch is predicted to be a cleavable signal peptide. Signal peptides facilitate the translocation of membrane proteins from ribosomes into the endoplasmatic reticulum (ER) for further transport to the plasma membrane. However, recently it has been suggested that the hydrophobic stretch ALAAALAAAAA in the N-tail of the rat α(2C)-adrenoceptor, rather than being part of a signal peptide, is an ER retention signal (Angelotti, 2010). Here, we have investigated the functionality of the N-terminal region of the human α(2C)-adrenoceptor further. The predicted signal peptide was found to be non-cleavable, as shown for a modified α(2C)-adrenoceptor construct equipped with a FLAG epitope. The influence of the N-terminal region on receptor translocation to the plasma membrane was investigated by rebuilding the N-tail and then by analyzing the expression level of binding-competent receptors in transfected COS-7 cell membranes. Truncated α(2C)-adrenoceptor constructs showed decreased expression levels as compared to the wild type α(2C)-adrenoceptor. Addition of, or exchange for, the influenza virus hemagglutinin signal peptide to the α(2C)-adrenoceptor had no effect, respectively decreased, the expression level of binding-competent receptor in the membranes. Our analysis supports the conclusions that the predicted signal peptide in the N-terminal tail of the α(2C)-adrenoceptor does not act as a cleavable signal peptide. In addition, the results indicate that the presence of an intact N-tail is augmenting the amount of binding-competent α(2C)-adrenoceptors at the cell surface.

The N-terminal region of the human 5-HT2C receptor has as a cleavable signal peptide.

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor has a single nucleotide polymorphism (SNP) site at amino acid position 23 in its N-terminal tail. The polymorphism involves conversion of a cysteine to serine. The site, designated C23S, is located within a 32 amino acid long predicted signal peptide. The aim of the present study was to investigate whether the 5-HT(2C) receptor indeed has a functional cleavable signal peptide. For this purpose, ten N-terminally modified 5-HT(2C) receptors were constructed. Modifications included addition of the influenza virus hemagglutinin signal peptide, addition of a FLAG epitope, truncation of the N-terminal tail, and combinations of these changes. The receptors were transiently expressed in COS-7 cells. The relative amounts of receptors expressed at the membranes were quantified by [(3)H]-mesulergine radioligand binding. In one of the receptor constructs the FLAG epitope was inserted just after the endogenous putative signal peptide. Immunostaining with the M1 antibody, which recognizes the FLAG epitope only as free N-terminal entity, was used to detect whether the putative signal peptide preceding the FLAG epitope was cleaved off. The results suggest the following conclusions. The predicted signal peptide in the N-terminal tail of the 5-HT(2C) receptor acts as a cleavable signal peptide. Cleaving of the signal peptide is important for translocation of the wild type receptor to the plasma membrane. The two amino acids differentially encoded by the C23S SNP are likely absent from the mature 5-HT(2C) receptor.

Proteochemometric analysis of small cyclic peptides' interaction with wild-type and chimeric melanocortin receptors.

The melanocortin (MC) system confines unique G-protein coupled receptor pathways, which include the MC(1-5) receptors and their endogenous agonists and antagonists, the MCs and the agouti and agouti-related proteins. The MC4 receptor is an important target for development of drugs for treatment of obesity and cachexia. While natural MC peptides are selective for the MC1 receptor, some cyclic pentapeptides, such as the HS-129 peptide, show high selectivity for the MC4 receptor. Here we gained insight into the mechanisms for its recognition by MC receptors. To this end we correlated the interaction data of four HS peptide analogues with four wild-type and 14 multiple chimeric MC receptors to the binary and physicochemical descriptions of the studied entities by use of partial least squares regression, which resulted in highly valid proteochemometric models. Analysis of the models revealed that the recognition sites of the HS peptides are different from the earlier proteochemometrically mapped linear MSH peptides' recognitions sites, although they overlap partially. The analysis also revealed important amino acids that explain the selectivity of the HS-129 peptide for the MC4 receptor.

Proteochemometric modeling reveals the interaction site for Trp9 modified alpha-MSH peptides in melanocortin receptors.

The interactions of alpha-MSH peptides with melanocortin receptors (MCRs) were located by proteochemometric modeling. Nine alpha-MSH peptide analogues were constructed by exchanging the Trp9 residue in the alpha-MSH core with the natural or artificial amino acids Arg, Asp, Cys, Gly, Leu, Nal, d-Nal, Pro, or d-Trp. The nine peptides created, and alpha-MSH itself, were evaluated for their interactions with the 4 wild-type MC(1,3-5)Rs and 15 multichimeric MCRs, each of the latter being constructed from three sequence segments, each taken from a different wild-type MC(1,3-5)R. The segments of the chimeric MCRs were selected according to the principles of statistical molecular design and were arranged so as to divide the receptors into five parts. By this approach, a set of 19 maximally diverse MC receptor proteins was obtained for which the interaction activity with the 10 peptides were measured by radioligand binding thus creating data for 190 ligand-protein pairs, which were subsequently analyzed by use of proteochemometric modeling. In proteochemometrics, the structural or physicochemical properties of both interaction partners, which represent the complementarity of the interacting entities, are used to create multivariate mathematical descriptions. (Here, physicochemical property descriptors of the receptors' and peptides' amino acids were used). A valid, highly predictive (Q2 = 0.74) and easily interpretable model was then obtained. The model was further validated by its ability to correctly predicting the affinity of alpha-MSH for new point and cassette-mutated MC4/MC1Rs, and it was then used to identify the receptor residues that are important for affording the high affinity and selectivity of alpha-MSH for the MC1R. It was revealed that these residues are located in several quite distant parts of the receptors' transmembrane cavity and must therefore cause their influence at various stages of the dynamic ligand-binding process, such as by affecting the conformation of the ligand at the vicinity of the receptor and taking part in the path of the ligand's entry into its binding pocket. Our study can be used as a template how to create high resolution proteochemometric models when there are a limited number of natural proteins and ligands available.

Prediction of indirect interactions in proteins.

BACKGROUND: Both direct and indirect interactions determine molecular recognition of ligands by proteins. Indirect interactions can be defined as effects on recognition controlled from distant sites in the proteins, e.g. by changes in protein conformation and mobility, whereas direct interactions occur in close proximity of the protein's amino acids and the ligand. Molecular recognition is traditionally studied using three-dimensional methods, but with such techniques it is difficult to predict the effects caused by mutational changes of amino acids located far away from the ligand-binding site. We recently developed an approach, proteochemometrics, to the study of molecular recognition that models the chemical effects involved in the recognition of ligands by proteins using statistical sampling and mathematical modelling. RESULTS: A proteochemometric model was built, based on a statistically designed protein library's (melanocortin receptors') interaction with three peptides and used to predict which amino acids and sequence fragments that are involved in direct and indirect ligand interactions. The model predictions were confirmed by directed mutagenesis. The predicted presumed direct interactions were in good agreement with previous three-dimensional studies of ligand recognition. However, in addition the model could also correctly predict the location of indirect effects on ligand recognition arising from distant sites in the receptors, something that three-dimensional modelling could not afford. CONCLUSION: We demonstrate experimentally that proteochemometric modelling can be used with high accuracy to predict the site of origin of direct and indirect effects on ligand recognitions by proteins.

Nandrolone treatment decreases the alpha1B-adrenoceptor mRNA level in rat kidney, but not the density of alpha1B-adrenoceptors in cultured MDCK-D1 kidney cells.

We have previously shown that treatment of rats with the anabolic androgen steroid nandrolone decreased the density of alpha1B-adrenoceptors in the rat kidney [Uhlén, S., Lindblom, J., Kindlundh, A., Muhisha, P., Nyberg, F., (2003). Nandrolone treatment decreases the level of rat kidney alpha1B-adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmacol. 368, 91-98]. This effect may have been caused either by decreased de novo synthesis of alpha1B-adrenoceptors or by increased degradation of alpha1B-adrenoceptors [corrected] In the present study, we show that treatment of rats with nandrolone decreases the level of mRNA for the alpha1B-adrenoceptor in the kidneys, implying decreased synthesis of alpha1B-adrenoceptors. On the other hand, nandrolone did not decrease the density of alpha1B-adrenoceptors in Madin-Darby Canine Kidney (MDCK) cells, even though the sub-cell line tested, MDCK-D1, expresses both the androgen receptor and the alpha1B-adrenoceptor. It is concluded that the regulation of alpha1B-adrenoceptor expression by anabolic androgenic steroids is intricate and cell-type specific.

Improved approach for proteochemometrics modeling: application to organic compound--amine G protein-coupled receptor interactions.

MOTIVATION: Proteochemometrics is a novel technology for the analysis of interactions of series of proteins with series of ligands. We have here customized it for analysis of large datasets and evaluated it for the modeling of the interaction of psychoactive organic amines with all the five known families of amine G protein-coupled receptors (GPCRs). RESULTS: The model exploited data for the binding of 22 compounds to 31 amine GPCRs, correlating chemical descriptions and cross-descriptions of compounds and receptors to binding affinity using a novel strategy. A highly valid model (q2 = 0.76) was obtained which was further validated by external predictions using data for 10 other entirely independent compounds, yielding the high q2ext = 0.67. Interpretation of the model reveals molecular interactions that govern psychoactive organic amines overall affinity for amine GPCRs, as well as their selectivity for particular amine GPCRs. The new modeling procedure allows us to obtain fully interpretable proteochemometrics models using essentially unlimited number of ligand and protein descriptors.

Addition of a signal peptide sequence to the alpha1D-adrenoceptor gene increases the density of receptors, as determined by [3H]-prazosin binding in the membranes.

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Proteochemometric mapping of the interaction of organic compounds with melanocortin receptor subtypes.

Proteochemometrics was applied in the analysis of the binding of organic compounds to wild-type and chimeric melanocortin receptors. Thirteen chimeric melanocortin receptors were designed based on statistical molecular design; each chimera contained parts from three of the MC(1,3-5) receptors. The binding affinities of 18 compounds were determined for these chimeric melanocortin receptors and the four wild-type melanocortin receptors. The data for 14 of these compounds were correlated to the physicochemical and structural descriptors of compounds, binary descriptors of receptor sequences, and cross-terms derived from ligand and receptor descriptors to obtain a proteochemometric model (correlation was performed using partial least-squares projections to latent structures; PLS). A well fitted mathematical model (R(2) = 0.92) with high predictive ability (Q(2) = 0.79) was obtained. In a further validation of the model, the predictive ability for ligands (Q(2)lig = 0.68) and receptors (Q(2)rec = 0.76) was estimated. The model was moreover validated by external prediction by using the data for the four additional compounds that had not at all been included in the proteochemometric model; the analysis yielded a Q(2)ext = 0.73. An interpretation of the results using PLS coefficients revealed the influence of particular properties of organic compounds on their affinity to melanocortin receptors. Three-dimensional models of melanocortin receptors were also created, and physicochemical properties of the amino acids inside the receptors' transmembrane cavity were correlated to the PLS modeling results. The importance of particular amino acids for selective binding of organic compounds was estimated and used to outline the ligand recognition site in the melanocortin receptors.

Investigations on the pharmacology of the cardioprotective guanidine ME10092.

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to alpha 1- and alpha 2-adrenoreceptors with moderate affinity (Ki values 1-4 microM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.

The methylester of gamma-butyrobetaine, but not gamma-butyrobetaine itself, induces muscarinic receptor-dependent vasodilatation.

Gamma-butyrobetaine (GBB) is known mostly as a bio-precursor of carnitine, a key molecule in the regulation of myocardial energy metabolism. The metabolites of carnitine and GBB were investigated for acetylcholine-like activity decades ago. The present study shows that the methylester of GBB (GBB-ME) exerts its biological activity by binding to muscarinic acetylcholine receptors. GBB-ME dose-dependently decreased the blood pressure in anaesthetised rats and also produced endothelium-dependent vasodilation in the isolated guinea-pig heart. The biological effects of GBB-ME were inhibited partially by the NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and abolished by the acetylcholine receptor antagonist atropine, thus supporting the hypothesis that GBB-ME acts as muscarinic agonist. Moreover, we have shown here for the first time that GBB-ME binds directly to transfected human muscarinic (m) acetylcholine receptors, the potency order being m2>m5> or =m4> or =m1>m3. GBB itself showed neither biological activity nor significant affinity for the m1-5 receptors. We conclude that GBB-ME, but not the parent GBB, possesses acetylcholine-like activity in vivo and in vitro.

Nandrolone treatment decreases the level of rat kidney alpha(1B)-adrenoceptors.

Abuse of anabolic androgenic steroids (AAS) is associated with serious side effects, such as hypertension and fluid retention. Renal alpha(1)- and alpha(2)-adrenoceptors are implicated in the regulation of blood pressure and fluid balance. In the present study, the levels of renal alpha(1A)-, alpha(1B)-, alpha(2A)- and alpha(2B)-adrenoceptors, and spleen alpha(1B)-adrenoceptors, were quantified in tissue membranes from rats treated with the AAS nandrolone decanoate (15 mg/kg) for 14 days. The radioligands used were [(3)H]-prazosin and [(3)H]-RX821002. The nandrolone treatment caused a 50% reduction of kidney alpha(1B)-adrenoceptors (from 15 fmol/mg protein in control rats to 6.5 fmol/mg protein in treated rats). In contrast, the levels of kidney alpha(1A)-, alpha(2A)- and alpha(2B)-, and spleen alpha(1B)-adrenoceptors were unaffected. These results raise the possibility that a decreased level of kidney alpha(1B)-adrenoceptors may cause some of the effects observed on blood pressure and fluid balance in heavy abuse of AAS.

Reduced natriuretic response to acute sodium loading in COMT gene deleted mice.

BACKGROUND: The intrarenal natriuretic hormone dopamine (DA) is metabolised by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Inhibition of COMT, as opposed to MAO, results in a potent natriuretic response in the rat. The present study in anaesthetized homozygous and heterozygous COMT gene deleted mice attempted to further elucidate the importance of COMT in renal DA and sodium handling. After acute intravenous isotonic sodium loading, renal function was followed. RESULTS: COMT activity in heterozygous mice was about half of that in wild type mice and was zero in the homozygous mice. MAO activity did not differ between the genotypes. Urinary sodium excretion increased 10-fold after sodium loading in wild type mice. In heterozygous and homozygous mice, the natriuretic effects of sodium loading were only 29 % and 39 %, respectively, of that in wild type mice. Arterial pressure and glomerular filtration rate did not differ between genotypes. Baseline norepinephrine and DA excretions in urine were elevated in the homozygous, but not in heterozygous, COMT gene deleted mice. Urinary DA excretion increased after isotonic sodium loading in the wild type mice but not in the COMT gene deleted mice. CONCLUSIONS: Mice with reduced or absent COMT activity have altered metabolism of catecholamines and are unable to increase renal DA activity and produce normal natriuresis in response to acute sodium loading. The results support the hypothesis that COMT has an important role in the DA-mediated regulation of renal sodium excretion.

Binding of [(3)H]prazosin to alpha(1A)- and alpha(1B)-adrenoceptors, and to a cimetidine-sensitive non-alpha(1) binding site in rat kidney membranes.

[(3)H]Prazosin bound to alpha(1A)- and alpha(1B)-adrenoceptors, as well as to a cimetidine-sensitive non-alpha(1)-adrenoceptor binding site in rat kidney membranes. An experimental design is presented where the alpha(1)-adrenoceptors are selectively exposed by blocking the non-alpha(1) binding site with 60 microM cimetidine. Conversely, the non-alpha(1) binding site can be selectively exposed by blocking the alpha(1)-adrenoceptors with 600 nM metitepine. The identity of the non-alpha(1) binding site for [(3)H]prazosin in the rat kidney, herein pharmacologically characterized by 33 competing substances, is still unknown.