Selectivity For Binding Sites Research Articles

Probing the Paradigm of Promiscuity for N‐Heterocyclic Carbene Complexes and their Protein Adduct Formation

AbstractMetal complexes can be considered a “paradigm of promiscuity” when it comes to their interactions with proteins. They often form adducts with a variety of donor atoms in an unselective manner. We have characterized the adducts formed between a series of isostructural N‐heterocyclic carbene (NHC) complexes with Ru, Os, Rh, and Ir centers and the model protein hen egg white lysozyme by X‐ray crystallography and mass spectrometry. Distinctive behavior for the metal compounds was observed with the more labile Ru and Rh complexes targeting mainly a surface l‐histidine moiety through cleavage of p‐cymene or NHC co‐ligands, respectively. In contrast, the more inert Os and Ir derivatives were detected abundantly in an electronegative binding pocket after undergoing ligand exchange of a chlorido ligand for an amino acid side chain. Computational studies supported the binding profiles and hinted at the role of the protein microenvironment for metal complexes eliciting selectivity for specific binding sites on the protein.

Homobivalent Conjugation Increases the Allosteric Effect of 9-aminoacridine at the α1-Adrenergic Receptors.

The α1-adrenergic receptors are targets for a number of cardiovascular and central nervous system conditions, but the current drugs for these receptors lack specificity to be of optimal clinical value. Allosteric modulators offer an alternative mechanism of action to traditional α1-adrenergic ligands, yet there is little information describing this drug class at the α1-adrenergic receptors. We have identified a series of 9-aminoacridine compounds that demonstrate allosteric modulation of the α1A- and α1B-adrenergic receptors. The 9-aminoacridines increase the rate of [3H]prazosin dissociation from the α1A- and α1B-adrenergic receptors and noncompetitively inhibit receptor activation by the endogenous agonist norepinephrine. The structurally similar compound, tacrine, which is a known allosteric modulator of the muscarinic receptors, is also shown to be a modulator of the α1-adrenergic receptors, which suggests a general lack of selectivity for allosteric binding sites across aminergic G protein-coupled receptor. Conjugation of two 9-aminoacridine pharmacophores, using linkers of varying length, increases the potency and efficacy of the allosteric effects of this ligand, likely through optimization of bitopic engagement of the allosteric and orthosteric binding sites of the receptor. Such a bivalent approach may provide a mechanism for fine tuning the efficacy of allosteric compounds in future drug design efforts.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Multisite Ion Models That Improve Coordination and Free Energy Calculations in Molecular Dynamics Simulations.

Current ion models in molecular mechanics are simple spheres, and their interactions are solely determined from the van der Waals radius of the sphere and the total charge. Here, we introduce a model where we distribute the total charge of the ion into n-dummy centers that are placed in the direction of the coordinating atoms. We have parametrized this model for two divalent cations, Ca(2+) and Mg(2+), and have tested the model's accuracy in a variety of simulations. With this model we are not only able to correctly predict the free energies and selectivity for cation binding sites in proteins and nucleic acids, but we achieve better coordination geometries and can capture more subtle effects such as the exchange of inner shell waters. Additionally, this model does not employ higher-order electrostatics and thus can be easily used with standard force fields.

A Multisite Ion Model that Improves Free Energy Calculations in Molecular Dynamics Simulations

Current ion models in molecular mechanics are simple spheres, and their interactions are solely determined from the radius of the sphere and the total charge. This set of parameters is chosen to closely reproduce the hydration free energy for the ion, but this exercise uses all the available degrees of freedom and our ability to reproduce the binding free energy to a protein or other thermodynamic quantities is therefore limited. Here we introduce a new model where we distribute the total charge of the ion into n-dummy centers that are placed in the direction of the coordinating atoms. We have parameterized this model for two divalent cations, Ca2+ and Mg2+, and have tested the model’s accuracy in a variety of simulations. With this model we are not only able to correctly predict the free energy and selectivity for cation binding sites, but we achieve better coordination geometries and can capture more subtle effects such as the exchange of inner shell waters. One further advantage of this model is that it does not use higher-order electrostatics and thus can be easily used with standard force fields.

Angiotensin-converting enzyme (ACE) inhibitors have different selectivity for bradykinin binding sites of human somatic ACE

The angiotensin-converting enzyme (ACE) has two natural substrates and two catalytic domains: one cleaving angiotensin I and one inactivating bradykinin. The aim of this study was to investigate the comparative binding affinity of ACE inhibitors for the two binding sites of human endothelial ACE. In vitro binding assays were performed to test the ability of bradykinin, angiotensin I, or various ACE inhibitors (enalaprilat, perindoprilat, quinaprilat, ramiprilat, and trandolaprilat) to displace a saturating concentration of [ 125I]351A, a radiolabeled lisinopril analogue, from ACE binding sites. The calculated IC 50 values for the ACE inhibitors were in the nanomolar range, while those for the natural substrates were in the micromolar range. The bradykinin/angiotensin I selectivity ratios calculated from double displacement experiments were: perindoprilat, 1.44; ramiprilat, 1.16; quinaprilat, 1.09; trandolaprilat, 1.08; enalaprilat, 1.00. The ACE inhibitors generally had higher affinity for the bradykinin than the angiotensin I binding sites, supporting the idea that these agents are primarily inhibitors of bradykinin degradation, and secondarily inhibitors of angiotensin II production. Perindoprilat had the highest selectivity for bradykinin versus angiotensin I binding sites, and enalaprilat has the lowest. These results indicate that there are differences in the affinity of ACE inhibitors toward sites for bradykinin degradation, which could lead to differences in efficacy in cardiovascular disease.

The ACE-inhibitor perindoprilat has high selectivity for bradykinin binding sites of human somatic ACE

The ACE-inhibitor perindoprilat has high selectivity for bradykinin binding sites of human somatic ACE

Behavioral Pharmacology of σ-Ligands

Sigma (sigma) receptors, first defined as a subclass of opioid receptors, later confounded with the high affinity phencyclidine (PCP) binding sites, now are regarded as unique binding sites, distinct from opiate and PCP receptors, and related to higher brain function. The investigation of functional significance of sigma receptors in the brain has been hampered for many years by relative lack of specific tool drugs and by the unavailability of their coherent classification into postulated agonists and antagonists. However, a potential involvement of sigma receptors in psychotic disorders was first suggested soon after their discovery. The sigma receptors are classified into two subtypes, sigma (1) and sigma (2) receptors, of which the first was recently cloned from rodent and human tissues while the second has not yet been fully characterized. Although the precise mechanism of the functional response of these receptors is still uncertain, it is accepted that sigma receptors can modulate a number of central neurotransmitter systems, including noradrenergic, glutamatergic and dopaminergic ones. The sigma receptors have been postulated to be involved in numerous pharmacological and physiological functions, including motor disorders, psychotic disorders, neuroprotective mechanisms. In the last years, a number of compounds with a high affinity and selectivity for sigma binding sites have been discovered and investigated for their therapeutic potential. In this review, we try to summarize the behavioral effects of sigma receptor ligands that have been described, and their activity in animal models related to some brain disorders, especially schizophrenia and affective disorders.

Selective binding of 2-[ 125I]iodo-nisoxetine to norepinephrine transporters in the brain

A radioiodinated ligand, ( R)-N-methyl-(2-[ 125I]iodo-phenoxy)-3-phenylpropylamine, [ 125I]2-INXT, targeting norepinephrine transporters (NET), was successfully prepared. A no-carrier-added product, [ 125I]2-INXT, displayed a saturable binding with a high affinity ( K d =0.06 nM) in the homogenates prepared from rat cortical tissues as well as from LLC-PK 1 cells expressing NET. A relatively low number of binding sties ( B max=55 fmol/mg protein) measured with [ 125I]2-INXT in rat cortical homogenates is consistent with the value reported for a known NET ligand, [ 3H]nisoxetine. Competition studies with various compounds on [ 125I]2-INXT binding clearly confirmed the pharmacological specificity and selectivity for NET binding sites. Following a tail-vein injection of [ 125I]2-INXT in rats, a good initial brain uptake was observed (0.56% dose at 2 min) followed by a slow washout from the brain (0.2% remained at 3 hours post-injection). The hypothalamus (a NET-rich region) to striatum (a region devoid of NET) ratio was 1.5 at 3 hours post-i.v. injection. Pretreatment of rats with nisoxetine significantly inhibited the uptake of [ 125I]2-INXT (70-100% inhibition) in locus coeruleus, hypothalamus and raphe nuclei, regions known to have a high density of NET; whereas escitalopram, a serotonin transporter ligand, did not show a similar effect. Ex vivo autoradiography of rat brain sections of [ 125I]2-INXT (at 3 hours after an i.v. injection) displayed an excellent regional brain localization pattern corroborated to the specific NET distribution in the brain. The specific brain localization was significantly reduced by a dose of nisoxetine pretreatment. Taken together, the data suggest that [ 123I]2-INXT may be useful for mapping NET binding sites in the brain.

I2-Imidazoline Binding Site Affinity of a Structurally Different Type of Ligands

Two families of compounds with affinity towards the I2 imidazoline binding sites are reported. The first is a family of compounds structurally related to agmatine with two guanidine or 2-aminoimidazoline groups at each end of an aliphatic chain of six, eight, nine or 12 methylene groups. Second, and following the model of clonidine, we propose another family of compounds also with two guanidine or 2-aminoimidazoline groups at each end of a chain consisting of two phenyl rings connected by groups such as CH2, CO, NH and SO2. The affinity of the compounds towards the I2 imidazoline binding sites was then evaluated in human brain tissues. In order to determine their pharmacological selectivity versus α2-adrenoceptors, the affinity for these receptors was also evaluated for the compounds with the highest affinities at I2 imidazoline binding sites. The results obtained show that many of the compounds exhibit a considerable affinity towards the I2 imidazoline binding sites. The aliphatic derivatives, in particular, present a very interesting selectivity for the I2 imidazoline binding sites versus the α2 adrenoceptors. To better understand these findings, mono-guanidinium analogues of the aliphatic derivatives were synthesised and tested showing poor affinity for I2 imidazoline binding sites. The importance of these results lies in the novelty of the chemical structures studied (dicationic aliphatic compounds particularly) because they are significantly different to those of the I2 imidazoline binding site ligands reported to date.

(+)-cis-N-ethyleneamino-N-normetazocine derivatives. Novel and selective sigma ligands with antagonist properties.

A series of (+)-cis-N-normetazocine derivatives has been described, and their affinities for sigma1, sigma2, and phencyclidine (PCP) sites and opioid, muscarinic (M2), dopamine (D2), and serotonin (5-HT2) receptors were evaluated. The effect of the N-substitution with a substituted ethylamino spacer was investigated. Compounds 8c-11c displayed high affinities for sigma1 sites and for opioid receptors. Substitution of the second basic nitrogen either with alkyl or cycloalkyl substituents give compounds (1a-6a) with high affinity and selectivity for sigma1 binding sites. Compounds 1a-5a were further characterized in vivo, and their agonist/antagonist activity was evaluated. In mouse, compound 1a and 2a as well as haloperidol suppressed in a dose-related manner the stereotyped behavior induced by (+)-SKF 10,047. Compounds 3a-5a and (+)-pentazocine do not affect the stereotyped behavior induced by ip injection of (+)-SKF 10,047. Therefore, from this series of compounds we identified potent and selective sigma1 ligands which might prove useful to unveil the functional role of sigma1 sites.

The 5-HT1D Receptor Antagonist GR-127,935 Prevents Inhibitory Effects of Sumatriptan but not CP-122,288 and 5-CT on Neurogenic Plasma Extravasation within Guinea Pig Dura Mater

The aim of this study was to examine whether GR-127,935, a 5-HT1B/1D receptor antagonist, blocks the inhibitory effects of sumatriptan, CP-122,288 and 5-carboxamidotryptamine (5-CT) on plasma protein extravasation, within guinea pig and rat dura mater, following electric stimulation of the trigeminal ganglion. Binding studies first established that GR-127,935 shows a 500-fold selectivity for 5-HT1D binding sites (labeled by [3H]L-694,247) versus 5-HT1F binding sites (labeled by 3H]sumatriptan in the presence of 50 nM 5-carboxamidotryptamine) in guinea pig forebrain homogenates (pKD ± SD = 7.0 ± 0.2 at 5-HT1F sites and 9.7 ± 0.1 at 5-HT1D sites). In guinea pigs, GR-127,935 showed partial agonist activity and inhibited dural plasma protein extravasation. Increasing doses of GR-127,935 reversed the effect of sumatriptan, but did not affect the action of 5-CT and CP-122,288 (at a dose as high as 2 μmol/kg). Sumatriptan, CP 122,288 and 5-CT dose-responsively inhibited plasma protein extravasation. At a dose of 2 μmol/kg (but not at 0.2 μmol/kg), GR-127,935 right-shifted the dose-response curve of sumatriptan. No significant rightward shift was observed in the dose-response of CP-122,288 and 5-CT. In rats, GR-127,935 did not show any significant partial agonist activity. A dose of 0.2 μmol/kg was sufficient to right-shift the dose-response curve of sumatriptan. These data suggest that sumatriptan inhibits neurogenic inflammation via 5-HT1Dα receptors in guinea pigs and 5-HT1Dβ (5-HT1B) receptors in rats. Additional receptor subtypes are likely to be involved in the inhibition of plasma extravasation by CP-122,288 and 5-CT. Pertussis toxin reduced the inhibitory effects of both sumatriptan and 5-CT, but not of muscimol, known to act at GABAA receptors. These results suggest that 5-CT, as well as sumatriptan, act at a receptor linked to an inhibitory G-protein. © 1997 Elsevier Science Ltd. All rights reserved.

Sigma ligands with subnanomolar affinity and preference for the sigma 2 binding site. 2. Spiro-joined benzofuran, isobenzofuran, and benzopyran piperidines.

Spiro[isobenzofuran-1(3H),4'-piperidines] and the corresponding benzofuran and benzopyran derivatives have been synthesized and evaluated as sigma ligands. The compounds are related to Lu 28-179 (1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1- butyl]spiro[isobenzofuran-1(3H),4'-piperidine]) that has been demonstrated to be a selective sigma 2 ligand with affinity in the subnanomolar range. The object of the study was to determine the structural factors governing sigma 1/sigma 2 affinity and selectivity within this class of compounds. The N-substituent in spiro[isobenzofuran-1(3H),4'-piperidines] is highly important, both for affinity and selectivity. Spiropiperidines with no or small N-substituents (H, Me, Et) exert very low affinity for both sigma 1 and sigma 2 binding sites (IC50(sigma 1, sigma 2) > 100 nM), whereas medium-sized substituents (e.g., Pr, Bu, Ph(CH2)2) result in potent, but unselective compounds (IC50(sigma 1, sigma 2) = 2-5 nM). Increasing the chain length and the lipophilicity of the N-substituent result in compounds in which high affinity for sigma 2 binding sites is retained and with selectivity for sigma 2 vs sigma 1 binding sites (e.g., 4-cyclohexyl-1-butyl: IC50-(sigma 1) = 1.5 nM, IC50(sigma 2) = 0.07 nM). Introduction of substituents in the benzene ring of the spiro[isobenzofuran-1(3H),4'-piperidine] ring system of Lu 28-179 mainly affects affinity for sigma 1 binding sites. Compounds with substituents (F, CF3) in the 4- or 7-position of the isobenzofuran display high affinity for sigma 2 binding sites (IC50(sigma 2) = 0.5-2 nM) and very low affinity for sigma 1 binding sites (IC50(sigma 1) > 100 nM). Compounds with substituents (F, CF3, Me) in the 5- or 6-position of the isobenzofuran exert increased affinity for sigma 1 binding sites (IC50(sigma 1) = 5-30 nM, IC50(sigma 2) = 0.3-7 nM), thus rendering unselective compounds. Exchanging the isobenzofuran moiety of Lu 28-179 with thioisobenzofuran, benzofuran, or benzopyran also has a pronounced effect on both affinity and selectivity for sigma binding sites. The position of the oxygen atom and the position of the spiroconnection with the 4-position of the piperidine ring were varied, and only compounds in which both the benzene ring and the heteroatom are attached directly to the piperidine ring retain high affinity and selectivity for sigma 2 binding sites (e.g., 3,4-dihydro-1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1- butyl]spiro[1H-2-benzopyran-1,4'-piperidine]: IC50(sigma 1) = 53 nM, IC50(sigma 2) = 0.9 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Differential ability of tachykinin NK-1 and NK-2 agonists to produce scratching and grooming behaviours in mice

Potent and selective NK-1 and NK-2 agonists as well as compounds with lower selectivity and affinity for NK-1 binding sites were compared in their ability to produce scratching and grooming behaviours when injected intracerebroventricularly in mice. Septide, an agonist with a low affinity for NK-1 binding sites, [Sar 9, Met(O 2) 11]SP and to a lesser extent [Pro 9]SP, two potent and selective NK-1 agonists were the most effective drugs in stimulating these behaviours. Only high doses of [Apa 9,10]SP and [Lys 5, Tyr 7, Pro 8]NKA(4–10), two agonists with low affinity for NK-1 binding sites, produced scratching and grooming responses. Similarly, only high doses of [Lys 5, MeLeu 9, NLe 10]NKA(4–10), a potent NK-2 agonist, produced grooming behaviour. When coinjected with the endopeptidase enzyme inhibitor phosphoramidon, the effects of [Apa 9,10]SP, [Lys 5, Tyr 7, Pro 8]NKA(4–10) and [Pro 9]SP were markedly enhanced. Analyses of the potency of the different agents to displace 3H-SP binding in mouse subcortical structures revealed that the affinities of the agonists for NK-1 receptors are similar to those previously reported in rat brain. The efficacy of the agonists at producing behavioural responses was not equivalent to their potency to bind to central NK-1 receptors. These findings therefore suggest that a stimulation of NK-1 but also non classical NK-1 receptors are involved in the induction of scratching and grooming behaviours.

5-HT1-like receptor mediated changes in porcine carotid haemodynamics: are 5-HT1D receptors involved?

5-Hydroxytryptamine (5-HT) reduces porcine arteriovenous shunting in the carotid vascular bed by stimulation of both 5-HT1-like and 5-HT2 receptors and increases capillary flow to some tissues, like the skin and ears, by different 5-HT1-like receptors. In view of the heterogeneous nature of the 5-HT1-like receptors and the relative selectivity for the 5-HT1D binding sites of sumatriptan, which also reduces porcine arteriovenous shunting and slightly increases capillary blood flow towards skin and ears by 5-HT1-like receptors, we have attempted to determine whether one or both of these carotid 5-HT1-like receptors belong to the 5-HT1D subtype. Pentobarbitone anaesthetized pigs, subjected to bilateral cervical vagosympathectomy, received either 5-HT (2 micrograms.kg-1.min-1) in the carotid artery or cumulative i.v. doses of sumatriptan (10, 30, 100 and 300 micrograms.kg-1). Their effect on the total carotid blood flow and its distribution into capillary and arteriovenous anastomotic parts was determined with radioactive microspheres. The effect of metergoline (1 mg.kg-1), a substance with a very high affinity for the 5-HT1D receptor as well as for the 5-HT1A, 5-HT1B, 5-HT1C and 5-HT2 receptors, was studied on the responses to 5-HT and sumatriptan. Both 5-HT and sumatriptan reduced carotid arteriovenous anastomotic blood flow. 5-HT and, to a lesser extent, sumatriptan also increased capillary blood flow towards some tissues. Metergoline by itself did not affect the distribution of porcine carotid blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Signal Transduction in Anesthetic Action

The molecular mechanism for general anesthetic action is not known. The alpha 2 adrenergic agonists represent a novel class of "anesthetic-like" agent because of their selectivity for receptor binding sites and because the transmembrane signaling systems mediating their biologic responses in non-CNS systems are known. We have begun to characterize the signal transduction pathway involved in the anesthetic-like action of the alpha 2 adrenergic agonists. The alpha 2 adrenergic agonists potently decrease both central noradrenergic neurotransmission and halothane anesthetic requirements (MAC). Since MAC is only reduced by 30-40% when noradrenergic neurotransmission is totally abolished and since the reduction in MAC with the highly selective alpha 2 adrenergic agonists exceeds 90%, factors in addition to noradrenergic neurotransmission must be contributing to the anesthetic action of the alpha 2 agonists. Studies with the superselective alpha 2 agonist dexmedetomidine confirmed this, as the alpha 2 agonist could still reduce the MAC for halothane in rats depleted of their central norepinephrine stores. The profound reduction in anesthetic requirements with dexmedetomidine raised the possibility that alpha 2 adrenergic agonists may be considered an anesthetic hypnotic agent by itself. This sole anesthetic hypnotic response was established together with the confirmation that a central alpha 2 adrenoceptor mediated this action. Subsequently, data using molecular biologic techniques suggested that the alpha 2 C4 isoreceptor was the probable receptor that mediated the anesthetic response. We further explored the postreceptor effector mechanism for the signal transduction pathway for alpha 2 anesthetic action and identified the participation of two other molecular components, namely, a pertussis-toxin-sensitive G protein and a 4-aminopyridine-sensitive ion channel. Whether the signal transduction pathway for alpha 2 anesthetic action mediates the further response to other non-alpha 2 anesthetic agents needs to be defined.

125I]Iodoclonazepam, a specific high affinity radioligand for the identification of BZ 1 and BZ 2 sites in rat brain

Binding of the radioligand [ 125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum B max = 1.49 ± 0.3 pmol/mg of protein, k d = 0.39 ± 0.06 nM; hippocampus B max = 1.5 ± 0.14 pmol/mg of protein, k d) = 0.38 ± 0.6 nM and striatum B max = 0.53 ± 0.1 pmol/mg of protein, k d = 0.34 ± 0.03 nm, n = 3). In all regions only one population of sites was apparent. However. competition for [ 125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The bz 1 selective compounds zolpidem and CIL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [ 125I]iodoclonazepam is a novel, high affinity ligand which recognises both bz 1 and BZ 2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.

Rilmenidine lowers arterial pressure via imidazole receptors in brainstem C1 area

We sought to determine the site of action and receptor type responsible for the antihypertensive actions of rilmenidine. an oxazoline analogue of clonidine. In anesthetized paralyzed rats decerebration did not alter the dose dependent reductions in arterial pressure and heart rate elicited by i.v. drug. Rilmenidine microinjected bilaterally into the C1 area of the rostral ventrolateral medulla (RVL), but not nucleus tractus solitarii (NTS) nor caudal ventrolateral medulla (CVL), elicited dose-dependent falls in arterial pressure and heart rate at doses an order of magnitude less than required systemically. Prior microinjection into the Cl area of the selective α 2-adrenoceptor antagonist SKF-86466, even at high doses, failed to modify the hypotension to i.v. rilmenidine. However, microinjection of 3- to 10-fold lower doses of idazoxan, a ligand for imidazole as well as α 2-adrenoceptors, blocked the effects. Rilmenidine also competed with the clonidine analogue [ 3H]p-aminoclonidine ([ 3H]pac) at specific binding sites in membranes of bovine ventrolateral medulla and frontal cortex. In RVL rilmenidine competed with binding to imidazole and α 2-adrenergic binding sites with a 30-fold selectivity for the imidazole binding sites. In frontal cortex binding was of lower affinity and restricted to α 2-adrenergic sites. We conclude that rilmenidine, like clonidine, acts to lower arterial pressure by an action on imidazole receptors in the Cl area of RVL. The higher selectivity of rilmenidine for imidazole to α 2-adrenoceptors as compared to clonidine may explain the lower sedative effects of rilmenidine. Rilmenidine; Oxazoline; α 2-Adrenoceptors; Imidazole receptors; Medulla (rostral ventrolateral); Arterial pressure

RTI-4614-4: An analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites

The objective of this study was to determine the binding affinities of (±)-cis-N-[1-(2-hydroxy-2- phenylethyl)-3-methyl-4-piperidyl]-N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl)2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid μ receptor.

Discriminative stimulus properties of flesinoxan

Different groups of rats were trained to discriminate either 0.3 mg/kg of flesinoxan (N = 13) or 0.1 mg/kg of 8-OH-DPAT (N = 7) from saline in a two-lever operant drug discrimination task using a fixed ratio 10 schedule of reinforcement. Once trained, animals in both groups displayed a dose-related decrease in discriminative performance upon administration of lower doses of the drug used in training. In generalization tests, flesinoxan generalized to 8-OH-DPAT in 8-OH-DPAT-trained animals and 8-OH-DPAT substituted for flesinoxan in flesinoxan-trained animals. Buspirone substituted partially for both the flesinoxan and the 8-OH-DPAT cue. The results of the present study indicate similarity between the discriminative stimulus effects of flesinoxan and the stimulus produced by the 5-HT1A agonist 8-OH-DPAT. These results, coupled with the finding that flesinoxan has a significant affinity and selectivity for 5-HT1A binding sites, suggest that the stimulus effects of flesinoxan are mediated by a 5-HT1A mechanism.