Michael J. Lew

Analysis of competitive agonist-antagonist interactions by nonlinear regression

The rigorous estimation of a dissociation constant (Kb) for antagonists in functional assays has been sought by pharmacologists using a variety of techniques ever since the regression method of Arunlakshana and Schild in 1959. Here, Michael Lew and James Angus describe a simplified global regression method with improved accuracy compared to Schild analysis. The method is suitable for personal computers with standard graphing and statistical software. The accuracy of the predicted pKb values and confidence intervals has been tested by comparing examples of published data, and by mathematical (bootstrap) simulations.

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand.

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M2 mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M2 mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[3H]methylscopolamine, but in functional assays of M2 mAChR-mediated ERK1/2 phosphorylation and guanosine 5′-3-O-([35S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M2 mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of “directed efficacy” whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.

STRUCTURE-FUNCTION RELATIONSHIPS OF OMEGA -CONOTOXIN GVIA : SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, ω-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

Background: Receptors activate channels of sensory nerves to cause inflammation and pain by unknown mechanisms. Results: Protease-activated receptor 2 (PAR2) stimulated transient receptor potential vanilloid 4 (TRPV4) by generation of channel agonists. This required a key TRPV4 tyrosine and induced inflammation. Conclusion: PAR2 opens TRPV4 by functional coupling. Significance: Antagonism of PAR2-TRPV4 coupling could alleviate inflammation and pain. G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR2), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR2 regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR2 agonist stimulated a transient increase in [Ca2+]i. TRPV4 expression led to a markedly sustained increase in [Ca2+]i. Removal of extracellular Ca2+ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A2 and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR2 generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR2-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR2 was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR2 signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR2-stimulated neurogenic inflammation. Thus, PAR2 activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR2.

Release of endogenous ATP during sympathetic nerve stimulation

1 Vas deferens from guinea‐pig was stimulated with a suction electrode and both contractions and release of endogenous ATP monitored 2 Release of ATP was tetrodotoxin‐sensitive and increased when the number of stimuli was increased. 3 Release of ATP was not due to contraction of the muscle and persisted following block of contractions with prazosin and α,β‐methylene ATP. 4 These results indicate that stimulation of the sympathetic nerves in the vas deferens releases endogenous ATP presynaptically, supporting a cotransmitter function for ATP with noradrenaline.

The assessment of antagonist potency under conditions of transient response kinetics

The muscarinic acetylcholine receptor antagonists, atropine and pirenzepine, produced an apparent insurmountable antagonism of muscarinic M(1) receptor-mediated intracellular Ca(2+) mobilization in Chinese hamster ovary (CHO) cells when tested against the agonists carbachol or xanomeline. Each antagonist caused a dextral shift of the agonist concentration-response curves with depression of the maximum response that was incomplete (i.e., saturated) and which varied with the pairs of agonist and antagonist. Equilibrium competition binding assays found no deviation from simple, reversible competitive behavior for either antagonist. The relative rates of dissociation of unlabeled atropine and pirenzepine were also assessed in radioligand kinetic studies and it was found that atropine dissociated from the receptor approximately 8-fold slower than pirenzepine. Numerical dynamic simulations suggested that the insurmountability of antagonism observed in the present study was probably a kinetic artifact related to the measurement of transient responses to a non-equilibrated agonist in the presence of a slowly dissociating antagonist. Importantly, the patterns of antagonism observed included a saturable depression of agonist maximal response, a mode of antagonism that is incompatible with the previously described phenomenon of hemi-equilibrium states. Monte Carlo simulations indicated that reasonable, semi-quantitative estimates of antagonist potency could be determined by a minor modification of standard methods, where equieffective agonist concentrations, rather than EC(50) values, are compared in the absence and presence of antagonist. Application of the latter approach to the functional data yielded estimates of antagonist potency that were in excellent agreement with those derived from the equilibrium binding assays, thus indicating that the present method can be useful for quantifying antagonist potency under non-equilibrium conditions.

Good statistical practice in pharmacology Problem 1

This paper is intended to assist pharmacologists in making the most of statistical analysis and in avoiding common errors that can lead to false conclusions.

Dynamic mechanisms of non-classical antagonism by competitive AT1 receptor antagonists

Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.

Wall thickness to lumen diameter ratios of arteries from SHR and WKY: comparison of pressurised and wire-mounted preparations.

Passive properties (diameter, wall-to-lumen ratio and axial length) of small mesenteric arteries from SHR and WKY rats were measured with the artery segments cannulated and pressurised, or mounted on wires in a myograph. The measurements were made with a range of distending pressures (or calculated equivalent distending pressures when wire-mounted) from 0 to 180 mm Hg. The axial length of artery segments increased with increasing distending pressure when cannulated, but not when wire-mounted. The axial extension was greater for arteries from WKY (up to 105%) than for arteries from SHR (up to 65%). The arteries from SHR had significantly smaller diameters and greater wall-to-lumen ratios than the arteries from WKY. However, the diameters calculated for the arteries when wire-mounted were less than the measured diameters, and the wall-to-lumen ratio was always greater when wire-mounted than when cannulated because of the underestimated diameter and the absence of axial extension. Wall-to-lumen ratios decreased with increased distending pressure; values at 180 mm Hg were only 18 and 25% of those at 0 mm Hg for WKY and SHR arteries, respectively. The large degree of variability of wall-to-lumen ratios obtained from the two different preparations and the large range of values that are obtained from a single artery at different distending pressures must call into question the validity of characterising vascular hypertrophy by any single estimation of this parameter.

Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens

The interaction between the cannabinoid agonists, WIN 55,212‐2 or CP 55,940 with the CB1 receptor‐selective antagonists, SR141716A or LY320135 was investigated using the rat electrically‐stimulated vas deferens bioassay. Tissues were stimulated by single‐field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 μM), agonists elicited a concentration‐dependent inhibition of the contractile response, with pEC50 values of 7.93 and 6.84 for WIN 55,212‐2 and CP 55,940, respectively. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration‐response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration‐dependent enhancement of basal (agonist‐independent) tissue contraction. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 μM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. In light of these findings, we propose a theoretical model of competitive agonist‐antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. This approach yielded pKB estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB1 receptors. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB1 receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.