Elsevier

Life Sciences

Volume 78, Issue 24, 8 May 2006, Pages 2812-2819
Life Sciences

Estimation of both the potency and efficacy of α7 nAChR agonists from single-concentration responses

https://doi.org/10.1016/j.lfs.2005.11.009Get rights and content

Abstract

The assessment of functional properties is a crucial step in the screening of potential new drug candidates. The development of moderate to high throughput electrophysiological recording systems such as OpusXpress (Molecular Devices) has facilitated the process of testing new drugs to a large degree. However, while the simple screening of multiple drugs at a single concentration identifies “hits” and “misses”, the generation of full concentration–response studies is still a bottleneck in drug development. The α7 nicotinic acetylcholine receptor displays a unique concentration dependence of response kinetics which permits estimates of EC50 and Imax values for experimental drugs to be generated from single-concentration responses. This method is based on the analysis of 13 different concentration–response studies utilizing either human or rat α7 nAChR. Each experimental response was first normalized to an ACh control, and then a transformation of the pooled data was generated which, based on the relationship between the net charge and peak current to their respective EC50 values defined the “functional concentration” (the test concentration relative to the EC50 for the given agonist). At low functional concentrations, net charge is large relative to peak current amplitude and at higher functional concentration this relationship reverses. For any single-concentration response, the ratio of net charge to peak current can be used to estimate functional concentration. Efficacy can then be estimated by comparing the observed (net charge) response to the expected value for a full agonist at the estimated functional concentration. This extended analysis, combined with automated recording methods, should greatly increase the efficiency with which promising new drug candidates can be characterized.

Introduction

The identification of new drug candidates most often begins with the screening of many experimental compounds. In this context, the concept of high throughput becomes of great importance. However, high throughput approaches often rely on either binding assays, which cannot distinguish agonists from antagonists, or fluorescence-based functional studies, which may not distinguish between full agonists and partial agonists. Therefore, for drugs that target ion channel receptors, electrophysiology, in particular voltage-clamp experiments, remains the gold standard for functional studies, and recent advances in automated recording systems have brought these methods into consideration for high throughput.

A common first pass in a screening procedure is to evaluate an array of compounds at a single test concentration. Such single-concentration testing can serve to distinguish between active and inactive compounds but provides little information about the actual potency and efficacy of the active compounds, since a potent partial agonist and a full agonist of low potency might give responses of similar amplitude. The α7-type nicotinic acetylcholine receptor (nAChR) has been acknowledged as a potential target for a number of diverse indications including Alzheimer's disease, schizophrenia, and even peripheral inflammation (Freedman et al., 2000, Kem, 2000, Wang et al., 2003). This paper describes a method for greatly increasing the efficiency of drug screening for this target by estimating both EC50 and efficacy values from single-concentration screens.

The α7-type nAChR exhibits a unique concentration-dependent form of desensitization such that when exposed to a high concentration of agonist, synchronous channel activation is maximal prior to the completion of the solution exchange. This is a consistent feature of α7-mediated responses, regardless of whether agonist concentration rises over the course of several seconds, as in an oocyte experiment (Papke and Thinschmidt, 1998), or over just a few milliseconds as in the case with rapid solution delivery to an acutely isolated neuron (Papke et al., 2000, Uteshev et al., 2002). We have suggested that this could arise from extremely rapid desensitization or deactivation of receptors fully saturated at their agonist binding sites. As a consequence of the preferential desensitization of fully liganded receptors, channels tend to be open only within a limited concentration range, corresponding to what would produce a relatively low fractional occupancy of the binding sites. The application of a relatively low concentration of agonist can sustain that condition and maintain channel activation for several seconds of agonist application, and the net charge evoked by such an agonist application can be relatively large, even though the peak current representing synchronous activation of channels is relatively small (Papke et al., 2000). These features of the α7 receptor responses account for qualitative differences in the concentration dependence for net charge and peak current responses (Papke and Papke, 2002). Of these two measurements, arguably, the net charge measure is of greater physiological significance and scientific validity (Papke and Papke, 2002). Nonetheless, peak current amplitudes, and more importantly, the relationship between net charge and peak current, can be used to define the functional concentration applied to a population of α7 receptors, functional concentration being defined as the concentration relative to the EC50 for the specific agonist being tested. In this paper, concentration–response data are pooled from 13 separate studies in order to generalize a method for estimating potency and efficacy based on comparisons of the net charge and peak currents of single-concentration responses.

Section snippets

Expression on Xenopus oocytes

The preparation of Xenopus laevis oocytes for RNA expression was conducted as previously described (Papke and Papke, 2002). In brief, mature (> 9 cm) female Xenopus laevis African frogs (Nasco, Ft. Atkinson, WI) were used as a source of oocytes. Prior to surgery, the frogs were anesthetized by placing the animal in a 1.5 g/l solution of MS222 (3-aminobenzoic acid ethyl ester) for 30 min. Oocytes were removed from an incision made in the abdomen.

In order to remove the follicular cell layer,

Comparisons of two response measures

The average ACh EC50 value reported in papers (Papke et al., 2005b, Papke and Papke, 2002, Placzek et al., 2004, Stokes et al., 2004) from our laboratory (n = 5) on either rat or human α7 receptors has been 26.3 μM. In spite of the fact that these papers spanned several years and utilized many different frogs as source for oocytes, the standard deviation in our EC50 estimates has been only 7.6 μM. As shown in Fig. 1, responses of α7 nAChR to increasing concentrations of agonist show

Discussion

The unique concentration-dependent desensitization of α7 nAChR permits every drug application to cells expressing this receptor to generate a dynamic analysis through the course of solution application and inherent concentration change. The fact that this quality of the α7 response is seen for all agonists, be they high potency or low, full agonist or partial, has allowed for rules to be defined to extend the analysis of single-concentration responses. We show that for two previously

Acknowledgements

This work was supported by NIH grants GM57481-01A2 and PO1 AG10485. The author thanks Drs. Alan Finkel and Cathy Smith-Maxwell of Molecular Devices for comments and support and Taiho Pharmaceuticals (Tokyo, Japan), Memory Pharmaceuticals and Dr. Peter Crooks for experimental agonists. Technical support was provided by Brian A. Jack, Clare Stokes and Joshua D. Buhr. Thanks to Maryanne Rose Papke for fuzzy lemons and careful algebra.

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