Competitive Antagonism of Anesthetic Action at the γ-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy

Abstract

Background The authors characterized the γ-aminobutyric acid type A receptor pharmacology of the novel etomidate analog naphthalene–etomidate, a potential lead compound for the development of anesthetic-selective competitive antagonists. Methods The positive modulatory potencies and efficacies of etomidate and naphthalene–etomidate were defined in oocyte-expressed α1β3γ2L γ-aminobutyric acid type A receptors using voltage clamp electrophysiology. Using the same technique, the ability of naphthalene–etomidate to reduce currents evoked by γ-aminobutyric acid alone or γ-aminobutyric acid potentiated by etomidate, propofol, pentobarbital, and diazepam was quantified. The binding affinity of naphthalene–etomidate to the transmembrane anesthetic binding sites of the γ-aminobutyric acid type A receptor was determined from its ability to inhibit receptor photoaffinity labeling by the site-selective photolabels [3H]azi-etomidate and R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. Results In contrast to etomidate, naphthalene–etomidate only weakly potentiated γ-aminobutyric acid–evoked currents and induced little direct activation even at a near-saturating aqueous concentration. It inhibited labeling of γ-aminobutyric acid type A receptors by [3H]azi-etomidate and R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid with similar half-maximal inhibitory concentrations of 48 μM (95% CI, 28 to 81 μM) and 33 μM (95% CI, 20 to 54 μM). It also reduced the positive modulatory actions of anesthetics (propofol > etomidate ~ pentobarbital) but not those of γ-aminobutyric acid or diazepam. At 300 μM, naphthalene–etomidate increased the half-maximal potentiating propofol concentration from 6.0 μM (95% CI, 4.4 to 8.0 μM) to 36 μM (95% CI, 17 to 78 μM) without affecting the maximal response obtained at high propofol concentrations. Conclusions Naphthalene–etomidate is a very low-efficacy etomidate analog that exhibits the pharmacology of an anesthetic competitive antagonist at the γ-aminobutyric acid type A receptor.

Etomidate and Etomidate Analog Binding and Positive Modulation of γ-Aminobutyric Acid Type A Receptors

“…The current studies were motivated by our previous observation that naphthalene-etomidate, an etomidate analog containing a bulky substituent group on its phenyl ring, exhibits pharmacologic properties that are quite distinct from those of etomidate. 11 Unlike etomidate, naphthalene-etomidate binds nonselectively to the GABA A receptor’s two classes of transmembrane anesthetic binding sites and produces relatively little positive modulation of GABA A receptors even at high, near-aqueous saturating concentrations. Consequently, it exhibits the pharmacology of an anesthetic competitive antagonist capable of reversing the GABA A receptor actions of anesthetics that bind to these sites and represents a potential lead compound for the development of anesthetic reversal agents.…”

“…To test that possibility, we transformed the peak electrophysiologic currents shown in figure 3B into P open values and fit the data as a function of drug concentration to equation 4. For all fits, we constrained the dissociation constant for the closed state (Kd closed ) to our previously determined value of 44 μM for etomidate 11 and the number of binding sites (n) to 2 to reflect positive modulation resulting only from drug binding to the two β + – α − sites as suggested by our mutation studies. Figure 9A plots the relationship between P open and drug concentration along with the fits to equation 4.…”

“…The line in each panel is a linear least squares fit of the dataset. Naphthalene-etomidate photoaffinity labeling data are from Ma et al 11…”

“… * (IC 50 forinhibiting 3 [H]azi-etomidate photolabeling) + (IC 50 for inhibiting R- 3 [H] m TFD-MPAB photolabeling). † Data from Ma et al 11 IC 50 = half-maximal inhibitory concentration; R- 3 [H] m TFD-MPAB = tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. …”

“…We recently reported that appending a phenyl ring substituent group onto etomidate’s existing phenyl ring (forming naphthalene-etomidate) essentially abolished conformational state selectivity and binding site selectivity, reducing etomidate’s intrinsic efficacy and turning the drug into an anesthetic-selective competitive antagonist at the GABA A receptor. 11 To explain this finding, we hypothesized that the binding pocket where etomidate’s phenyl ring lies becomes smaller as the receptor isomerizes from closed to open, sterically hindering naphthalene-etomidate from binding to the open state (fig. 1).…”