Carboetomidate

Abstract

Background Carboetomidate is a pyrrole etomidate analog that is 3 orders of magnitude less potent an inhibitor of in vitro cortisol synthesis than etomidate and does not inhibit in vivo steroid production. Although carboetomidate's reduced functional effect on steroid synthesis is thought to reflect lower binding affinity to 11β-hydroxylase, differential binding to this enzyme has never been experimentally demonstrated. In the current study, we tested the hypothesis that carboetomidate and etomidate bind with differential affinity to 11β-hydroxylase by comparing their abilities to inhibit photoaffinity labeling of purified enzyme by a photoactivatable etomidate analog and to modify the enzyme's absorption spectrum in a way that is indicative of ligand binding. In addition, we made a preliminary exploration of the manner in which etomidate and carboetomidate might differentially interact with this site using spectroscopic methods as well as molecular modeling techniques to better understand the structural basis for their selectivity. Methods The ability of azi-etomidate to inhibit cortisol synthesis was tested by assessing its ability to inhibit cortisol synthesis by H295R cells. The binding affinities of etomidate and carboetomidate to 11β-hydroxylase were compared by assessing their abilities to (1) inhibit photoincorporation of the photolabile etomidate analog [3H]azi-etomidate into the enzyme and (2) modify the absorption spectrum of the enzyme's heme group. In silico docking studies of etomidate, carboetomidate, and azi-etomidate binding to 11β-hydroxylase were performed using the computer software GOLD. Results Similar to etomidate, azi-etomidate potently inhibits in vitro cortisol synthesis. Etomidate inhibited [3H]azi-etomidate photolabeling of 11β-hydroxylase in a concentration-dependent manner. At a concentration of 40 μM, etomidate reduced photoincorporation of [3H] azi-etomidate by 96% ± 1% whereas carboetomidate had no experimentally detectable effect. On addition of etomidate to 11β-hydroxylase, a type 2 difference spectrum was produced indicative of etomidate complexation with the enzyme's heme iron; carboetomidate had no effect whereas azi-etomidate produced a reverse type 1 spectrum. Computer modeling studies predicted that etomidate, carboetomidate, and azi-etomidate can fit into the heme-containing pocket that forms 11β-hydroxylase's active site and pose with their carbonyl oxygens interacting with the heme iron and their phenyl rings stacking with phenylalanine-80. However, additional unique poses were identified for etomidate and azi-etomidate that likely account for their higher affinities. Conclusions Carboetomidate's reduced ability to suppress in vitro and in vivo steroid synthesis as compared with etomidate reflects its lower binding affinity to 11β-hydroxylase and may be attributed to carboetomidate's inability to form a coordination bond with the heme iron located at the enzyme's active site.

γ-Aminobutyric Acid Type A Receptor Modulation by Etomidate Analogs

“…33–36 In the CoMFA approach, there are no a priori assumptions regarding the nature of the protein binding site. Instead, the molecular (i.e.…”

Minimum infusion rate and adrenocortical function after continuous infusion of the novel etomidate analog ET-26-HCl in rats

“…The safe dosage range of etomidate has been diminishing owing to its inhibition of adrenocortical function, as the suppression following even a single bolus may last 72 h ( Molenaar et al, 2012 ). This inhibition is mainly the result of the high-affinity binding between the basic nitrogen in the imidazole ring of etomidate and the heme iron on 11β-hydroxylase ( Den Brinker et al, 2008 ; Fellows et al, 1983 ; Shanmugasundararaj et al, 2013 ). In the 1980s, etomidate was used as a sedative for critically ill patients; however, in 1983, Watt and colleagues found that the continuous infusion of etomidate may increase mortality, and they speculated that the increased mortality is mainly caused by adrenocortical suppression.…”

Sedative-hypnotic Binding to 11β-hydroxylase

“…12,33 This inhibition is remarkably potent, occurring even at nanomolar etomidate concentrations. 14,34,35 Computational docking studies using homology models of 11β-hydroxylase and spectroscopic studies using heterologously-expressed and purified enzyme indicate that etomidate’s high inhibitory potency arises primarily from its ability to engage in a coordination bond with the heme iron located within the enzyme’s active site. 14 This site is believed to be hydrophobic, a feature that would also enhance the binding of etomidate and other hydrophobic drugs.…”

“…8–11 Etomidate is believed to suppress adrenocortical function primarily by binding within a heme-containing cavity that forms the active site of 11β-hydroxylase, thus inhibiting the enzyme’s function. 12–14 …”

“…14,22 In carboetomidate, the basic nitrogen that is found in etomidate’s imidazole ring and is hypothesized to be required for high affinity binding to 11β-hydroxylase has been replaced with a methylene group. 14 Although carboetomidate’s binding affinity for 11β-hydroxylase has never been quantified, in vitro studies in an adrenocortical cell line revealed that it is three orders of magnitude less potent than etomidate at inhibiting cortisol synthesis and in vivo studies in animals showed that it does not suppress adrenocortical function when administered at hypnotic doses. 22,23 …”