Relationship between aryl hydrocarbon receptor-affinity and the induction of EROD activity by 2,3,7,8-tetrachlorinated phenothiazine and derivatives.

Reported herein are semi-empirical calculations of the molecular geometry of TCDD, TCPT, TCPT-sulfoxide (TCPT-O), TCPT-sulfone (TCPT-O(2)), N-methyl-TCPT (Me-TCPT), N-methyl-TCPT-sulfoxide (Me-TCPT-O), and N-methyl-TCPT-sulfone (Me-TCPT-O(2)), the characterization of their AhR binding affinity in rat hepatic cytosol, and their ability to induce EROD activity in a rat hepatoma cell line in vitro. Semi-empirical calculations yielded detailed information about the stereochemistry and the preferred conformation of each of these compounds. These results in combination with observations reported in this paper were used to determine structure-activity relationships. In vitro displacement of (3)H-TCDD was measured by increasing concentrations of the respective ligands. This assay revealed a strong binding affinity of TCPT to the AhR with a K(i) value of 1.08 nM. TCDD had a K(i) value of 0.54 nM. The affinity of TCPT derivatives for the AhR decreased with increasing degree of oxidation. Moreover, N-methylation further lowered the affinity, so that the N-methyl sulfone derivative of TCPT displayed the highest K(i) at approximately 1200 nM (=460.4 ng/ml). A corresponding trend was observed regarding the potency of TCPT and derivatives to induce EROD activity in vitro. However, the potencies were considerably lower than that of TCDD. Enzyme induction was measured in a rat hepatoma cell line H4IIEC/T3 by quantification of ethoxyresorufin-O-deethylase (EROD) activity. Induction was measured at 12, 24, 48 and 72 h to determine time dependence. Sulfoxidated and N-methylated phenothiazines displayed a lower potency than their respective parent compounds. TCPT and all derivatives induced enzyme activity at an efficacy similar to TCDD at all time points measured. The reported findings clearly separate the induction of EROD activity by TCPT and derivatives from their binding affinities to the AhR. In contrast, a direct correlation between the two is generally assumed in drug development, leading to - in our view - unwarranted termination of drug candidates. Therefore, a lack of such a correlation for TCPT and derivatives in fact supports their further development as possible drug leads.