An Approach to Greater Specificity for Glucocorticoids

Abstract

Glucocorticoid steroids are among the most prescribed drugs each year. Nonetheless, the many undesirable side effects, and lack of selectivity, restrict their greater usage. Research to increase glucocorticoid specificity has spanned many years. These efforts have been hampered by the ability of glucocorticoids to both induce and repress gene transcription and also by the lack of success in defining any predictable properties that control glucocorticoid specificity. Correlations of transcriptional specificity have been observed with changes in steroid structure, receptor and chromatin conformation, DNA sequence for receptor binding, and associated cofactors. However, none of these studies have progressed to the point of being able to offer guidance for increased specificity. We summarize here a mathematical theory that allows a novel and quantifiable approach to increase selectivity. The theory applies to all three major actions of glucocorticoid receptors: induction by agonists, induction by antagonists, and repression by agonists. Simple graphical analysis of competition assays involving any two factors (steroid, chemical, peptide, protein, DNA, etc.) yields information (1) about the kinetically described mechanism of action for each factor at that step where the factor acts in the overall reaction sequence and (2) about the relative position of that step where each factor acts. These two pieces of information uniquely provide direction for increasing the specificity of glucocorticoid action. Consideration of all three modes of action indicate that the most promising approach for increased specificity is to vary the concentrations of those cofactors/pharmaceuticals that act closest to the observed end point. The potential for selectivity is even greater when varying cofactors/pharmaceuticals in conjunction with a select class of antagonists.

Keywords: antiglucocorticoids; glucocorticoid specificity; induction; kinetic mechanism of cofactor action; mathematical model; repression; selective glucocorticoid receptor modulators; site of cofactor action.

Figure 1

Theory of glucocorticoid-regulated gene expression. (A) Glucocorticoid induction and repression is assumed to obey a series of interconnected reaction steps. The first step is steroid (S) binding to receptor (R) to give the receptor–steroid complex (RS). The subsequent steps involve the possible input of various factors (A, B, C, D, etc.) to produce intermediates [M, concentration limiting step (CLS), N, O, etc.] and the possible loss of other factors (F, G, H, I, etc.). The dashed curve from intermediate “O” to the observed product “Z” is to indicate the presence of yet additional steps. One of the steps before “Z” is the CLS (see below in text), which may be anywhere but is shown, only for the purposes of illustration, as being between intermediates M and N. (B) In the mathematical model, each step in the sequence in (A) is represented by a set of enzymatic reactions where Y_i is the reaction product of step i, X_i is an accelerator, activating cofactor, or activator, and D_i is a decelerator, inhibiting cofactor, or inhibitor. The labels on the reactions represent association constants for reversible reactions and reaction rates for non-reversible reactions. As in enzyme kinetics, we denote the case of α = 0 to be competitive inhibition, γ = 0 to be uncompetitive inhibition, α = γ to be noncompetitive inhibition, and α and γ both non-zero to be mixed inhibition. The case of β = 0 is called linear inhibition, and β > 0 is called partial inhibition. In general, computing the dose–response curve for such a reaction sequence would be analytically intractable. However, imposing the experimentally observed constraint that the dose–response curve has a Hill-coefficient of one yields a closed-form expression for the dose–response curve in terms of the parameters of all the reactions.

Figure 2

Competition assay with glucocorticoid receptor (GR) and GREtkLUC during GR-mediated induction in 293 cells. All combinations of four concentrations each of GR and GREtkLUC plasmids for a total of 16 sets, all in triplicate, were used to cotransfect 293 cells, which were then treated with ethanol, or three subsaturating concentrations of Dex in ethanol, before determining the amounts of induced luciferase. Exact fits of these data to a first-order Hill plot yielded the A_max and EC₅₀ for each combination [for details, see Ref. (34)]. Graphs of 1/EC₅₀ vs. GR (A), 1/EC₅₀ vs. GREtkLUC (B), A_max/EC₅₀ vs. GR (C), and A_max/EC₅₀ vs. GREtkLUC (D) are the averages of three independent experiments (34).

Figure 3

Ordering of factors in reaction scheme for induction of luciferase activity from synthetic reporter (GREtkLUC, MMTVLUC) by steroid-bound receptor [glucocorticoid receptor (GR), progesterone receptor (PR)]. The position of the concentration limiting step (CLS), which is the site of action of the reporter, and positions of action of various factors relative to the CLS and other factors are indicated. Abbreviations: A, accelerator; C, competitive decelerator; C,2, competitive decelerator at two sites; C,2*, competitive decelerator at two sites for BRD4 only with relatively high concentrations of CDK9.

Figure 4

Flow chart of actions of factors in glucocorticoid receptor (GR)-regulated gene repression. Schematic diagram of PMA induction of Luciferase activity from synthetic reporter (AP1LUC) by AP1 that is repressed by steroid-bound receptor (GR). The position of the concentration limiting step (CLS), and sites of action of TIF2, NU6027, phenanthroline, and GR are indicated. A′ and A″ represent unknown, post-CLS steps, each of which can lead to Luciferase activity but the efficiency from A″ is much less than A′ [from Ref. (37)].

Figure 5

Effect of changing concentrations of glucocorticoid receptor (GR) without (A) and with (B) Ubc9 on partial agonist activity (PAA) of antiglucocorticoids. Experiments were conducted with 1 μM antisteroid. Luciferase activities were determined and the PAA of each steroid was calculated relative to 1 μM Dex under the same conditions. The values of four independent experiments were averaged and plotted ± SEM. The thin horizontal line at 50% is only for reference [from Ref. (11)].