Kinetic analysis of estrogen receptor/ligand interactions

Abstract

Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants. On average, antagonist ligands (such as tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists (such as estriol and beta-estradiol). This finding is consistent with these antagonists binding to an altered conformation of the receptor. The biosensor assay also could identify subtle differences in how the same ligand interacted with two different isoforms of the receptor (alpha and beta). The biosensor's ability to determine kinetic rate constants for small molecule/protein interactions provides unique opportunities to understand the mechanisms associated with complex formation as well as new information to drive the optimization of drug candidates.

Figure 1

Panel of compounds studied. (A) Estrogen agonists. (B) Non-estrogen agonists. (C) Antagonists. (D) Nonbinding compounds included as controls.

Figure 2

Design of the receptor/ligand assay. His₄ mAb was covalently immobilized on the biosensor surface, followed by capture of the estrogen-receptor construct (≈28 kDa) via its His tag. Compounds were injected across the surface, and the ER/ligand interaction was monitored. The ER/ligand complex was readily stripped from the antibody, and fresh receptor was captured before the next ligand-binding test.

Figure 3

Sensorgrams of the complete antibody/receptor/ligand binding cycle. As noted in the figure, ER was made to flow over the anti-His₄ mAb surface (yielding a response of ≈1800 RU), the surface was washed with buffer, ligand was injected over the captured receptor (yielding a response of ≈30 RU), and mild acid was injected over the surface to disrupt the receptor/antibody interaction. Reproducibility of the capturing assay is demonstrated by the overlay of 40 replicate binding cycles shown here in different colors.

Figure 4

Interaction of weak-affinity binders with ER. (A) Representative sensorgrams obtained from injections of prasterone at concentrations of 0, 0.12, 0.37, 1.1, 3.3, 10, and 30 μM over ER captured on the His₄ antibody surface. (B) Sensorgram responses at equilibrium (t ≈ 25 s) were plotted against prasterone concentration and fit to a simple binding isotherm to yield an equilibrium dissociation constant. (C) Representative sensorgrams (black lines) obtained from triplicate injections of bisphenol A at concentrations of 0, 0.037, 0.11, 0.33, 1.0, and 3.0 μM over ER captured on the anti-His₄ antibody surface. Red lines depict the global fit of the data to a simple 1:1 bimolecular interaction model, yielding k_a = 1.3 (1) × 10⁶ M⁻¹ s⁻¹ and k_d = 0.27 (1) s⁻¹.

Figure 5

Screen of drug panel for binding to ER-α. Responses were generated from the injection of 1 μM each of non-estrogen agonists (green), estrogen agonists (red), antagonists, (blue), and control compounds (black).

Figure 6

Representative data sets (black lines) for kinetic analysis of ligand–ER interactions. The estrogen agonists 17β-estradiol (A) and estriol (B) were injected at concentrations of 0, 0.0041, 0.012, 0.037, 0.11, 0.33, and 1.0 μM for 60 s, and dissociation was monitored for 3 min. Another estrogen agonist, estrone (C), was injected at concentrations of 0, 0.69, 2.0, 6.2, 19, 56, and 170 nM for 60 s, and dissociation was monitored for 3 min. The non-estrogen agonist, diethylstilbestrol (D), was injected at concentrations of 0, 0.012, 0.037, 0.11, 0.33, and 1.0 μM for 30 s, and dissociation was monitored for 3 min. The antagonists, nafoxidine (E) and tamoxifen (F), were injected at concentrations of 0, 0.032, 0.063, 0.13, 0.25, and 0.50 μM for 3 min, and dissociation was monitored for 10 min. Red lines represent the global fits of the data to a 1:1 bimolecular interaction model. The kinetic parameters obtained from each interaction are reported in Table 1.

Figure 7

Kinetic analysis of estriol binding to different estrogen-receptor isoforms. Estriol was injected at concentrations of 0, 0.019, 0.056, 0.17, 0.50, and 1.5 μM over captured α (A) and β (B) ER for 30 s, and dissociation was monitored for 6 min. Colored lines represent the global fits of the sensorgrams (black lines) to a 1:1 bimolecular interaction model. From the data shown here, the kinetic parameters obtained for each interaction are k_a = 1.0 × 10⁶ M⁻¹ s⁻¹, k_d = 1.8 × 10⁻² s⁻¹, k_d =18 nM for α and k_a = 5.7 × 10⁵ M⁻¹ s⁻¹, k_d = 3.2 × 10⁻³ s⁻¹, K_D = 5.6 nM for β.