Kinetic Analysis of Estrogen Receptor Ligand Interactions

Kinetic analysis of estrogen receptor͞ ligand interactions

Rebecca L. Rich*, Lise R. Hoth†, Kieran F. Geoghegan†, Thomas A. Brown†, Peter K. LeMotte†, Samuel P. Simons†, Preston Hensley†, and David G. Myszka*‡

*Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132; and †Pﬁzer Global Research and Development, Eastern Point Road, Groton, CT 06340

Communicated by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, May 14, 2002 (received for review February 18, 2002) Surface plasmon resonance biosensor technology was used to receptor͞ligand interactions. In a qualitative screening mode, directly measure the binding interactions of small molecules to the receptor binders and nonbinders were identified. In a quantita- ligand-binding domain of human estrogen receptor. In a screening tive high-resolution mode, precise kinetic and affinity parame- mode, specific ligands of the receptor were easily discerned from ters were obtained for compounds across a wide dynamic range. nonligands. In a high-resolution mode, the association and disso- These mechanistic data complement static structural informa- ciation phase binding responses were shown to be reproducible tion about ER (10–14), revealing how radically different rate and could be fit globally to a simple interaction model to extract constants govern the binding of agonist and selected antagonist reaction rate constants. On average, antagonist ligands (such as ligands. tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists Methods (such as estriol and ␤-estradiol). This finding is consistent with Reagents. Ligand-binding domains of human ER-␣ and -␤ were these antagonists binding to an altered conformation of the expressed in Escherichia coli as His-tagged proteins and purified receptor. The biosensor assay also could identify subtle differences by metal-affinity chromatography. The ER-␣ fragment was in how the same ligand interacted with two different isoforms of expressed as a 21 amino acid tag sequence (MGSSHHHHHH- the receptor (␣ and ␤). The biosensor’s ability to determine kinetic SSGLVPRGSHM) followed by residues 305–548 of human rate constants for small molecule͞protein interactions provides ER-␣ with the mutations Cys3813Ser, Cys4173Ser, and unique opportunities to understand the mechanisms associated Cys5303Ser. Conversion of these free Cys residues to Ser helps with complex formation as well as new information to drive the stabilize the protein, but does not change the ligand-binding optimization of drug candidates. activity (unpublished results). Electrospray mass spectrometry demonstrated that the initiator Met was absent in the purified urface plasmon resonance (SPR) biosensor technology has product (theoretical mass of 29,990 Da; observed mass of 29,993 Da), and that Ϸ50% of the product was modified by N-terminal Sadvanced to the point where it is possible to measure directly ␤ small molecules interacting with immobilized macromolecular gluconoylation, adding 178 Da to the mass (15). The ER- targets (1, 2). This development suggests that biosensor analysis fragment had the same tag followed by residues 258–498 of the receptor, with the mutations Cys3343Ser, Cys3693Ser, and will become an important secondary screening tool in drug 3 discovery, confirming hits from primary screens and providing Cys481 Ser [theoretical mass (without the Met initiator) of detailed kinetics for lead optimization (3, 4). To illustrate the 29,319 Da; observed mass of 29,317 Da]. utility of current SPR technology, the binding properties of small His4 mAb was purchased from Qiagen (Chatsworth, CA), coupling reagents (EDC, NHS, and ethanolamine HCl) were compounds (200–500 Da) interacting with human estrogen ͞ receptor (ER) were analyzed. purchased from Biacore (Uppsala, Sweden), and gentle Ag Ab elution buffer was purchased from Pierce. Corticosterone, dexa- Ligand binding to ER is responsible for controlling the basic ␤ biology of estrogen-sensitive tissues. Using selective agonists or methasone, diethylstilbestrol, 17 -estradiol, estriol, estrone, antagonists to modulate this biology is the focus of significant 4-hydroxytamoxifen, nafoxidine, prasterone (also known as de- activity in the pharmaceutical industry (5–9). To date, a ligand’s hydroisoandrosterone), testosterone, and tamoxifen were pur- binding properties for ER have mainly been determined by chased from Sigma. Bisphenol A was purchased from Fluka. The equilibrium binding assays that often employ radiolabeled com- compounds were prepared as 1 mM stock solutions in DMSO to pounds and require overnight incubations. Here, we demon- ensure complete dissolution. Immediately before analysis, each strate how optical biosensors may be used to determine both compound was diluted in 20 mM sodium phosphate, 150 mM sodium chloride, 1 mM DTT, pH 7.4 to yield a final DMSO kinetic and equilibrium binding constants for compounds inter- ␮ acting with ER in real time without labeling either binding concentration of 3% and a compound concentration of 30 M. partner. Concentration series were prepared by serial dilution of the 30 ␮M solutions with 20 mM sodium phosphate, 150 mM sodium SPR biosensor experiments require immobilizing one reactant ͞ on a surface and monitoring its binding to a second reactant in chloride, 1 mM DTT, 3% (vol vol) DMSO, pH 7.4. solution. An antibody-capturing method was used to study the Instrumentation. dynamics of ER͞ligand interactions. This assay format created a Surface plasmon resonance analyses were per- chemically homogenous receptor surface and allowed us to formed by using Biacore 2000 and Biacore 3000 optical biosen- determine rapidly the binding properties of a variety of com- sors equipped with research-grade CM5 sensor chips (Biacore). pounds. We examined the binding of 12 compounds (shown in Immobilization of His mAb. Fig. 1) having differing receptor activities: both estrogen and 4 His4 mAb surfaces were prepared by non-estrogen agonists, SERMs (selective ER modulators, which using standard amine-coupling procedures (16) and HBS for this discussion are referred to as antagonists), and nonbinding control compounds that possess core structures similar to Abbreviations: SPR, surface plasmon resonance; ER, estrogen receptor. those of the estrogen agonists. ‡To whom reprint requests should be addressed at: Center for Biomolecular Interaction These results illustrate that current biosensor technology can Analysis, School of Medicine 4A417, 50 North Medical Drive, Salt Lake City, UT. E-mail: be used both qualitatively and quantitatively to monitor nuclear [email protected].

8562–8567 ͉ PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.142288199 Downloaded by guest on September 29, 2021 Kinetic Analysis of Ligand͞ER Interactions. To collect detailed kinetic data, a concentration series of each compound was injected at a flow rate of 50 ␮l͞min over antibody-captured ER and the His4 mAb reference surface at 20°C. Triplicate injections of each ligand concentration were analyzed in random order, with buffer blanks injected periodically for double referencing. For analysis of bisphenol A and prasterone, both the association and dissociation phases were 30 s; for diethylstilbestrol, 17␤- estradiol, estriol, and estrone, the association and dissociation phases were 30 and 180 s, respectively; and for 4-hydroxytamoxifen, nafoxidine, and tamoxifen, the association and dissociation phases were 3 and 10 min, respectively. When necessary, buffer samples containing 2.9–3.1% (vol͞vol) DMSO were injected to construct a DMSO calibration plot to correct for bulk refractive index shifts (17). The antibody surface was regenerated between binding cycles with two 18-s injections of 2:1 gentle Ab͞Ag elution buffer:10 mM H3PO4. The complete kinetic analysis of each ligand͞receptor interaction was performed three or more times over different His4 mAb surfaces. Data were collected at a rate of 2.5 Hz.

Data Processing and Analysis. All sensorgrams were processed by using double referencing (18). First, the responses from the reference surface were subtracted from the binding responses collected over the reaction surfaces to correct for bulk refractive index changes. Second, the response from an average of the blank injections was subtracted to remove any systematic artifact observed between the reaction and reference flow cells. To obtain kinetic rate constants, corrected response data were then fit in CLAMP, a data analysis program that combines numerical integration and nonlinear global curve fitting routines (19). A kinetic analysis of each ligand͞receptor interaction was obtained by fitting the response data to a reversible 1:1 bimolecular Fig. 1. Panel of compounds studied. (A) Estrogen agonists. (B) Non-estrogen ϭ interaction model that includes a term for mass transport (Ao agonists. (C) Antagonists. (D) Nonbinding compounds included as controls. ϩ ϭ A B AB) (20). The equilibrium dissociation constant (KD) ͞ BIOCHEMISTRY was determined by the quotient kd ka. Constants reported in Table 1 represent the average of three or more independent (Hepes-buffered saline: 20 mM Hepes, 150 mM sodium chloride, analyses of each receptor͞ligand interaction. 0.005% P20, pH 7.4) as the running buffer. Flow cells were activated for 7 min by injecting 140 ␮lof50mMN- Results hydroxysuccinimide (NHS):200 mM ethyl-3(3-dimethylamino) Ligand-Binding Assay. A capturing system was used to monitor ␮ ͞ propyl carbodiimide (EDC). Fifty microliters of a 10 g ml His4 ligand binding to ER on the SPR biosensor (Fig. 2). First, ER was mAb solution (in 10 mM sodium acetate, pH 5.0) was injected captured by His4 mAb immobilized on the sensor chip surface. for 5 min at 10 ␮l͞min, followed by a 70-␮l injection of Second, ligand was made to flow across the captured receptor, ethanolamine to block any remaining activated groups on the and binding events were monitored for several minutes. Third, surface. Typically, this method resulted in Ϸ10,000 RU antibody the ligand͞receptor complex was stripped from the antibody immobilized. The stability of the antibody surface was demon- surface by a short injection of mild acid. An example of the strated by the flat baseline achieved at the beginning (0–100 s) primary data collected for the capture and ligand-binding assay ␮ of each sensorgram. is shown in Fig. 3. Injection of 2 M ER across the His4 mAb surface for 5 min resulted in Ϸ1,800 RU of captured receptor.

Capture of ER by His4 mAb. Receptor surfaces were prepared by The receptor was bound tightly, as indicated by the minor loss in Ϸ using 20 mM sodium phosphate, 150 mM sodium chloride, 1 mM signal during the wash phase ( 450–600 s). At 600 s, ligands DTT, 3% (vol͞vol) DMSO, pH 7.4 as the running buffer. ER (2 were injected across the captured receptor to collect binding ␮M) in this buffer was injected at 10 ␮l͞min for 5 min across data. Because the SPR responses are proportional to mass, ligand-binding signals are small in comparison to the large signal immobilized His antibody, resulting in the capture of Ϸ1,800 RU corresponding to the mAb capturing of ER. In this instance, an receptor. The receptor͞antibody complex was stable over the Ϸ100-fold mass difference between the receptor and the ligand time course of each ligand-binding cycle. It is likely that ER is exists, so an Ϸ100-fold difference in responses between the dimerized at the surface, providing enhanced stability due to an receptor-capturing step and the ligand-binding step is ex- avidity effect. pected (16). ␮ After ligand binding to the captured receptor was monitored, Screening of Ligand Binding to ER. In a screening format, 1 Mof the surface was washed with mild acid, stripping the captured ␮ ͞ each compound was injected at a flow rate of 50 l min over ER͞ligand complex from the antibody surface and returning the antibody-captured ER. An His4 mAb surface served as a refer- signal to baseline (t Ͼ 960 s). The data shown in Fig. 3 were ence. Ligands were allowed to associate with the receptor for 30 s obtained from 40 repeated cycles. The excellent overlay of these and dissociation was monitored for 2 min. Ligand͞receptor sensorgrams demonstrates the ability to capture reproducibly the complexes were stripped from the antibody surface by two 18-s same density of receptor, which is critical when comparing the ͞ injections of 2:1 gentle Ab Ag elution buffer:10 mM H3PO4. responses generated by different ligands (or a concentration

Rich et al. PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 ͉ 8563 Downloaded by guest on September 29, 2021 Table 1. Afﬁnity and rate constants for estrogen receptor͞ligand interactions

KD (nM) from biosensor b c (ligand-binding KD (nM) from literature KD (nM) from literature Ϫ1 Ϫ1 a Ϫ1 a a Interaction ka (M s ) kd (s ) domain) (wild type, full-length) (ligand-binding domain)

Agonist ligand prasterone —d —d 4400 (2000) 235–5400ef bisphenol A 1.3 (1) ϫ 106 2.7 (1) ϫ 10Ϫ1 210 (10) 195–32000g 17␤-estradiol 1.3 (6) ϫ 106 1.2 (2) ϫ 10Ϫ3 0.9 (4) 0.05–15.0h 0.92,ik 0.3l estriol 1.0 (2) ϫ 106 1.3 (3) ϫ 10Ϫ2 13 (5) 1.4–9.3em estrone 1.1 (2) ϫ 106 9 (2) ϫ 10Ϫ3 8 (3) 0.3–626en diethylstilbestrol 6.0 (7) ϫ 106 5 (2) ϫ 10Ϫ5 0.009 (3) 0.04–11o Antagonist ligand tamoxifen 4.5 (1) ϫ 103 1.0 (1) ϫ 10Ϫ3 220 (20) 3.4–423p 4-hydroxytamoxifen 2.3 (1) ϫ 103 4.1 (1) ϫ 10Ϫ5 18 (1) 0.1–96q 0.22l nafoxidine 6.3 (2) ϫ 103 1.6 (1) ϫ 10Ϫ4 25 (2) 0.3–125er

aExperimental error is reported in parentheses and was obtained from three or more independent analyses using different biosensors, sample preparations, and receptor densities on the ﬂow cell surfaces. b Range of literature values reported (KD, KI,IC50,EC50, and RBA) for full-length wild-type human estrogen receptor-␣. c Range of literature values reported (KD, KI,IC50,EC50, and RBA) for wild-type human estrogen receptor-␣ ligand-binding domain. dNot determined. eFrom both human and nonhuman estrogen receptor-␣. fRefs. 21 and 22. gRefs. 21 and 23–26. hRefs. 10, 21, 23, 24, 27–29, and 32–36. iDetermined using a three Cys3Ser mutant ER construct. kRef. 10. lRef. 33. mRefs. 21, 37, and 38. nRefs. 21, 23, and 37. oRefs. 21, 23–26, 28, and 29. pRefs. 21, 23, 24, 26, 35, and 39. qRefs. 8, 21, 24, 26, 28, 29, 33, 35, and 40. rRefs. 21, 37, 41, and 42.

series of one ligand) binding to the receptor. Having demon- concentration-dependent responses rapidly reach equilibrium strated the reliability of receptor capture as an assay method, we and return to baseline within seconds, indicating a weak inter- could examine ligand binding with confidence that the receptor action between prasterone and the receptor. To extract a binding surfaces were identical. constant for this interaction, the responses at equilibrium were plotted against prasterone concentration and fit to a simple Prasterone Equilibrium Analysis. As an initial test for ligand bind- binding isotherm (Fig. 4B). To test the reproducibility of the ing, we monitored prasterone (288 Da) interacting with the assay, the entire analysis was performed by using nine different Ϯ receptor surface. Prasterone is a small ER agonist with a densities of captured receptor to yield an average KD of 4.4 2.0 ␮ reported weak affinity of 0.2–5.4 ␮M (21, 22). Injecting a M (Table 1). concentration series of prasterone (0.12–30 ␮M) across the captured ER yielded the sensorgrams shown in Fig. 4A. The

Fig. 3. Sensorgrams of the complete antibody͞receptor͞ligand binding Fig. 2. Design of the receptor͞ligand assay. His4 mAb was covalently immo- cycle. As noted in the ﬁgure, ER was made to ﬂow over the anti-His4 mAb bilized on the biosensor surface, followed by capture of the estrogen-receptor surface (yielding a response of Ϸ1800 RU), the surface was washed with construct (Ϸ28 kDa) via its His tag. Compounds were injected across the buffer, ligand was injected over the captured receptor (yielding a response of surface, and the ER͞ligand interaction was monitored. The ER͞ligand complex Ϸ30 RU), and mild acid was injected over the surface to disrupt the receptor͞ was readily stripped from the antibody, and fresh receptor was captured antibody interaction. Reproducibility of the capturing assay is demonstrated before the next ligand-binding test. by the overlay of 40 replicate binding cycles shown here in different colors.

8564 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.142288199 Rich et al. Downloaded by guest on September 29, 2021 Fig. 5. Screen of drug panel for binding to ER-␣. Responses were generated from the injection of 1 ␮M each of non-estrogen agonists (green), estrogen agonists (red), antagonists, (blue), and control compounds (black).

nists (prasterone, bisphenol A, and diethylstilbestrol) were dra- matic. Although each of these ligands displayed rapid association to the ER surface, the dissociation rates varied tremendously. Prasterone and bisphenol A dissociated from the receptor within seconds (as expected from the results described in Fig. 4 A and C). Diethylstilbestrol, however, formed a stable complex, as indicated by its very slow dissociation from the surface during the Fig. 4. Interaction of weak-affinity binders with ER. (A) Representative washout phase. The greater response intensity and stable com- sensorgrams obtained from injections of prasterone at concentrations of 0, plex formation observed for diethylstilbestrol is consistent with 0.12, 0.37, 1.1, 3.3, 10, and 30 ␮M over ER captured on the His4 antibody surface. (B) Sensorgram responses at equilibrium (t Ϸ 25 s) were plotted it having a high affinity (0.04–11 nM, based on literature values) against prasterone concentration and fit to a simple binding isotherm to yield for human ER (21, 23–26, 28, 29). an equilibrium dissociation constant. (C) Representative sensorgrams (black The three estrogen metabolites, 17␤-estradiol, estriol, and lines) obtained from triplicate injections of bisphenol A at concentrations of estrone, all displayed rapid association to ER, but differences in 0, 0.037, 0.11, 0.33, 1.0, and 3.0 ␮M over ER captured on the anti-His4 antibody their respective dissociation rates. Visually comparing the ki- surface. Red lines depict the global fit of the data to a simple 1:1 bimolecular netics for the estrogen metabolites illustrates the biosensor’s ϭ ϫ 6 Ϫ1 Ϫ1 ϭ Ϫ1 interaction model, yielding ka 1.3 (1) 10 M s and kd 0.27 (1) s . ability to detect subtle differences in dissociation rates of ligands having similar core structures. In contrast, the antagonist ligands screened here (tamoxifen, 4-hydroxytamoxifen, and nafoxidine) BIOCHEMISTRY Bisphenol A Kinetics. Bisphenol A is one of the smallest of the ER’s all demonstrated relatively slow association to the receptor, but agonist ligands and binds ER with a reported affinity of 0.2–32 once bound, formed stable complexes. ␮M (21, 23–26). Fig. 4C depicts triplicate injections of 0.037–3.0 ␮ M bisphenol A (228 Da) to the receptor surface. The binding High-Resolution Kinetic Analysis. A detailed kinetic analysis for the responses were reproducible and concentration-dependent. agonists and antagonists was performed to determine the rate From the visual comparison of Fig. 4 A and C, however, it is constants associated with each compound. Fig. 6 depicts the evident that bisphenol A dissociates slower than prasterone from ␤ ͞ binding responses obtained for concentration series of 17 - the receptor surface. In fact, the bisphenol A receptor reaction estradiol, estriol, estrone, diethylstilbestrol, tamoxifen, and is slow enough to provide sufficient information for the deter- nafoxidine injected across the ER-␣ surface. From inspection, mination of kinetic rate constants. The binding responses were the differences in the six ligands’ dissociation rates are apparent. globally fit to a simple 1:1 interaction model (shown as red lines Each of the binding responses shown in Fig. 6 were well described in Fig. 4C). To establish the reliability of the kinetic assays, the by a 1:1 interaction model. As shown in Table 1, the kinetic rate entire bisphenol A analysis was performed by using nine differ- constants observed for the antagonists (2–6 ϫ 103 MϪ1 sϪ1 for ϭ ent densities of captured receptor (1.7–2.3 kRU) to yield ka 1.3 tamoxifen, 4-hydroxytomaxifen, and nafoxidine) were on aver- ϫ 6 Ϫ1 Ϫ1 ϭ Ϫ1 Ϫ Ϫ (1) 10 M s and kd 0.27 (1) s . An equilibrium age Ϸ500-fold slower than the agonist (1–6 ϫ 106 M 1 s 1 for Ϯ dissociation constant (KD)of210 10 nM was calculated from 17␤-estradiol, estriol, estrone, and diethylstilbestrol) rate the ratio of the association and dissociation rates. constants.

Ligand Screening. To compare the binding of diverse compounds Selectivity Assays. The results described to this point were ob- to ER, the biosensor assay was performed in a qualitative tained from the analysis of the ␣ isoform of ER only. Current screening mode. In this assay, the panel of compounds shown in Biacore 2000 and 3000 instruments are capable of monitoring Fig. 1 was tested for receptor binding. All of the compounds were the interactions within four flow cells simultaneously. Therefore, assayed at the same concentration (1 ␮M) to rank binding to ER to collect information on receptor selectivity, both ␣ and ␤ by response intensity as well as by association and dissociation isoforms of ER were immobilized onto the same sensor chip, but rates. Three compounds (testosterone, dexamethasone, and within different flow cells. The data presented in Fig. 7 illustrate corticosterone) were included as controls in the analysis because the differences observed when estriol was injected over both they are not recognized by ER, even though their core structures receptor surfaces simultaneously. It is clear from a visual in- resemble those of the estrogen metabolites (21, 26, 27). As shown spection of the data that estriol dissociates from the ␣ isoform in Fig. 5, these control compounds did not show any binding to faster than from the ␤ isoform. Both data sets could be described ER. Receptor binding of the other nine compounds was evident, by using a simple 1:1 interaction model yielding the following ϭ ϫ 6 Ϫ1 Ϫ1 even for the weak binders, bisphenol A and prasterone. rate constants for each interaction: ka 1.0 10 M s , kd ϭ ϫ Ϫ2 Ϫ1 ␣ ϭ ϫ 5 Ϫ1 Ϫ1 ϭ ϫ The differences in binding kinetics of the non-estrogen ago- 1.8 10 s for , and ka 5.7 10 M s , kd 3.2

Rich et al. PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 ͉ 8565 Downloaded by guest on September 29, 2021 Fig. 6. Representative data sets (black lines) for kinetic analysis of ligand–ER interactions. The estrogen agonists 17␤-estradiol (A) and estriol (B) were injected at concentrations of 0, 0.0041, 0.012, 0.037, 0.11, 0.33, and 1.0 ␮M 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 ␮M 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 ␮M 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.

10Ϫ3 sϪ1 for ␤. The kinetics provide a detailed picture of the Discussion threefold difference in affinity observed for estriol binding to ␤ The results presented here illustrate that it is possible to observe ϭ ␣ ϭ (KD 5.6 nM) and (KD 18 nM) isoforms of the receptor. small ligands (Ͻ300 Da) binding to a captured receptor surface by using SPR biosensors. The use of a capturing system has several advantages over covalent immobilization of the receptor. Because the His tag is engineered into the receptor at a site spatially distant from the ligand-binding site, receptor immobilization does not interfere with ligand binding. Receptor molecules are homogeneously bound to the surface, thus minimizing surface heterogeneity. Capturing levels are reproducible and easily controlled. Disrupting the antibody͞receptor interaction is simpler than determining regeneration conditions that effec- tively remove a tightly bound ligand from the receptor, yet leave the receptor undamaged. The immobilized antibody is robust under these conditions, for hundreds of receptor-capturing͞ ligand-binding cycles can be performed on a single antibody surface. The validity and sensitivity of the biosensor assay was demonstrated by its ability to discriminate between estrogen- receptor ligands and nonligands (including the detection of prasterone as a ligand, even though it was screened at a concentration below its KD), as well as to yield equilibrium dissociation constants that fall within the range previously reported for each ligand͞receptor pair (Table 1). Prior functional and structural analyses of ligand-bound receptor provided the groundwork for understanding ER regula- tion by agonist and antagonist ligands: (i) the two ligand types bind to ER at the same site, and with similar affinities, but (ii) Fig. 7. Kinetic analysis of estriol binding to different estrogen-receptor the receptor exists in different conformations when it is com- isoforms. Estriol was injected at concentrations of 0, 0.019, 0.056, 0.17, 0.50, plexed with agonists or antagonists (10–14). Structural studies and 1.5 ␮M over captured ␣ (A) and ␤ (B) ER for 30 s, and dissociation was monitored for 6 min. Colored lines represent the global ﬁts of the sensorgrams demonstrated that when an agonist is bound, the C-terminal (black lines) to a 1:1 bimolecular interaction model. From the data shown here, helix of the estrogen-receptor ligand-binding domain folds back 6 Ϫ1 the kinetic parameters obtained for each interaction are ka ϭ 1.0 ϫ 10 M atop the ligand-binding pocket in a manner that facilitates Ϫ1 Ϫ2 Ϫ1 5 Ϫ1 Ϫ1 s , kd ϭ 1.8 ϫ 10 s , kd ϭ18 nM for ␣ and ka ϭ 5.7 ϫ 10 M s , kd ϭ 3.2 ϫ coactivator binding and the subsequent activation of transcrip- Ϫ3 Ϫ1 10 s , KD ϭ 5.6 nM for ␤. tion. When an antagonist is bound, its bulky side chain prevents

8566 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.142288199 Rich et al. Downloaded by guest on September 29, 2021 the C-terminal helix from occupying its normal packing site, and binding by the ␣ and ␤ isoforms of the receptor. Although estriol coactivator binding is not possible (11–12). Although the struc- bound more rapidly to the ␣ isoform, it also dissociated more tural data provide a detailed snapshot of conformation states of rapidly, giving it an overall weaker affinity than for the ␤ isoform. the receptor, they do not provide any dynamic information on These results illustrate the important insights that can be ob- complex formation. tained from binding kinetics rather than performing purely The biosensor analysis revealed clear mechanistic differences equilibrium-based measurements. The high resolution available in how agonist and antagonist ligands bind to ER. As a group, with the biosensor may provide the basis for the design of ligands agonists all displayed relatively fast association rate constants, that specifically target only one receptor isoform, thereby in- Ϫ Ϫ which averaged around 2 ϫ 106 M 1 s 1. In contrast, the creasing tissue selectivity and decreasing undesirable effects of antagonists possessed association rates that were nearly 500-fold new therapeutic agents. Ϫ Ϫ slower, averaging 4.4 ϫ 103 M 1 s 1. The slower association rates Our results demonstrate that it is possible to detect small are consistent with conformational adaptation accompanying ligands binding to large proteins captured on the biosensor, as antagonist complex formation. It is likely that the antagonists well as to obtain reaction rate and affinity information, even for recognize a conformation of the receptor that is normally low in weak binders. The biosensor assay is robust, requires no labels, population, leading to the observed slow association rate. and current instruments have a throughput of Ϸ100–200 assays Whether or not the divergence in rate constants observed for ER per day. The kinetic information accessible with the biosensor trends with other known agonist and antagonist ligands and offers new insight into receptor–ligand interactions, as well as other nuclear receptor systems is currently under investigation. new criteria for lead optimization. We are also investigating how ligands bind ER in the presence of coactivator proteins (27, 30, 31). This research was supported by National Science Foundation Grant NSF By taking advantage of the multiple reaction surfaces available MCB 00-78143 (to D.G.M.) and National Institutes of Health Fellowship in the optical biosensor, we simultaneously examined ligand F32 DK10150 (to R.L.R.).

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Rich et al. PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 ͉ 8567 Downloaded by guest on September 29, 2021