Receptor Binding Kinetics Research Articles

The duration of sleep promoting efficacy by dual orexin receptor antagonists is dependent upon receptor occupancy threshold

BackgroundDrugs targeting insomnia ideally promote sleep throughout the night, maintain normal sleep architecture, and are devoid of residual effects associated with morning sedation. These features of an ideal compound are not only dependent upon pharmacokinetics, receptor binding kinetics, potency and pharmacodynamic activity, but also upon a compound’s mechanism of action.ResultsDual orexin receptor antagonists (DORAs) block the arousal-promoting activity of orexin peptides and, as demonstrated in the current work, exhibit an efficacy signal window dependent upon oscillating levels of endogenous orexin neuropeptide. Sleep efficacy of structurally diverse DORAs in rat and dog was achieved at plasma exposures corresponding to orexin 2 receptor (OX2R) occupancies in the range of 65 to 80%. In rats, the time course of OX2R occupancy was dependent upon receptor binding kinetics and was tightly correlated with the timing of active wake reduction. In rhesus monkeys, direct comparison of DORA-22 with GABA-A modulators at similar sleep-inducing doses revealed that diazepam produced next-day residual sleep and both diazepam and eszopiclone induced next-day cognitive deficits. In stark contrast, DORA-22 did not produce residual effects. Furthermore, DORA-22 evoked only minimal changes in quantitative electroencephalogram (qEEG) activity during the normal resting phase in contrast to GABA-A modulators which induced substantial qEEG changes.ConclusionThe higher levels of receptor occupancy necessary for DORA efficacy require a plasma concentration profile sufficient to maintain sleep for the duration of the resting period. DORAs, with a half-life exceeding 8 h in humans, are expected to fulfill this requirement as exposures drop to sub-threshold receptor occupancy levels prior to the wake period, potentially avoiding next-day residual effects at therapeutic doses.

Measuring ligand–receptor binding kinetics and dynamics using k-space image correlation spectroscopy

Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.

A Simple Kinetic Model with Explicit Predictions for Nuclear Transport

Molecular exchange between the cell nucleus and cytoplasm is one of the most fundamental features of eukaryotic cell biology. The nuclear pores act as a conduit of this transport, both for cargo that crosses the pore autonomously as well as that whose translocation requires an intermediary receptor. The major class of such receptors is regulated by the small GTPase Ran, via whose interaction the nucleo-cytoplasmic transport system functions as a selective molecular pump. We propose a simple analytical model for transport that includes both translocation and receptor binding kinetics. The model is suitable for steady-state kinetics such as fluorescence recovery after photobleaching. Time constants appear as a combination of parameters whose effects on measured kinetics are not separable. Competitive cargo binding to receptors and large cytoplasmic volume buffer the transport properties of any particular cargo. Specific limits to the solutions provide a qualitative insight and interpretation of nuclear transport in the cellular context. Most significantly, we find that under realistic conditions receptor binding, rather than permeability of the nuclear pores, may be rate-limiting for nucleo-cytoplasmic exchange.

Cooperative Roles of SDF-1α and EGF Gradients on Tumor Cell Migration Revealed by a Robust 3D Microfluidic Model

Chemokine-mediated directed tumor cell migration within a three dimensional (3D) matrix, or chemoinvasion, is an important early step in cancer metastasis. Despite its clinical importance, it is largely unknown how cytokine and growth factor gradients within the tumor microenvironment regulate chemoinvasion. We studied tumor cell chemoinvasion in well-defined and stable chemical gradients using a robust 3D microfluidic model. We used CXCL12 (also known as SDF-1α) and epidermal growth factor (EGF), two well-known extracellular signaling molecules that co-exist in the tumor microenvironment (e.g. lymph nodes or intravasation sites), and a malignant breast tumor cell line, MDA-MB-231, embedded in type I collagen. When subjected to SDF-1α gradients alone, MDA-MB-231 cells migrated up the gradient, and the measured chemosensitivity (defined as the average cell velocity along the direction of the gradient) followed the ligand – receptor (SDF-1α – CXCR4) binding kinetics. On the other hand, when subjected to EGF gradients alone, tumor cells increased their overall motility, but without statistically significant chemotactic (directed) migration, in contrast to previous reports using 2D chemotaxis assays. Interestingly, we found that the chemoinvasive behavior to SDF-1α gradients was abrogated or even reversed in the presence of uniform concentrations of EGF; however, the presence of SDF-1α and EGF together modulated tumor cell motility cooperatively. These findings demonstrate the capabilities of our microfluidic model in re-creating complex microenvironments for cells, and the importance of cooperative roles of multiple cytokine and growth factor gradients in regulating cell migration in 3D environments.

Probing the Cytoadherence of Malaria Infected Red Blood Cells under Flow

Malaria is one of the most widespread and deadly human parasitic diseases caused by the Plasmodium (P.) species with the P.falciparum being the most deadly. The parasites are capable of invading red blood cells (RBCs) during infection. At the late stage of parasites’ development, the parasites export proteins to the infected RBCs (iRBC) membrane and bind to receptors of surface proteins on the endothelial cells that line microvasculature walls. Resulting adhesion of iRBCs to microvasculature is one of the main sources of most complications during malaria infection. Therefore, it is important to develop a versatile and simple experimental method to quantitatively investigate iRBCs cytoadhesion and binding kinetics. Here, we developed an advanced flow based adhesion assay to demonstrate that iRBC’s adhesion to endothelial CD36 receptor protein coated channels is a bistable process possessing a hysteresis loop. This finding confirms a recently developed model of cell adhesion which we used to fit our experimental data. We measured the contact area of iRBC under shear flow at different stages of infection using Total Internal Reflection Fluorescence (TIRF), and also adhesion receptor and ligand binding kinetics using Atomic Force Microscopy (AFM). With these parameters, we reproduced in our model the experimentally observed changes in adhesion properties of iRBCs accompanying parasite maturation and investigated the main mechanisms responsible for these changes, which are the contact area during the shear flow as well as the rupture area size.

Coupled particulate and continuum model for nanoparticle targeted delivery

Prediction of nanoparticle (NP) distribution in a vasculature involves transport phenomena at various scales and is crucial for the evaluation of NP delivery efficiency. A combined particulate and continuum model is developed to model NP transport and delivery processes. In the particulate model ligand–receptor binding kinetics is coupled with Brownian dynamics to study NP binding on a microscale. An analytical formula is derived to link molecular level binding parameters to particulate level adhesion and detachment rates. The obtained NP adhesion rates are then coupled with a convection–diffusion–reaction model to study NP transport and delivery at macroscale. The binding results of the continuum model agree well with those from the particulate model. The effects of shear rate, particle size and vascular geometry on NP adhesion are investigated. Attachment rates predicted by the analytical formula also agree reasonably well with the experimental data reported in literature. The developed coupled model that links ligand–receptor binding dynamics to NP adhesion rate along with macroscale transport and delivery processes may serve as a faster evaluation and prediction tool to determine NP distribution in complex vascular networks.

Direct Actions of Granulocyte-Colony Stimulating Factor on Human Neuronal and Monocytic Cell Lines

Introduction: Granulocyte colony stimulating factor (G-CSF) administration produces beneficial effects in rodent models of stroke, trauma and neurodegenerative diseases by acting on both bone marrow-derived and neuronal cells. The aim of the study was to elucidate cellular mechanism(s) of G-CSF action by direct application to neuronal and monocytic cell lines. Method: Cell culture models of monocytes (THP-1) and neurons (SH-SY5Y) cells were incubated with G-CSF. The following parameters were measured: G-CSF receptor binding kinetics; DNA synthesis; signal transduction, in particular expression of alternatively spliced protein kinase C (PKCδVIII) and the anti-apoptotic protein Bcl-2; changes in adhesiveness and migratory properties induced by G-CSF in the monocytic cells. Results: G-CSF receptor binding kinetics in the two lines differed, with Kd in the neuronal line being significantly higher than that of the monocytic cells. Despite higher affinity of G-CSF for receptors on the monocytic cells, G-CSF treatment increased Bcl-2 expression in the neuronal line at lower concentrations than that required in the monocytic cell line. G-CSF did not increase either cellular adhesiveness or migration through a semi-permeable membrane, whereas monocyte chemotactic protein (MCP-1) significantly improved migration. Conclusions: The cellular and molecular responses to G-CSF treatment of monocytic cells suggest that neither changes in adhesiveness nor migratory capacity are responsible for the beneficial effects of G-CSF administration in models of neurologic diseases. G-CSF induction of anti-apoptotic signaling in neurons is an important component of its neuroprotective effects in models of brain injury.

Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.

Slow receptor dissociation is not a key factor in the duration of action of inhaled long‐acting β2‐adrenoceptor agonists

β(2) -Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on β(2) -adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of β(2) -adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic. [(3) H]-DHA was used to label β(2) -adrenoceptors expressed in CHO-cell membranes (K(d) of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled β(2) agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ×K(i) ) of the unlabelled compound were employed against a fixed concentration of [(3) H]-DHA (0.6 nM). The clinically used β(2) -adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic K(d) and the competition K(i) values of the eight β(2) -adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate k(off) and k(on) rates. The kinetic on-rate was highly correlated with equilibrium binding affinity. Although the β(2) -adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing).

Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific sites

The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by an increase of both rise- and decay-time constants. A particularly large increase of rise times reflects initial formation of non-symmetric complexes by protein binding to non-specific sites at DNA ends. The increase of the hydrodynamic dimensions continues up to ~1s, before a decrease of time constants reflects transition to compact states with bent DNA up to the time range of ~10(3)s. The slow approach to CAP-induced DNA bending is due to non-specific complexes, which are formed initially and are converted slowly to the specific complex. At the salt concentration of 13.5mM, conversion to specific complexes with bent DNA is completed after ~40s at pH 8 compared to >10(3)s at pH 7, resulting from a higher affinity of CAP to non-specific sites at pH 7 than 8 by a factor of ~100. Thus, under the given conditions non-specific sites delay rather than facilitate formation of the specific complex with bent DNA. Experimental data obtained for a non-specific DNA clearly indicate the impact of pseudo-sites. The different electro-optical parameters have been combined in global fits.

Binding Kinetics Redefine the Antagonist Pharmacology of the Corticotropin-Releasing Factor Type 1 Receptor

Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF₁ receptor antagonist pharmacology was investigated by measuring the association rate constant (k₁), dissociation rate constant (k₋₁), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k₋₁/k₁), 170-fold range of k₋₁, and 13-fold range of k₁. The k₋₁ values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k₋₁ t(1/2) of 1.6-7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k₁, approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF₁ receptor antagonists.

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF(1)) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF(1) antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic-pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF(1) receptor binding of novel compounds can be predicted on the basis of in vitro assays. The non-competitive antagonist behaviour appeared to be correlated to the CRF(1) receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF(1) receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF(1) receptor antagonists in an efficient manner in a drug discovery setting.

Mechanism-Based Pharmacokinetic–Pharmacodynamic Modeling of the Dopamine D2 Receptor Occupancy of Olanzapine in Rats

PurposeA mechanism-based PK-PD model was developed to predict the time course of dopamine D2 receptor occupancy (D2RO) in rat striatum following administration of olanzapine, an atypical antipsychotic drug.MethodsA population approach was utilized to quantify both the pharmacokinetics and pharmacodynamics of olanzapine in rats using the exposure (plasma and brain concentration) and D2RO profile obtained experimentally at various doses (0.01–40 mg/kg) administered by different routes. A two-compartment pharmacokinetic model was used to describe the plasma pharmacokinetic profile. A hybrid physiology- and mechanism-based model was developed to characterize the D2 receptor binding in the striatum and was fitted sequentially to the data. The parameters were estimated using nonlinear mixed-effects modeling .ResultsPlasma, brain concentration profiles and time course of D2RO were well described by the model; validity of the proposed model is supported by good agreement between estimated association and dissociation rate constants and in vitro values from literature.ConclusionThis model includes both receptor binding kinetics and pharmacokinetics as the basis for the prediction of the D2RO in rats. Moreover, this modeling framework can be applied to scale the in vitro and preclinical information to clinical receptor occupancy.

Bronchodilatory effects of NVA237, a once daily long-acting muscarinic antagonist, in COPD patients

NVA237 is a novel, once daily inhaled long-acting muscarinic antagonist administered via a dry powder inhaler. This study aimed to assess the 24-h bronchodilatory effect following 14 days of treatment with inhaled NVA237 in patients with mild, moderate or severe COPD. This was a randomized, double-blind, placebo-controlled, two-period, crossover, multicenter study. A total of 33 patients (≥ 40 years; smoking history of ≥ 10 pack-years) were randomized to receive NVA237 50 μg once daily followed by placebo or placebo followed by NVA237 50 μg for 14 days. Treatment periods were separated by a 7-14 day washout period. The primary variable was the mean forced expiratory volume in 1 s (FEV(1)) derived from the area under the curve (AUC) between 0 and 24 h post-dose on Day 14. The 24-h FEV(1) profiles showed a consistent bronchodilator effect for NVA237 versus placebo on Day 14. Least square (LS) mean difference in FEV(1) AUC(0-24 h) values between NVA237 and placebo was 163 mL (P < 0.001). There were significant increases in mean FEV(1) AUC(0-12 h) (LS mean difference 165 mL, P = 0.001) and FEV(1) AUC(12-24 h) (161 mL, P < 0.001) versus placebo. NVA237 significantly improved peak FEV(1) (by 208 mL, P < 0.001) and trough FEV(1) (by 154 mL, P = 0.003) versus placebo on Day 14. NVA237 was well tolerated; all adverse events were mild or moderate in intensity and not related to study drug. NVA237 50 μg once daily was well tolerated and showed significant and sustained 24-h bronchodilation in patients with COPD.

Two Independent Histidines, One in Human Prolactin and One in Its Receptor, Are Critical for pH-dependent Receptor Recognition and Activation

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

Diffusion-Dependent Mechanisms of Receptor Engagement and Viral Entry

Enveloped viruses attach to host cells by binding to receptors on the cell surface. For many viruses, entry occurs via membrane fusion after a sufficient number of receptors have engaged ligand proteins on the virion. Under conditions where the cell surface receptor densities are low, recruitment of receptors may be limited by diffusion rather than by receptor-ligand interactions. We present a receptor-binding model that includes the effects of receptor availability at the viral binding site. The receptor binding and unbinding kinetics are coupled to receptor diffusion across the cell membrane. We find numerical solutions to our model and analyze the viral entry probabilities and the mean times to entry as functions of receptor concentration, receptor diffusivity, receptor binding stoichiometry, receptor detachment rates, and virus degradation/detachment rates. We also show how entry probabilities and times differ when receptors bind randomly or sequentially to the binding sites on the viral glycoprotein spikes. Our results provide general insight into the biophysical transport mechanisms that may arise in viral attachment and entry.

MATHEMATICAL MODELS OF RECEPTOR-MEDIATED TRANSPORT OF MORPHOGENS

Two models of receptor-mediated morphogen transport in a biological tissue are proposed to investigate morphogen gradient formation. The first model concerns intracellular transport of morphogen molecules, while the second describes transport along the cell surface. Both models couple via diffusivity a quasilinear degenerate parabolic equation describing the transport of the morphogens with an ordinary differential equation describing reversible binding kinetics of receptors. A detailed study of the steady states is provided, together with numerical tests which compare the models with experimental data.

Structure–activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4–12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

Obligate Ordered Binding of Human Lactogenic Cytokines

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.

Measuring Binding Kinetics of Ligands with Tethered Receptors by Fluorescence Polarization and Total Internal Reflection Fluorescence

Binding kinetics of nuclear receptors and their specific ligands was measured using polarization anisotropy complemented with total internal reflection fluorescence. Binding affinities of tethered full length human estrogen receptor alpha (ERalpha) with 17beta-estradiol, diethylstilbestrol, raloxifene, 4-hydroxytamoxifen, tamoxifen, and genistein were measured to be 100 (as reference), 100, 35, 21, 8, and 1.5, respectively. They agreed with published results. For the first time, rate constants were measured, and off rates were 1.5, 1.5, 1.3, 1.6, 1.7, and 2.3 x 10(-3) s(-1) while on rates were 11, 10, 3.3, 2.4, 1.0, and 0.26 x 10(5) M(-1)s(-1), respectively. For the antiestrogen drugs, their comparable off-rates correlated well with their equally similar potency. Eleven ginsenosides were screened as potential ligands. None were found to bind to ERalpha, but Rb1(S) and 20(S)-Rg3 were shown to bind to peroxisome proliferator-activated receptor gamma. The latter finding corroborated strongly with the therapeutic effects of ginsenosides on diabetic mice observed in a separate study. Our method would complement surface plasmon resonance assay for small ligands in the mass range of tens to hundreds of Daltons.