Relative Receptor Binding Affinity Research Articles

The emerging Omicron variant spike mutation: the relative receptor-binding domain affinity and molecular dynamics

The emerging Omicron variant spike mutation: the relative receptor-binding domain affinity and molecular dynamics

Drug-Drug Interactions (DDIs) in Psychiatric Practice, Part 9: Interactions Mediated by Drug-metabolizing Cytochrome P450 Enzymes.

This column is the ninth in a series exploring drug-drug interactions (DDIs) with a special emphasis on psychiatric medications. The first 3 columns in this DDI series discussed why patients being treated with psychiatric medications are at increased risk for taking multiple medications and thus experiencing DDIs, how to recognize such DDIs, strategies for avoiding and/or minimizing adverse outcomes from such DDIs, and pharmacokinetic considerations concerning DDIs in psychiatric practice. The fourth and fifth columns in this series presented a pair of parallel tables, one of which outlined the primary, known mechanism(s) of action of all commonly used psychiatric medications and one of which summarized major types of pharmaco-dynamic DDIs based on mechanism of action. Clinicians can use these 2 tables together to predict pharmacodynamically mediated DDIs. The sixth column discussed key pharmacodynamic interactions involving ethanol, opioids, and monoamine oxidase inhibitors. The seventh and eighth columns presented the concept of relative receptor binding and included tables summarizing the relative receptor binding affinity of currently available antipsychotics and antidepressants, respectively. This ninth and final column in this series discusses pharmacokinetic DDIs with a focus on psychiatric medications and contains 3 tables. The first table is an abbreviated version of a table available online showing which drugs are substrates for which cytochrome P450 (CYP) enzymes and which drugs are inhibitors or inducers of specific CYP enzymes. The abbreviated version of the table presented in this column focuses on psychiatric medications. This table and the larger website version can allow prescribers to anticipate which drug combinations may pose the risk of a CYP enzyme-mediated DDI. The second table summarizes which antidepressants inhibit specific CYP enzymes and which antidepressants do not or are unlikely to inhibit specific CYP enzymes. The third table presents psychiatric medications whose clearance is not principally dependent on CYP enzyme-mediated oxidative metabolism as a necessary step in their clearance from the body. The latter 2 tables inform prescribers as to which drugs they may prefer to use to avoid CYP enzyme-mediated DDIs. The overall goal of this series of columns is to present a simple way of conceptualizing neuropsychiatric medications in terms of their pharmacodynamics and pharmacokinetics to allow prescribers to take these facts into consideration when they need to use ≥2 drugs in combination to optimally treat a patient.

Drug-Drug Interactions (DDIs) in Psychiatric Practice, Part 8: Relative Receptor Binding Affinity as a Way of Understanding the Differential Pharmacology of Currently Available Antidepressants.

This column is the eighth in a series exploring drug-drug interactions (DDIs) with a special emphasis on psychiatric medications. The first 3 columns in this DDI series discussed why patients being treated with psychiatric medications are at increased risk for taking multiple medications and thus experiencing DDIs, how to recognize such DDIs, strategies for avoiding and/or minimizing adverse outcomes from such DDIs, and pharmacokinetic considerations concerning DDIs in psychiatric practice. The fourth and fifth columns in this series presented a pair of parallel tables, one of which outlined the primary, known mechanism(s) of action of all commonly used psychiatric medications and one of which summarized major types of pharmacodynamic DDIs based on mechanism of action. Clinicians can use these 2 tables together to predict pharmacodynamically mediated DDIs. The sixth column discussed key pharmacodynamic interactions involving ethanol, opioids, and monoamine oxidase inhibitors. That column focused particularly on hypertensive crises and serotonin syndrome with monoamine oxidase inhibitors and also DDIs involving psychiatric medications with adverse effects on the cardiovascular system and on the central nervous system. The seventh column presented the concept of relative receptor binding and included a table summarizing the relative receptor binding affinity of antipsychotics including all of the newer agents as well as some of the older agents such as haloperidol. This eighth column in this series presents a parallel table to the one in the seventh column summarizing the relative receptor binding affinity of currently available antidepressants. The overall goal of this series of columns is to present a simple way of conceptualizing neuropsychiatric medications in terms of their pharmacodynamics and pharmacokinetics to allow prescribers to take these facts into consideration when they need to use ≥2 drugs in combination to optimally treat a patient.

Drug-Drug Interactions (DDIs) in Psychiatric Practice, Part 7: Relative Receptor Binding Affinity as a Way of Understanding the Differential Pharmacology of Currently Available Antipsychotics.

This column is the seventh in a series exploring drug-drug interactions (DDIs) with a special emphasis on psychiatric medications. The first 3 columns in this DDI series discussed why patients being treated with psychiatric medications are at increased risk for taking multiple medications and thus experiencing DDIs, how to recognize such DDIs, strategies for avoiding and/or minimizing adverse outcomes from such DDIs, and pharmacokinetic considerations concerning DDIs in psychiatric practice. The fourth and fifth columns in this series presented a pair of parallel tables, one of which outlined the primary, known mechanism(s) of action of all commonly used psychiatric medications and one of which summarized major types of pharmacodynamic DDIs based on mechanism of action. Clinicians can use these 2 tables together to predict pharmacodynamically mediated DDIs. The sixth column discussed key pharmacodynamic interactions involving ethanol, opioids, and monoamine oxidase inhibitors. That column focused particularly on hypertensive crises and serotonin syndrome with monoamine oxidase inhibitors and also DDIs involving psychiatric medications with adverse effects on the cardiovascular system and on the central nervous system. This seventh column in this series presents the concept of relative receptor binding and includes a table summarizing the relative receptor binding affinity of commonly used antipsychotic medications, including all of the newer agents as well as some of the older agents such as haloperidol. The overall goal of this series of columns is to present a simple way of conceptualizing neuropsychiatric medications in terms of their pharmacodynamics and pharmacokinetics to allow prescribers to take these facts into consideration when they need to use ≥2 drugs in combination to optimally treat a patient.

Reintroduction of quazepam: an update on comparative hypnotic and adverse effects.

Insomnia is a prevalent disorder that affects over one-third of the U.S. population to varying degrees and is highly disruptive towards quality of life. Pharmacological treatments for insomnia include benzodiazepines (BZs) and the non-BZ 'Z-drugs' (zolpidem, zaleplon, eszopiclone, zopiclone), which are amongst the most widely prescribed medications. Yet, these agents can produce adverse effects such as tolerance to the hypnotic effect, rebound insomnia, next-day residual drowsiness, as well as amnesia and complex behaviours such as sleep-walking, sleep-eating and sleep-driving. Quazepam, one of the five BZ approved for treatment of insomnia, was recently relaunched to the U.S. market in 2016 and is distinguished amongst hypnotic BZ by unique pharmacological characteristics including selectivity for sleep-promoting α1-subunit containing γ-aminobutyric acid (GABA-A) receptors and a significantly lower relative receptor binding affinity. These features likely drive the decreased rate of adverse events seen clinically with quazepam, such as tolerance, rebound insomnia and amnesic behaviours, compared with other BZ. Given the recent reintroduction of quazepam as a pharmacotherapeutic option, and the lack of head-to-head comparative trials against newer agents, the purpose of this review is to provide an update on distinguishing features of quazepam with regard to its pharmacology, pharmacokinetics, sleep efficacy and potential adverse effects compared to other agents used for insomnia.

Evaluation of Biological Activity and Computer-Aided Design of New Soft Glucocorticoids.

Soft glucocorticoids are compounds that are biotransformed to inactive and non-toxic metabolites and have fewer side effects than traditional glucocorticoids. A new class of 17β-carboxamide steroids has been recently introduced by our group. In this study, local anti-inflammatory activity of these derivatives was evaluated by use of the croton oil-induced ear edema test. Glucocorticoids with the highest maximal edema inhibition (MEI) were pointed out, and the systemic side effects of those with the lowest EC50 values were significantly lower in comparison to dexamethasone. A 3D-QSAR model was created and employed for the design of 27 compounds. By use of the sequential combination of ligand-based and structure-based virtual screening, three compounds were selected from the ChEMBL library and used as a starting point for the design of 15 derivatives. Molecular docking analysis of the designed derivatives with the highest predicted MEI and relative glucocorticoid receptor binding affinity (20, 22, 24-1, 25-1, 27, VS7, VS13, and VS14) confirmed the presence of interactions with the glucocorticoid receptor that are important for the activity.

Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR

ObjectiveHuman insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11− (IR-A) and exon 11+ (IR-B) isoforms. DesignThree IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICIN and IGF-IICIC contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. ResultsThe IGF-IICIN analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICIN to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICIC bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICIS contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. ConclusionsTogether, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

Estrogen receptor affinity chromatography: A new method for characterization of novel estrogenic disinfection by-products

To identify the unknown estrogenic disinfection by-products (DBPs) from the chlorination extract, an effective method based on affinity chromatography with immobilized human recombinant estrogen receptor α (ERα) was developed, which has an advantage in targeting different potential estrogenic compounds from mixed sample simultaneously by comparing their relative binding activities to ER. The new method worked well for six known environmental estrogens. To further test the validity of this method for unknown chemicals, six DBPs of diethylstilbestrol (DES) with relatively strong ER binding affinity after chlorination were isolated and identified. It was found that except for 2-chloro-DES which showed 1.36 times stronger binding affinity than DES, most of the by-products bound to ER much more weakly than DES. All these seven by-products induced a dose-dependent transcriptional activation in two-hybrid-yeast assays. Z,Z-dienestrol (DE) and 2-chloro-DES, which exhibiting the weakest and the strongest binding affinity, were further tested for their transcriptional potential as 0.00243 and 0.014 compared to DES, respectively. However, they were still potential harmful environmental estrogenic disruptors as their estrogenic activities were much stronger than that of bisphenol A (BPA). These results demonstrated that the new method can help to screen unknown estrogenic compounds from mixture more efficiently.

Impact of a functionalized olive oil extract on the uterus and the bone in a model of postmenopausal osteoporosis

The Mediterranean diet rich in fruits, vegetables and olive oil has been related to a lower osteoporosis incidence and accordingly to a reduced fracture risk. These observations might be mediated by the active constituents of extra virgin olive oil, and especially polyphenols. In the context of exploring the features of olive oil active constituents on postmenopausal osteoporosis, an extra virgin olive oil total polyphenolic fraction (TPF) was isolated and its effect on the bone loss attenuation was investigated. Female Lewis rats were ovariectomized and fed a diet enriched with a total phenolic extract of extra virgin olive oil in a concentration of 800mg/kg diet. Oleocanthal, one compound of the polyphenolic fraction, showed a higher relative estrogen receptor binding affinity to the ERα compared to the ERβ. While the TPF only slightly induced the uterine wet weight (490.7±53.7 vs. 432.7±23, p=0.058), TPF regulated estrogen response genes in the uterus (progesterone receptor, antigen identified by monoclonal antibody Ki67, complement C3). Comparing the quantified bone parameters, the oral TPF substitution did not attenuate the ovariectomy-induced bone loss. The administration of extra virgin olive oil polyphenols regulated uterine estrogen response marker genes in an E2-agonistic manner. The bone loss induced by estrogen ablation was not mitigated by treatment with the polyphenolic extract.

Relative receptor affinity comparisons among inhaled/intranasal corticosteroids: perspectives on clinical relevance

BackgroundPharmacokinetic properties, dosing regimen, and potency at the site of action are among the factors that influence activity of a corticosteroid. The potency of a corticosteroid at the site of action is determined significantly by its affinity to the glucocorticoid receptor. Recent literature on topical corticosteroids reveals an increasing emphasis on comparative relative receptor affinity values as a key method of differentiating among various corticosteroid compounds, particularly with regard to clinical efficacy.MethodsA response was formulated to: Valotis A, Högger P: Human receptor kinetics and lung tissue retention of the enhanced-affinity glucocorticoid fluticasone furoate. Respir Res 2007, 8:54.ResultsRelative receptor binding affinities, while often showing significant variability across different laboratories, are a valid parameter when a comparison of the pharmacological activity of various glucocorticoids at the site of action is desired. Unfortunately within this context, scientific literature including the article from Valotis and Högger, confuse differences in potency (concentration or dose necessary to achieve a certain effect) with differences in efficacy (a quantitative difference in the overall maximum effect, even if all the receptors are occupied). All glucocorticoids will show the same efficacy as long as the selected dose will occupy the same number of receptors.ConclusionWhile relative receptor affinities are useful for comparing in vitro potencies of corticosteroids, these data are not representative of physiologic conditions and should not be used as a basis for comparing the presumed effectiveness of compounds in a clinical situation.

Clinical Relevance of Relative Receptor Binding Affinity: Quetiapine and Ziprasidone as Examples

Clinical Relevance of Relative Receptor Binding Affinity: Quetiapine and Ziprasidone as Examples

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERα and ERβ and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERα and ERβ than ZEN did. The IC 50 values of ZEN and ZOL for binding to human ERα were 240.4 and 21.79 nM, respectively, and the IC 50 values for binding to ERβ were 165.7 and 42.76 nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses ≥2 mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (≥0.5 mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERα and ERβ, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERα and ERβ. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERα and ERβ in a strikingly similar manner as 17β-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17β-estradiol. The primary reason that ZOL and ZEN is less potent than 17β-estradiol is likely because 17β-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).

The Effect of Protein Binding on Adrenal Suppression Potential of Inhaled Corticosteroids

RATIONALE: High protein-binding levels associated with some inhaled corticosteroids (ICS) may predict low levels of cortisol suppression. METHODS: An integrated pharmacokinetic/pharmacodynamic model based on the circadian rhythm of cortisol secretion was developed to accurately predict ICS-related cortisol suppression (% decrease in 24h cortisol area under the curve) using individual pharmacokinetic profiles and relative receptor binding affinities. The model was applied to several ciclesonide, fluticasone propionate and budesonide studies. Model-predicted cumulative cortisol suppressions (p) were compared with experimentally measured values (m). RESULTS: Accurate predictions were obtained for ciclesonide (metered-dose inhaler [MDI]; protein binding of the active metabolite desisobutyryl-ciclesonide=98.3%) 320μg twice daily (ex-actuator; bid; 4.9%p, 6.5%m), 640μg once daily (qd) at 7AM (6.5%p, 6.5%m) and 640μg qd at 7PM (2.9%p, 1.9%m). Predictions for budesonide (Turbuhaler®; protein binding=88%) also compared well with study observations: 400 μg bid (19.3%p, 19%m) and 1000μg bid (33.5%p, 36%m). Assuming a 90% protein binding (p90) for fluticasone propionate, the model slightly overpredicted measured values. However, the model greatly underpredicted cortisol suppression utilizing a recently reported 99.3% (p99.3) protein binding. Predictions for fluticasone propionate (p90 and p99.3, respectively) were 20.5%p and 1.9%p for 200 μg bid (Diskus®), 38.1%p and 4.5%p for 500 μg bid (Diskus®) and 62.2%p and 11.7%p for 880μg bid (pressurized MDI) compared with measured values of 14%m, 27%m and 59%m, respectively. CONCLUSIONS: ICS protein binding levels appear to correlate well with degrees of cortisol suppression. The recently reported high protein binding of fluticasone propionate is not consistent with the predicted and observed degree of cortisol suppression.

Significant receptor affinities of metabolites and a degradation product of mometasone furoate.

Mometasone furoate (MF) is a highly potent glucocorticoid used topically to treat inflammation in the lung, nose and on the skin. However, so far no information has been published on the human glucocorticoid receptor activity of the metabolites or degradation products of MF. We have now determined the relative receptor binding affinities of the known metabolite 6β-OH MF and the degradation product 9,11-epoxy MF to understand their possible contribution to undesirable systemic side effects. In competition experiments with human lung glucocorticoid receptors we have determined the relative receptor affinities (RRA) of these substances with reference to dexamethasone (RRA = 100). We have discovered that 6β-OH MF and 9,11-epoxy MF display RRAs of 206 ± 15 and 220 ± 22, respectively. This level of activity is similar to that of the clinically used inhaled corticosteroid flunisolide (RRA 180 ± 11). Furthermore we observed that 9,11-epoxy MF is a chemically reactive metabolite. In recovery experiments with human plasma and lung tissue we found a time dependent decrease in extractability of the compound. Hence, we provide data that might contribute to the understanding of the pharmacokinetics as well as the clinical effects of MF.

Ciclesonide: A novel inhaled corticosteroid for asthma

The adoption of inhaled corticosteroids for the treatment of asthma has resulted in significant improvements in asthma symptoms and pulmonary function, and has reduced the incidence of asthma exacerbations, emergency room visits and hospitalizations. Although inhaled corticosteroids are generally safe and well tolerated compared with oral corticosteroids, concerns remain about potential side effects. Clinically significant complications include local effects such as dysphonia and oral candidiasis and potentially serious systemic effects associated with cortisol suppression including growth retardation, glucose intolerance and increased risk for osteoporosis and fractures. These side effects are more likely to occur with long-term use and are dose related. Over the years, attempts have been made to develop corticosteroids with potent local antiinflammatory activity and an improved safety profile. Ciclesonide ([R]-11 beta,16 alpha,17,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetal with cyclohexanecarboxaldehyde 21-isobutyrate) was designed to achieve these objectives. Ciclesonide is a parent compound that is converted locally in airways by esterases to produce the active metabolite, desisobutyryl-ciclesonide (des-CIC). The active metabolite, des-CIC, has a 100-fold greater relative glucocorticoid receptor binding affinity than ciclesonide itself (relative glucocorticoid receptor binding affinities are 1200 and 12, respectively; dexamethasone reference is 100). If any ciclesonide enters the circulation, it is highly protein bound (99%) and extensively metabolized by liver oxidases, resulting in very low systemic exposure. Ciclesonide is delivered in solution form via a hydrofluoroalkane metered-dose inhaler (MDI) with a once-daily dosing schedule, which facilitates patient compliance. Clinical studies demonstrate that ciclesonide is as effective as existing "gold standard" inhaled corticosteroids for control of asthma and has a good safety profile.

Fluorescent cellular sensors of steroid receptor ligands.

Steroid hormone receptors comprise a major class of therapeutic drug targets that control gene expression by binding steroid hormone ligands. These small molecule-protein interactions are typically characterized in living cells by quantification of ligand-mediated reporter gene expression. As an alternative, non-transcriptional approach, we constructed fluorescent cellular sensors by expressing yellow fluorescent protein (YFP) fused to the ligand binding domains (LBDs) of estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), androgen receptor (AR), and the glucocorticoid receptor (GR). These proteins were tethered through a short two amino acid linker and expressed in S. cerevisiae yeast. Recombinant yeast treated with cognate steroid receptor ligands exhibited dose-dependent fluorescence enhancements that were correlated with known relative receptor binding affinity values. These effects generally paralleled ligand-mediated receptor dimerization quantified with analogous yeast two-hybrid transcriptional assays, suggesting that the majority of the observed fluorescence enhancements were conferred by conformational changes coupled with receptor dimerization, such as ligand-mediated stabilization of protein folding. Remarkably, certain interactions such as the binding of cortisol, progesterone, and dexamethasone to the GR were undetectable with yeast two-hybrid assays. However, these interactions were detected with the fluorescent cellular sensors, indicating the sensitivity of this system to subtle ligand-induced conformational effects. These sensors provide a novel, non-transcriptional, and high-throughput method to identify and analyze ligands of nuclear hormone receptors.

Pharmacology of AT 1 -receptor Blockers

Angiotensin II mediates its haemodynamic effects by binding to specific cell-surface receptors. In humans, two receptor subtypes have been identified, designated AT 1 and AT 2 . Because all major deleterious effects of angiotensin II are produced via binding to AT 1 -receptors, selective blockade of this receptor subtype should confer haemodynamic benefits, while allowing stimulation of the potentially beneficial effects mediated by AT 2 -receptors. Experimental studies using various models have consistently revealed marked differences in the receptor binding properties of different AT 1 -receptor blockers. The relative receptor binding affinities of currently available AT 1 -receptor blockers is candesartan > irbesartan > valsartan/EXP-3174/telmisartan > tasosartan > losartan > eprosartan. Candesartan is also released from the receptor more slowly than other available AT 1 -receptor blockers, with a half-life of approximately 152 min for the receptor-blocker complex, compared with 31 min for EXP-3174, 17 min for irbesartan and 5 min for losartan. Candesartan therefore binds to the AT 1 -receptor more tightly and more persistently than other AT 1 -receptor blockers.

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.

2-Phenylphenanthridinone and Related Compounds: Aryl Hydrocarbon Receptor Agonists and Suicide Inactivators of P4501A1

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 μM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a >80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.

Cumulative effect of double-site mutations of human epidermal growth factor on receptor binding.

Using site-directed mutagenesis, it was previously found that mutation of the individual residues Tyr13, Tyr22, Ile23, or Leu26 in the amino-terminal domain or of the highly conserved Leu47 in the carboxyl-terminal domain of human epidermal growth factor (hEGF), resulted in significantly decreased receptor binding affinity. In the present study, the single-site hEGF mutants Tyr13-->His, Tyr22-->Asp, Ile23-->Thr, and Leu26-->Gly were genetically combined with the Leu47-->Ala hEGF mutant to produce a series of double-site mutant hEGF gene products having alterations simultaneously at two sites, in separate domains, within the same hEGF molecule. Similarly, the combination of the single-site hEGF mutants Tyr13-->His and Ile23-->Thr generated a double-site mutant having two mutations within the same domain. Finally, combination of the hEGF mutation Ile23-->Ala with Leu23-->Ala altered two side chains located in close proximity within the large beta-sheet region of the molecule. Analysis of the relative receptor binding affinities, determined by radioreceptor competition assays of the various single- and double-site hEGF mutants, demonstrated that mutation at any one site does not substantially alter the effect of mutation at the second site in the molecule. The cumulative effect of simultaneous mutations on relative receptor binding affinity confirms the importance of residues, including those in the large amino-terminal beta-sheet, in receptor binding, and indicates that each of the separate sites functions essentially independently in the interaction of the hEGF molecule with its receptor.