Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin

Beate Illek1, Mike E. Lizarzaburu2, Vivien Lee1, Michael H. Nantz2, Mark J. Kurth2, and Horst Fischer1

1 Children's Hospital Oakland Research Institute, Oakland 94609; and 2 Department of Chemistry, University of California, Davis, California 95616


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Apigenin (4',5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl- currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation (Ks) was 9.1 ± 1.3 µM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4'-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 ± 11 µM. Blocked Cl- currents were structurally determined by 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, Ks was 4.1 ± 0.9 µM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl- current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.

flavonoids; resveratrol; binding site; chloride transport; epithelia; cystic fibrosis transmembrane conductance regulator


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EPITHELIAL CHLORIDE TRANSPORT stimulated by cAMP is mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in the apical membrane of epithelia. Salt and water transport across tight epithelia, such as in the airways and the large intestine, are significantly dependent on CFTR. Mutations in the CFTR gene cause cystic fibrosis, which is the most common genetic disease among Caucasians (18). There has been a surge of interest in discovering small molecular activators of CFTR that could potentially be used alone or in combination with genetic approaches for cystic fibrosis therapy (14). A number of chemically unrelated compounds have been introduced, which showed activity as activators of both wild-type and mutant CFTR (reviewed in Ref. 11).

The identification of specific CFTR blockers is of pharmaceutical interest as a potential antidiarrhetic drug. Enterotoxin-induced secretory diarrhea is primarily caused by toxin-induced elevated cAMP levels in enterocytes, which cause an irreversible activation of CFTR and concomitant water loss (3, 15). Presently, two types of molecules are used as CFTR blockers in vitro: arylaminobenzoates, such as N-phenylanthranilic acid (DPC) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (5, 24), and sulfonylureas, such as glibenclamide or tolbutamide (20). However, both compound classes are nonspecific CFTR blockers and have to be used at relatively high concentrations.

Flavonoids are a group of small molecules derived from plant-based compounds of the common flavone (2-phenyl-gamma -benzopyrone) structure. Using quantitative kinetic analysis of drug effects on CFTR-mediated Cl- currents, we selected apigenin (4',5,7-trihydroxyflavone) as a CFTR activator from a small group of homologous flavonoids (10). Apigenin activated CFTR according to measurements performed in single cells, epithelial tissues, and in humans in vivo in the low micromolar range, indicating that apigenin is a positive lead for CFTR drug development. In addition to stimulation, apigenin blocked CFTR at high concentrations (10). Recently, a plant extract containing flavone-related compounds was shown to block CFTR-mediated currents in T84 cells and is currently in clinical trial for AIDS-related diarrhea (7, 9).

The current study was designed to identify the structural components of the apigenin molecule responsible for activation and block of CFTR-mediated currents. The results allow predictions about interactions in the binding sites and about the molecular structures necessary for stimulation or block of CFTR activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cells

Calu-3 cells, a human airway cell line of adenocarcinoma origin, were cultured as described (12). Calu-3 cells have functional characteristics of serous airway gland cells, including high expression of CFTR in their apical membranes and few detectable types of other Cl- channels, as determined in patch-clamp experiments (8, 10) and by the pharmacological profile, the ion selectivity, and the physiological regulation of apical membrane Cl- currents (10, 12, 13, 19). No transepithelial Na+ transport was found in Calu-3 monolayers (19). Tissues were grown on permeable filter supports (Falcon, Becton Dickinson, Franklin Lakes, NJ) and used 1-5 days after seeding.

Transepithelial Measurements

Short-circuit current (Isc) measurements were done as described (13). Briefly, filters were mounted in circulation-type Ussing chambers (World Precision Instruments, Sarasota, FL) and short circuited (Physiologic Instruments, San Diego, CA); the resulting Isc was amplified, digitized, and recorded to a computer. Every 50 s (in some experiments every 20 s), 2-mV 1-s pulses were applied to continuously monitor the transepithelial resistance. Experiments were done with a serosal-to-mucosal Cl- gradient to generate a driving force for Cl- over the apical membrane and to amplify the signal. We previously found that, in the presence of a Cl- gradient, the measured transepithelial current is composed of a DPC-insensitive component (likely paracellular) and a DPC-sensitive transcellular component (12). For the measured transcellular current, the apical Cl- conductance is the rate-limiting step, whereas the basolateral K+ conductance had little effect. This was determined using serosal K+ channel blockers. Figure 1 shows the lack of effect of serosal addition of Ba2+, lidocaine, or quinidine. On average, addition of 1 mM barium acetate blocked 0.15 ± 0.66 µA/cm2 (n = 4), 1 mM lidocaine blocked 0.9 ± 0.83 µA/cm2 (n = 3), and 500 µM quinidine blocked 0.5 ± 0.58 µA/cm2 (n = 4). Effects of K+ channel blocker were not significantly different from zero (one-sample t-test), indicating that the basolateral K+ conductance had no significant effect on measured Cl- currents. Measured Cl- currents showed the typical blocker profile of CFTR-mediated currents. First, currents were blocked by apical DPC but not by DIDS (Fig. 1C). On average, DIDS blocked 0.79 ± 0.70 µA/cm2 (n = 7, not different from 0). Second, we have shown previously that Cl- currents were blocked by glibenclamide and not by 4,4'-dinitro-2,2'-stilbenedisulfonic acid (13). These data indicate that the measured currents across Calu-3 monolayers in the presence of a Cl- gradient were largely CFTR mediated. DPC, but not DIDS, also blocked a portion of unstimulated currents (55 ± 4%, n = 38) across Calu-3 monolayers (Fig. 1C, bottom trace), suggesting that a fraction of the unstimulated current is mediated by basal CFTR activity.


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Fig. 1.   Effects of blockers on gradient-driven Cl- currents across Calu-3 monolayers. A and B: forskolin-stimulated tissues; 500 µM quinidine, 1 mM barium acetate, or 500 µM lidocaine was added to the serosal side. C: effects of 100 µM DIDS and 4 mM N-phenylanthranilic acid (DPC) on forskolin-stimulated (top trace) or control (bottom trace) tissues. Residual current after DPC was 10.0 ± 0.9 µA/cm2 (control, n = 82) and 13.3 ± 1.3 µA/cm2 (forskolin, n = 58). Isc, short-circuit current.

The serosal solution contained (in mM) 120 NaCl, 20 NaHCO3, 5 KHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.6 glucose. In the mucosal solution, all Cl- salts were exchanged for gluconate salts. Both chamber compartments were gassed with 95% O2-5% CO2 (pH 7.4). Solution volume in each chamber compartment was 5.0 ml. Chamber aperture was 0.6 cm2, and Isc was normalized to 1 cm2. Positive currents were defined as Cl- movement from serosa to mucosa. Experiments were done at 37°C.

Analysis of Drug Effects

The effects of flavonoids were determined from dose-response curves. For each experiment, steady-state currents for each drug concentration were measured and normalized to the maximal current (100%) and the initial current (0%), and kinetics of drug effects were quantified by fitting a Michaelis-Menten (MM) function
I<SUB>sc</SUB><IT>=</IT>(<IT>I</IT><SUB>s</SUB><IT>×</IT>c<SUP><IT>n</IT><SUB>s</SUB></SUP>)<IT>/</IT>(<IT>K</IT><SUP><IT>n</IT><SUB>s</SUB></SUP><SUB>s</SUB><IT>+</IT>c<SUP><IT>n</IT><SUB>s</SUB></SUP>)
by nonlinear regression. Is is the maximally stimulated current (which was read directly from the data), and c is the drug concentration. The half-maximal concentrations (Ks) and the Hill coefficient (ns) were the fitted parameters that describe the kinetics of current stimulation. Drugs that caused both stimulation of current (at low concentrations) and block (at high concentrations) were fitted with a double MM function of the form
I<SUB>sc</SUB><IT>=</IT>(<IT>I</IT><SUB>s</SUB><IT>×</IT>c<SUP><IT>n</IT><SUB>s</SUB></SUP>)<IT>/</IT>(<IT>K</IT><SUP><IT>n</IT><SUB>s</SUB></SUP><SUB>s</SUB><IT>+</IT>c<SUP><IT>n</IT><SUB>s</SUB></SUP>)<IT>+</IT>(<IT>I</IT><SUB>b</SUB><IT>×</IT>c)<IT>/</IT>(<IT>K</IT><SUB>b</SUB><IT>+</IT>c)
which estimates, in addition, the half-maximal concentrations for the blocker kinetics (Kb). No Hill coefficient for the blocker reaction was estimated because, in most cases, there were not enough data points in the high-concentration range to reliably estimate this parameter. The maximally blocked current (Ib) was read directly from the data. Fits were performed under visual graphic control. Because of limited solubility at high concentrations, Kb and Ib could not be determined for all drugs. Therefore, we used the current blocked at 300 µM (I300), measured as a percentage of maximal current, as a determinant of the blocking reaction. Estimated parameters were averaged (given as means ± SE) and compared statistically using factorial ANOVAs followed by unpaired t-tests. One-sample t-tests were used to determine significant differences from zero or from one (for ns). Statistics were calculated with StatView (version 4.57, Abacus Concepts, Berkeley, CA).

Drugs

Stock solutions of test drugs were made in DMSO at concentrations of 1, 10, and 100 mM and used at final concentrations of 0.1-1,000 µM. All compounds were soluble up to 1,000 µM except for 4',7-dihydroxyflavone and flavone, which precipitated at >300 µM. Apigenin and resveratrol were from Sigma Chemical (St. Louis, MO); all other flavonoids were from Indofine (Somerville, NJ). According to the manufacturers' declarations, purities were 95% (apigenin), 97-98% (4',7-dihydroxyflavone, 5,7-dihydroxyflavone, 4',5-dihydroxyflavone, 7-methoxyapigenin, and trimethoxyapigenin), and >= 99% (flavone, resveratrol, and 4'-methoxyapigenin). All test drugs were added to the mucosal side. The adenylate cyclase activator forskolin (Calbiochem, La Jolla, CA) was made as a 20 mM stock in DMSO and used at 10 µM added to the serosal side; the Cl- channel blocker DPC (Aldrich, Milwaukee, WI) was made as a 200 mM stock in ethanol and used at 4 mM (mucosal) at the end of each experiment. Barium acetate and lidocaine were made as 1 M stock in water and used at 1 mM; quinidine was made as 100 mM stock in DMSO and used at 500 µM.

General Procedure for the Synthesis of 5-Methoxyapigenin

The synthesis of 5-methoxyapigenin (5Me) was accomplished in three steps from apigenin via selective bis-protection, 5-hydroxy methylation, and subsequent deprotection. The 7-hydroxy and 4'-hydroxy groups were selectively protected by refluxing an acetonitrile solution of apigenin with p-methoxybenzyl chloride (5 equivalents), NaHCO3 (3 equivalents), and catalytic tetrabutylammonium iodide. There was no detectable benzylation of the less reactive 5-hydroxyl moiety, which was methylated by deprotonation with NaH (3 equivalents) in DMSO-tolulene (1:1) followed by addition of excess methyl iodide. Standard trifluoroacetic acid deprotection conditions yielded 5Me (yellow-brown crystals; Ref. 26). The product was purified by silica gel chromatography using CH2Cl2-methanol (9:1) as eluent. Purity was >95% (by NMR and single thin-layer chromatographic analysis). NMR and infrared spectra were consistent with the structure of 5Me and with previously reported data (21).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the roles of the structural components of the apigenin molecule, we used apigenin derivatives that had 1) single hydroxyls either removed (compounds 4'H, 5H, 7H) or methoxylated (4'Me, 5Me, 7Me), 2) the central pyrone ring of apigenin removed (resveratrol), 3) or all three hydroxyls removed (flavone) or methoxylated (4',5,7-timethoxy apigenin; TMe) as shown in Table 1. The methoxylated derivatives were used to distinguish effects of the respective hydroxyl as an H-bond donor or acceptor in the drug binding site, since methoxyls can act as H-bond acceptors but not as H donors. This approach was used to deduce the molecular interactions of apigenin in the binding site. Drug kinetics were significantly affected by stimulation of the tissue with forskolin. Therefore, we investigated kinetics separately in unstimulated and forskolin-stimulated tissues.

                              
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Table 1.   Apigenin derivatives

Effects on Control Tissues

Kinetics of apigenin. Stimulation of Cl- currents by apigenin has been reported previously (10). Apigenin dose dependently stimulated Cl- currents across Calu-3 monolayers (Fig. 2A) with a Ks = 9.1 ± 1.3 µM (n = 14) and an ns significantly larger than 1 (ns = 1.6 ± 0.1), indicative of positive cooperative binding during stimulation. The ns was not affected by any of the tested derivatives (P = 0.37, ANOVA), and the total average was ns = 1.7 ± 0.1 (n = 63, different from one, P < 0.0001). In the presence of a Cl- gradient, the baseline current was 20.9 ± 2.6 µA/cm2 (n = 13). Apigenin stimulated maximal currents at ~30 µM, which were on average 64% (60.8 ± 5.9 µA/cm2, n = 13) of current stimulated with 10 µM forskolin (93.2 ± 5.1 µA/cm2, n = 58). Concentrations larger than 30 µM blocked currents dose dependently with a Kb of 74 ± 11 µM (n = 6). Dose-response kinetics for apigenin are shown in Fig. 3A.


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Fig. 2.   Drug effects under control conditions. A-J show representative Isc measurements of dose-response relations for the drug indicated in each graph. All y-axes are Isc in µA/cm2. Bar in each graph represents 30 min. Arrows show time point of drug application; numbers are cumulative drug concentrations in µM present at that time point. Continuous line shows Isc; pulses show current elicited by a 2-mV pulse. Abbreviations of drug names are given in Table 1. DPC (4 mM) was added at the conclusion of every experiment to fully block Cl- transport; forskolin (Fsk) was added in some experiments to determine responsiveness of tissue. Note that limited water solubility of some drugs may have limited effects at high concentrations (5H, flavone).



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Fig. 3.   Kinetics of current activation and inactivation. Steady-state currents were normalized and fitted with single or double Michaelis-Menten kinetics (lines); y-axes are normalized Cl- current in percentage of maximal current, and x-axes are drug concentrations in µM. A-D show kinetics in control tissues; E-H show kinetics in forskolin-stimulated tissues. A: note right shift of dose-response relation of 4'H compared with 4'Me, suggesting that the 4-hydroxyl of apigenin is an H-bond acceptor during binding. B: both 5H and 5Me show similarly reduced half-maximal binding affinity of apigenin for current stimulation (Ks) compared with apigenin, suggesting that the 5-hydroxyl serves as an H-bond donor. C: 7Me showed little change in kinetics (compared with apigenin), whereas 7H showed no current activation (not shown in this plot), suggesting that the 7-hydroxyl is an H-bond acceptor. D: both activation and inactivation kinetics of resveratrol (Res) were right shifted compared with apigenin, indicating that the pyrone ring of apigenin significantly contributed to binding to both the stimulatory and inhibitory site. Flavone and TMe show further reduced affinities (i.e., are right shifted in this plot). E: in forskolin-stimulated tissues, 4'H showed activation only, whereas 4'Me showed only block of current. Of all tested drugs, 4'Me was the blocker with the highest affinity when used on forskolin-stimulated tissues. F: 5H showed kinetics similar to apigenin, whereas 5Me showed little effects on current activation. G: both 7H and 7Me showed unchanged Ks compared with apigenin but reduced block. H: flavone showed right shift of dose-response relation compared with apigenin; TMe and resveratrol showed little activation and reduced blocker affinities compared with apigenin. In some graphs, the 100% point of different drugs overlapped.

Roles of single structures of apigenin on current stimulation. Transepithelial experiments with compounds that had single hydroxyls removed or methoxylated are shown in Fig. 2, B-G. The respective dose-response kinetics and fits are shown in Fig. 3, A-C, and average data are given in Table 2. Removal of the 4'-hydroxyl (compound 4'H) significantly reduced the binding affinity (compared with apigenin; Fig. 3A, Table 2). However, 4'-methoxylation (4'Me) of apigenin had no effect on Ks (Fig. 3A, Table 2), indicating that the 4'-methoxyl can functionally replace the 4'-hydroxyl; i.e., it acts as an H-bond acceptor during binding. Removal of the 5-hydroxyl (5H) or 5-methoxylation (5Me) showed a similar reduction of Ks values as shown by their similar dose-response relations (Fig. 3B). Binding affinities were not different between 5H and 5Me (Table 2); therefore, the 5-hydroxyl likely serves as an H-bond donor. Compared with apigenin, 5H and 5Me showed only a small change of Ks, indicating that the 5H contributes only little to the total binding affinity and forms a weak H bond in the binding site. The 5H is unique because of its close proximity to the C==O group at position 4, with which it forms an intramolecular H bond. Thus its ability to serve as an H-bond donor in a binding site is markedly reduced. Removal of the 7-hydroxyl (7H) rendered an ineffective compound (Fig. 2F). No significant currents were stimulated by 7H (Table 2). In contrast, 7-methoxylation (7Me; Fig. 2G) resulted in current activation with unchanged affinity for both stimulation and block (compared with apigenin, Fig. 3C) and thus acts as an H-bond acceptor. Resveratrol showed a fourfold increased Ks (Fig. 3D), indicating that the central pyrone ring of apigenin is a significant contributor to the binding affinity.

                              
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Table 2.   Kinetic parameters in control tissues

In summary, the contribution of the individual structural components to the binding affinity of apigenin, as measured by the Ks values (Table 2), resulted in a rank order of 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl, where the 7-hydroxyl is the most significant and the 5-hydroxyl the least significant contributor.

Several of the tested compounds showed high binding affinities but small stimulated currents, for example, 4'Me, 5Me, and 7Me (Table 2). This indicates a kinetic model in which drug binding and current activation are separate steps, which is most simply modeled in a linear and reversible three-state activation model of the form
where initial drug binding (step I) is followed by a secondary conformational change to an active state (step II). Assuming that the step II reaction into the active state is fast compared with drug binding, then the measured Ks value describes the kinetics of the step I reaction, and the maximally stimulated current is a measure for the efficacy of the step II reaction to the active state. 7H and 7Me showed reduced currents compared with apigenin (despite unchanged binding affinity of 7Me), indicating that the step II reaction is significantly dependent on the 7-hydroxyl. In addition, activated currents were reduced in 4'Me and 5Me (Is, Table 2) despite unchanged binding affinities (Ks, Table 2), indicating that the step II reaction is inhibited by the single methoxyls.

Role of single structures of apigenin on current block. Inhibition of Cl- current was observed at high drug concentrations. The amount of blocked current was quantified by the percentage of current blocked at 300 µM drug concentration (I300, Table 2). Removal of hydroxyls at either positions 4' or 7 largely reduced the drugs' ability to block currents, whereas methoxylation at 4' and 7 showed smaller effects consistent with the notion that the 4'H and 7H act as H acceptors in the blocking site. Removal of the 5-hydroxyl reduced the blocking ability to a lesser extent, suggesting a weaker interaction of the 5-hydroxyl in the binding site, where it likely serves as an H-bond donor (because 5Me does not recover blocker effects). Resveratrol blocked currents less effectively, with a sixfold higher Kb value (435 ± 45 µM, n = 6) than apigenin. The resulting rank order for the molecular components of apigenin for current block (ranked by I300) was 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl, which is distinguished from the rank order during activation by the significantly increased role of the 4'-hydroxyl during block.

Combined effects of all three hydroxyls of apigenin. Removal of all hydroxyls (flavone, Fig. 2I) resulted in an ~10-fold increase in Ks (Fig. 3D, Table 2), but maximally stimulated currents were not different from apigenin-stimulated currents. In addition, methoxylation of all hydroxyls (TMe, Fig. 2J) caused a similar low affinity but effective stimulation. Because TMe showed a significantly higher affinity than flavone but lower affinity than any other singly methoxylated compound (Table 2), H bonding to TMe is weak.

Effects of Apigenin on Forskolin-Stimulated Tissues

Kinetics of apigenin. Kinetics of all drugs were significantly changed in tissues that were prestimulated with forskolin. Drug-stimulated currents (P < 0.0001) and affinities (P = 0.0004) were significantly different from controls (Table 3; ANOVA, tested for condition by drug). In forskolin-stimulated tissues, apigenin additionally activated Cl- currents by 72.5% with a Ks of 4.1 ± 0.9 µM, which was significantly lower than in unstimulated controls (P = 0.007). As in control tissues, maximal currents were stimulated at ~30 µM apigenin (Fig. 4A) and blocking kinetics were unaffected by forskolin stimulation, as judged by Kb and I300 (Table 3).

                              
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Table 3.   Kinetic parameters in forskolin-stimulated tissues



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Fig. 4.   Drug effects after forskolin stimulation. A-J show representative dose-response effects of the drugs indicated. Calu-3 monolayers were prestimulated with 10 µM forskolin. Labels are as in Fig. 2.

Role of apigenin structures on current stimulation after forskolin activation. Removal of any single hydroxyl of apigenin did not significantly affect Ks values (Table 3, Fig. 3, E-G, and Fig. 4, B, D, and F). Removal of all three hydroxyls caused a 10-fold reduction of the binding affinity (flavone, Table 3, Fig. 3H and Fig. 4I), whereas methoxylation of all three hydroxyls did not change the Ks value compared with apigenin (TMe, Table 3). Thus, after forskolin stimulation, the stimulatory drug binding site is less discriminatory and at least two of the hydroxyls serve as H-bond acceptors (because Ks of TMe < flavone). Removal of the pyrone ring totally prevented stimulation (resveratrol, Table 3 and Fig. 4H), indicating that the pyrone ring is the most significant part of apigenin for stimulation after forskolin treatment. After pretreatment of tissues with forskolin, the rank order of the contribution of structural components to Ks was pyrone > all hydroxyls > single hydroxyls.

Role of apigenin structures on current block after forskolin activation. Current block after forskolin stimulation was similar to block under control conditions (Table 3). Kb values could be determined more consistently after forskolin stimulation because currents were larger. Kb values resulted in the same rank order for the structural components (Table 3) as determined under control by I300 values. Note that the removal of the 7-hydroxyl or the 4'-hydroxyl largely inhibited the blocking effect. Compared with control, several compounds showed greatly different effects on current stimulation after forskolin (e.g., 4'H, 5H, and 7H), whereas the blocking reaction for these compounds was little changed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated the roles of structural components of apigenin on activation and on block of CFTR-mediated Cl- currents. We found that the stimulatory and the blocking reaction showed significantly different characteristics distinguished by their binding affinities and by the molecular interactions of apigenin during binding. Activation, but not blocking kinetics of apigenin, and the molecular interactions were changed by cAMP-dependent activation. This suggests that stimulation and block by apigenin were mediated by different binding sites, and forskolin stimulation of the tissue changed the structural interactions in the stimulatory binding site.

Molecular Characteristics of the Apigenin Binding Sites

Under control conditions, the 4'-hydroxyl and 7-hydroxyl of apigenin act as H-bond acceptors in the stimulatory site, indicating that the corresponding amino acid residues of the binding site donate hydrogens (i.e., serine, threonine, or tyrosine). The 5-hydroxyl acts as an H-bond donor, indicating that the binding site presents a residue that accepts an H-bond (for example, asparagine). The 5-hydroxyl formed the weakest H bond, likely because of the close proximity of the 4-keto group, which is the primary H-bond acceptor of the 5-hydroxyl. The central pyrone ring structure stabilized binding of apigenin, probably by aromatic pi ,pi -stacking with an amino acid residue such as phenylalanine or tyrosine.

Drug binding and effect were distinguished by the selective contribution of substituents of apigenin. We used a two-step reaction model in which reversible drug binding (step I in Scheme 1) is followed by reversible activation (step II in Scheme 1) to describe the activation of Cl- currents by apigenin. Assuming that the reaction rate into the active state is fast, then the measured Ks values describe the step I binding reaction and the measured Is values the step II reaction. For the stimulation of control current, binding was governed by 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. For comparison, the step II activation was largely dependent on the 7-hydroxyl, with little effects of the other substituents (see Table 2, Is values). Methoxylation of any single hydroxyls inhibited the step II but not the step I reaction (Table 2). Because 4'H and 5H showed unchanged Is values, this effect appears not related to H bonding. Thus binding (step I) and activation (step II) relied on different molecular substituents.

For both CFTR block and stimulation, the 7-hydroxyl was the most significant substituent. The 4'-hydroxyl contributed quantitatively similarly as the 7-hydroxyl to the blocking reaction, whereas it had a smaller role during stimulation. Thus the interaction of the 4'-hydroxyl in its sites is a key difference between the stimulating and blocking sites. The data that describe the blocking reaction did not consistently allow determination of Kb and Ib values because of the high concentrations needed for maximal block; thus the contributions of single substituents to a two-step reaction comparable to that shown in Scheme 1 cannot be deduced from the current data. However, the blocking reaction of some drugs was well fitted by MM kinetics, for example, apigenin (Fig. 3A), resveratrol (Fig. 3D), and 4'Me (Fig. 3E), indicating MM-typical saturation kinetics during current block.

When all hydroxyls, including the 7-hydroxyl, were removed (or methoxylated), large currents were activated at high concentrations. This was in contrast to the observation that the removal of the single 7-hydroxyl totally inhibited CFTR activation. A possible explanation is that the hydroxyls sterically align apigenin during binding in its site by way of the directionality of H bonds; when only the 7-hydroxyl was removed, the molecular alignment remained unchanged but the step II reaction was inhibited. In contrast, binding of flavone is likely governed by hydrophobic interactions and/or aromatic pi ,pi -stacking. Thus flavone can be expected to bind sterically differently from the binding site, which resulted in an activation of the step II reaction (i.e., current activation) at high concentrations. Binding to the same site is supported by the qualitatively similar kinetics of flavone and apigenin (i.e., both showed CFTR activation and block, similar ns, and similar regulation of kinetics by forskolin stimulation). This is consistent with the notion that drug binding in the stimulatory binding site allows more than one steric arrangement that leads to CFTR activation.

Stimulation of tissues with forskolin drastically changed activation kinetics and the effects of substituents on binding. The binding affinity to the stimulatory site increased, which was largely governed by the central pyrone ring with little contribution of the hydroxyls. Recently, accumulating evidence has shown that the apigenin isomer genistein binds directly to the CFTR rather than to regulatory proteins (4, 6, 17, 23, 25). Physiologically, CFTR is regulated through phosphorylation (2, 22). Thus the observed changes in the binding kinetics of apigenin possibly reflect a phosphorylation-induced conformational change of the stimulatory apigenin binding site. Our observations suggest that phosphorylation changes the configuration of the binding site so that the formation of H bonds is largely reduced and interaction with the central pyrone is significantly strengthened. As a result, under forskolin-stimulated conditions, the binding site discriminated much less between the molecules used in this study.

Characteristics of a Flavonoid-Based CFTR Activator or Blocker

Apigenin was shown to exert several additional effects on cellular metabolism, including block of mitogen-activated protein kinase (at 25 µM; Ref. 16) and phosphatidylinositol 3-kinase (at 12 µM; Ref. 1). This report outlines the molecular characteristics to guide the molecular design of CFTR drugs with a higher affinity and specificity. CFTR stimulation by apigenin was largely governed by the electron-donating substituent at position 7 and by the pyrone ring. By exploring molecular space and charge distribution around position 7 and by modifying the central aromatic structure, we hope to identify drugs with improved binding and probably higher specificity. By using a target-oriented combinatorial chemistry approach, it should be possible to develop a high-affinity CFTR activator.

In contrast to CFTR stimulation, block of CFTR was largely dependent on the 4' substituent in which an electron-donating substituent was critical for blocker activity. In addition, one compound (4'Me) showed significant CFTR block under both control and forskolin-stimulated conditions with little (control) or no stimulation (forskolin) of current. Thus the 4' position of apigenin appears to be a key position for a flavonoid-based CFTR blocker.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant 1P50-HL-60288-01 and Cystic Fibrosis Foundation Grant FISCHE99G0.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609-1673 (E-mail: hfischer{at}chori.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 February 2000; accepted in final form 5 July 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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