Anal Bioanal Chem DOI 10.1007/s00216-013-7560-3
RESEARCH PAPER
A fluorescence-based high throughput assay for the determination of small molecule−human serum albumin protein binding Megan M. McCallum & Alan J. Pawlak & William R. Shadrick & Anton Simeonov & Ajit Jadhav & Adam Yasgar & David J. Maloney & Leggy A. Arnold
Received: 4 October 2013 / Revised: 26 November 2013 / Accepted: 6 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the library of pharmacological active compounds (LOPAC) small molecule library of 1,280 compounds identifying known high protein binders. The small molecule competition of HSA−Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3 and 24 μM. The compound affinity toward HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding.
Keywords Drug–protein binding . High throughput screening . Red Mega 500 . Human serum albumin M. M. McCallum : A. J. Pawlak : W. R. Shadrick : L. A. Arnold (*) Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, 3210 N. Cramer St, Milwaukee, WI 53211, USA e-mail: [email protected] A. Simeonov : A. Jadhav : A. Yasgar : D. J. Maloney NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892-3370, USA
Introduction Drug−plasma protein binding is a major determinant of drug distribution and elimination. Thus, the pharmacological effect of a drug is directly related to its extent of plasma protein binding. Only the unbound drug is capable of passing through membranes, interact with membrane proteins, and bind to freely circulating proteins. Thus, non-specific plasma protein binding of small molecules decreases the free drug concentration available to the site of action. The interactions between drug and plasma protein include ionic, hydrogen bond, and Van der Waals interactions. Although highly reversible, drug−plasma protein binding can be regarded as temporary storage of small molecules with a significant influence on pharmacokinetics, pharmacodynamics, and drug metabolism. Human serum albumin (HSA) is one of the most abundant blood proteins and carrier for many neutral and weak acidic metabolites and drugs [1]. The lack of specificity toward particular ligands, multiple HSA binding sites, and allosteric effects complicate the studies of plasma protein binding. Nevertheless, several preclinical in vitro methods to determine drug−plasma protein binding have been developed [2]. Separative methods to determine plasma protein binding such as equilibrium dialysis are commonly performed in pharmaceutical industry. This process is labor intensive, costly, time consuming, and difficult to automate. The application of 96-well dialysis blocks improves the throughput of equilibrium dialysis, but long incubation times are still required to reach equilibrium. In addition, small molecule binding to the apparatus can greatly affect the results [3]. Ultra-filtration methods have also been employed for the determination of plasma protein binding. It is a relatively fast and simple method that has been shown to have a good correlation to other methods. However, non-specific binding to the filtration device has been a major issue for this technique [4]. In effort to
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increase throughput of plasma protein binding techniques, LCMS methods in conjunction with immobilized HSA columns [5], capillary electrophoresis, or silica beads with immobilized HSA have been applied [6]. The main disadvantage of separative methods is the disturbance of the drug−protein equilibrium by the separation of the free drug. In addition, some methods assume that immobilized albumin retains its full binding characteristics, which is also relevant for surface plasmon resonance-based protein binding assays [7]. Non-seperative methods include calorimetric methods for plasma protein binding such as isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) [8]. Although the direct compound−HSA equilibrium constants and heat of binding can be determined, there is a lack of automation and throughput affiliated with these methods. Higher throughput can be achieved with spectroscopic methods such as circular dichroism (CD) and fluorescence. Fluorescent molecules with high protein affinity have been used as responsive probes to quantify and analyze proteins [9]. Usually, these probes, such as 1-anilinonaphthalene-8-sulfonate (ANS), are very sensitive to their environment so that the presence of proteins will cause a blue-shift of their emission spectrum [10]. The fluorescence change is due to ionic, hydrogen bond, and Van der Waals interactions between the fluorophore and the macromolecule altering the rates of non-radiative decay. ANS and its dimeric form, 4,4′-bis-1-anilinonaphthalene-8-sulfonate (Bis-ANS) have been most frequently used for protein characterization. The anilinonaphthalene analog Prodran has been applied to characterize the interaction between warfarin and HSA [11]. A fluorescence polarization method to determine small molecule−plasma protein binding was developed using danslyamide and dansylsarcosine [12]. In contrast to fluorescence intensity, fluorescence polarization (FP) is dependent on the fluorophore movement, which in turn is size-dependent. Thus, ratios of fluorescent molecule and fluorescent molecule in complex with plasma protein can be distinguished by FP. Recently, a high throughput FP plasma protein binding assay was introduced by Yasgar et al. using dansyl sarcosine and dipyridamole to determine the small molecule binding to α1-acid glucoprotein and HSA, respectively [13]. The assay was carried out in a 1,536well plate format but suffered like all fluorescence-based small molecule assays from false positives and false negatives hits due to small molecule auto-fluorescence and fluorescence quenching especially at short excitation wavelengths. Other potential problems of fluorescence-based assays can occur through compound aggregation in the absence of detergent [14]. Herein, we report a high throughput method to quantify the HSA protein binding of small molecules using fluorescence intensity detection with a novel fluorophore Red Mega 500. The assay employs low concentrations of probe and HSA and tolerates the presence of detergent NP-40 to suppress compound aggregation. The inhibition of HSA−Red Mega 500
binding by known high protein binders is concentrationdependent and similar to the direct HSA binding determined by isothermal titration calorimetry. The high throughput capability of this assay was demonstrated by determining the ability of 1,280 pharmacological compounds to inhibit the interaction between HSA and Red Mega 500.
Materials and methods Materials All materials were used as received with no further purification. The following small molecules were used as standards: diethylstilbestrol (Spectrum Chemical Mfg. Corp.), caffeine (Alfa Aesar), piroxicam (MP-Biomedicals), metoprolol tartarate (LKT Laboratories), naproxen (MP-Biomedicals), atenolol (MP-Biomedicals), ranitidine hydrochloride (Alfa Aesar), lansoprazole (Sigma Aldrich), omeprazole (Sigma Aldrich), nadolol (Sigma Aldrich), linezolid (Sigma Aldrich), antipyrine (Sigma Aldrich), and ofloxacin (Sigma Aldrich). Each of the small molecules was dissolved in DMSO (Acros, Spectroscopic Grade 99.9+ %) to make a 10 mM solution. Solutions From lyophilized powder, fatty acid free, globulin free, and ≥99 % human serum albumin (HSA) (Sigma Aldrich) were made in buffer. HSA solutions were stored for no longer than 1 week at 4 °C. Phosphate buffered saline (PBS) was prepared in 1 L batches using 18 MΩ water with 3.23 mM K2HPO4 7H2O (J.T. Baker), 7.84 mM KH2PO4 (J.T. Baker), 5 mM KCl (Fisher), 150 mM NaCl (Fisher), and adjusted to pH 7.2 with HCl (Mallinckrodt) and NaOH (Fisher). Nonylphenyl polyethylene glycol (NP-40) 0.01 % (v/v) surfactant (Boston BioProducts) and glycerol (Fisher) were used as buffer additives. In the study of binding dependence on buffer composition, a 200 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) (Acros) at pH 7.2 was used. Fluorescent molecule Red Mega 500 (Sigma) was dissolved to 10 mM in DMSO upon receiving and stored at −20 °C until used. The absorbance readings were completed in a 384-well UV plate (Greiner Bio-One, 781801). The Red Mega 500 assay was performed in a 384-well, flat bottom, black assay plate (Corning, 3573). Instrumentation All of the absorbance and fluorescence readings were performed with a Tecan Infinite M1000 plate reader. A total of 100 nL transfers were carried out with the Tecan Freedon EVO liquid handling system using a slotted stainless steel pin tool (V & P Scientific). A MicroFlo Select instrument
A fluorescence-based serum protein binding assay
(BioTek) was used for the addition of solutions to the assay plate. TA Instruments low volume nano isothermal titration calorimeter (ITC) was used for the determination of binding constants of standard molecules to HSA. The analysis of ITC data were completed using a Thermo NanoAnalyze software. High throughput protein binding assay In the preparation of the ‘compound plate’, 15 μL of the 10 mM solution of small molecules in DMSO were dispensed in a 384-well polystyrene plate filling columns 1–20. A second 384-well polystyrene plate had column 21 filled with 15 μL of control compounds naproxen and piroxicam (10 mM) and 22–24 filled with 15 μl DMSO to determine the vehicle control. A total of 19.6 μL assay buffer containing PBS, 0.20 mg/mL HSA (3 μM) and 500 nM Red Mega 500, 10 % glycerol, and 0.01 % NP-40 and was dispensed in columns 1–23. A 500 nM solution of Red Mega 500 in assay buffer was used as further control, and 20 μL was dispensed into column 24 of the assay plate. With the Tecan liquid handling system, 200 nL from the compound plate and 200 nL from the control plate were transferred into each assay plate. The assay plate was then centrifuged for 2 min at 2,500 rpm and put on the plate shaker for agitation for about 5 min followed by fluorescence detection. An excitation and emission wavelength of 514 and 532 nm with a band width of 5 nm was used for Red Mega 500 with 100 flashes, 20 μs integration time, optimized gain, and z-position. The Z' values for the assay were calculated using DMSO as negative control (0 % binding) and high protein binder piroxicam as the positive control (100 % binding) [15].
Results In order to determine the extent of small molecule HSA binding, the fluorescent molecule Red Mega 500 was chosen because of the drug-like structure, high excitation and emission wavelength, large fluorescence intensity in the presence of proteins, and low background fluorescence in pure water Fig. 1 a Structure of Red Mega 500 with wavelengths of maximal fluorescence excitation and emission in PBS with 0.5 mg/mL HSA. b Absorbance and fluorescence spectra of Red Mega 500 (500 nM) in PBS with 0.5 mg/mL HSA
[16]. Red Mega 500 is a charged molecule that contains a conjugated coumarin moiety, which demonstrates the ability to form ionic and Van der Waals interactions with proteins (Fig. 1a). The fluorescence properties of Red Mega 500 are governed by a twisted intramolecular charge transfer (TICT) in which an electron transfers from the diethylamino group to the electronwithdrawing aromatic system similar to that of fluorescent compound Nile-Red [17]. In polar solvents, the TICT state is non-radiative causing the low quantum yield in buffer. In apolar environments with HSA, the TICT process is thermodynamically unfavorable, which results in a significant increase in fluorescence lifetime and quantum yield. The absorbance and fluorescence spectra of Red Mega 500 were determined and depicted in Fig. 1b. The maximal absorbance wavelength is 509 nm, and the maximal fluorescence occurs at an emission wavelengths of 532 nm when exited at 510 nm. Because of the small Stokes shift, a narrow bandwidth (BW) of 5 nm was used for excitation and emission. The optimal setting for this assay with a maximal fluorescence intensity signal window between Red Mega 500 in the presence and absence of HSA was 514 nm (5 nm BW) for the excitation wavelength and 532 nm (5 nm BW) for the emission wavelength. The relationship between concentrations of Red Mega 500 and HSA was determined using a serial dilution of Red Mega 500 (1, 3, 9, 27, 81, 243, 729, and 2,187 nM) and 0, 0.2, 0.5 and 1.0 mg/mL of HSA in PBS (Fig. 2). A linear increase in fluorescence intensity was observed for Red Mega 500 concentrations up to 729 nM for each HSA concentration. The fluorescence intensity at a constant Red Mega 500 concentration saturated at an HSA concentration of 0.5 mg/mL. In order to minimize the presence of unbound HSA, a concentration of 0.2 mg/mL was selected. With respect to the fluorescence intensity, we observed that the background fluorescence was less than 5 % when a Red Mega 500 concentration of at least 500 nM was used in the presence and absence of 0.2 mg/mL HSA. Based on these findings, a concentration of 500 nM Red Mega 500 and 0.2 mg/mL (3 μM) HSA was used for all consecutive experiments.
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Fig. 2 Fluorescence intensity of varying concentrations of Red Mega 500 and HSA in PBS
In order to optimize the assay buffer, Red Mega 500 and HSA were analyzed in the presence and absence of high protein binder naproxen (100 μM). Several buffer additives were investigated, and the results are summarized in Fig. 3. For the assay, the gain of the TECAN M1000 reader was optimized to give 48,300 fluorescence intensity units for 500 nM Mega Red 500 and 0.2 mg/mL HSA in PBS (10 mM phosphate, 150 mM NaCl, pH 7.2). In the presence of naproxen (100 μM), the fluorescence intensity dropped to 30,302 units caused by the competition between naproxen and Red Mega 500 for HSA binding. The change of the ionic strength of the buffer from 150 mM to 500 mM NaCl resulted in a very limited competition between Red Mega 500 and naproxen, possibly due to a stronger interaction between HSA and Red Mega 500 under these conditions. To obtain further insight on the effect of ionized groups, the pH of the buffer was changed to 8. A small increase in HSA−naproxen binding was observed. The change to a buffer with zwitterionic HEPES at 20 mM and pH 7.2 showed no improvement of naproxen−HSA binding. Increasing amounts of DMSO in order to enhance the solubility of naproxen was investigated. Fig. 3 Inhibition of Red Mega 500−HSA binding in the presence and absence of naproxen (100 μM) using different buffer and additives
No significant fluorescence intensity changes were observed for concentrations of up to 5 % by volume. The addition of glycerol, typically 10 % by volume, is sometimes added to protein buffers to enhance the solubility and stability proteins especially upon freezing. NP-40 and other surfactants have been shown to increase the solubility of hydrophobic molecules and circumvent aggregation [14]. The HSA−Red Mega 500 binding in the presence of naproxen was determined at varying percentages of glycerol, 5, 10, and 20 by volume, with and without DMSO, and with 0.01 or 0.001 % NP-40. The buffer with 5 % glycerol did not change the degree of naproxen binding. However, higher amounts of glycerol (10 and 20 %) in concert with 0.01 % NP-40 showed a drastic increase of HSA−naproxen binding. About 10 % glycerol gave the best results. Using 0.001 % NP-40 or no surfactant with 10 % glycerol showed a decreased naproxen binding. Finally, the addition of 2 % DMSO to the buffer with 10 % glycerol showed an increase of BSA−naproxen binding. The optimized assay buffer giving a signal window of more 30,000 fluorescence intensity units consists of PBS at pH 7.2, 150 mM NaCl, 10 % glycerol, 0.01 % NP-40, and 1 % DMSO, which was added together with the test compound naproxen. Next, we assembled a set of 13 drug compound with different protein binding constants reported in the literature. This include the low plasma protein binder atenolol [18], metoprolol [18], and nadolol [19] of the β receptor antagonist family (beta-blocker), the analgesic antipyrine [20], the histamine H2-receptor antagonist ranitidine [21], the antibiotics linezolid [22], and ofloxacin [23], and caffeine. The literature plasma binding constants of these molecules range between 4 and 31 % (Fig. 4). Diethylstilbestrol [24] is a medium plasma protein binder, whereas proton pump inhibitors omeprazole [25] and lansoprazole [25] have a high protein binding, probably due to the reactivity toward nucleophilic residues such as cysteine moieties. The COX inhibitors naproxen and piroxicam have high plasma protein binding [26]. Figure 4 compares the
A fluorescence-based serum protein binding assay Fig. 4 Average drug literature values for in vivo plasma protein binding in comparison with fluorescence intensity values for 100 μM compound in the presence of 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA
Compound Antipyrine Atenolol Metoprolol Ranitidine Nadolol Ofloxacin Caffeine Linezolid Diethylstilbestrol Omeprazole Lansoprazole Naproxen Piroxicam
Plasma Fluorescence Binding (%) Intensity 4.0 ± 2.0 10.0 ± 5 11.0 ± 1 15.0 ± 3 15.5 ± 11.5 19.0 ± 11.0 30.0 ± 5 31.0 ± 1.0 65.0 ± 15 95.0 95.5 ± 1.5 99.7 ± 0.1 99.0 ± 0.3
average in vivo plasma binding of these compounds with the reduced fluorescence intensity determined in the presence of HSA and Red Mega 500. A good inverse correlation between both values was observed, which is graphically presented in the scatter plot of Fig. 4. Further investigations to determine the specific disruption of the HSA−Red Mega 500 complex by drug compounds included the dose–response analysis. Therefore, compounds were serial diluted and added to 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA. The results for piroxicam, diethylstilbestrol, and naproxen are presented in Fig. 5. The inhibition constants determined for piroxicam and diethylstilbestrol were similar with IC50 values of 4.2± Fig. 5 Dose–response analysis of piroxicam, diethylstilbestrol, and naproxen. a, b, c Quantification of dose-dependent inhibition of HSA−Red Mega 500 binding in the presence of piroxicam, diethylstilbestrol, and naproxen using 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA by fluorescence intensity; d determination of naproxen−HSA binding by isothermal titration calorimetry (ITC)
47797 ± 1743 48374 ± 1267 48498 ± 2419 48057 ± 872 48167 ± 2125 46833 ± 2301 46573 ± 2028 48009 ± 911 37610 ±2803 25162 ± 970 23081 ± 658 15879 ± 482 12011 ± 347
1.1 μM and 4.4±1.9 μM, respectively. Interestingly, the efficacy of inhibition is significantly different between the medium plasma protein binder diethylstilbestrol and the high plasma protein binder piroxicam. The high plasma protein binder naproxen showed an IC50 value of 24.6±8.3 μM, and the efficacy was comparable with piroxicam. In addition, we determined the KD value of the HSA−naproxen interaction using isothermal titration calorimetry (ITC), which was 58.2± 5 μM. A significant heat of 221±9 KJ/mol was generated upon binding. The Library of Pharmacologically Active Compounds1280 (LOPAC, Sigma) was used to determine the HTS quality of the assay and the ability to identify compounds that inhibit the interaction between HSA and Red Mega 500. Each
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Fig. 6 HTS small molecule assay using the LOPAC-1280 compounds screening collection at a concentration of 100 μM in the presence of 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA. DMSO was used as negative control and piroxicam and naproxen as positive control compounds
LOPAC compound was measured in triplicate at a single concentration of 100 μM, and the results are depicted in Fig. 6. The majority of the compounds exhibited a fluorescence intensity ranging between 44,000 and 56,000 units. All negative control DMSO values fell in between this range. For the positive control compounds piroxicam and naproxen fluorescence, intensities between 16,000 and 24,000 were observed for all assay plates. The average Z' factor for controls DMSO and piroxicam was 0.69. The normalization of all compound data to DMSO (0 % HSA binding) and piroxicam (100 % HSA binding) resulted in the identification of 97 compounds with an HSA binding of more than 50 % as indicated as the horizontal line in Fig. 6. Of these hit compounds, 92 % was determined to be moderately to highly lipophilic (>1
calculated logP(o/w)). In comparison, 73 % of all compounds of the LOPAC library have a calculated logP(o/w) of greater than 1. A composition map of the LOPAC compounds in respect to drug classes is depicted in Fig. 7a. The pie diagrams in Fig. 7 reflect the number of compounds that interact with a particular drug targets. For the LOPAC library (Fig. 7a), the largest groups of compounds are modulators of serotonin receptors, kinases, glutamate receptors, acetylcholine receptors, and adrenoceptors. In comparison, the pie diagram for the 97 hit compounds is given in Fig. 7b. The largest groups of HSA binders are COX inhibitors (prostaglandin synthesis), kinases inhibitors (phosphorylation), anionic P2 receptors antagonists and glutamate receptor modulators. Three for the four groups (COX, kinases and P2 receptors modulators) are significantly enlarge in Fig. 7b in comparison with 7A due numerous Red Mega 500 assay hit compounds with high HSA binding. Usually, compounds interacting with the same biological target have a common pharmacophore thus their chemical structure is related. Figure 8 presents the chemical structure of hit compounds from three classes (COX, kinases, and P2 receptors modulators) identified by presenting assay compounds in a pie diagram (Fig. 7). The pharmacophore of the anionic P2 receptor antagonists is a linear core structure capped with anionic carboxylate or sulfate groups (Fig. 8a). All members of this drug class inhibited the interaction between Red Mega 500 and HSA as least as good as piroxicam. The group of kinase inhibitors is structurally diverse due to the different enzyme inhibition modes that include direct ATP inhibition, allosteric modulation, reversible binding, and irreversible inhibition (Fig. 8b). The largest groups within B are tyrphostins and other tyrosine
Fig. 7 a Composition of LOPAC 1280 library in regard of drug classes; b Red Mega 500−HSA inhibitors in regard to drug classes
A fluorescence-based serum protein binding assay
Fig. 8 Structures of compounds identified with the LOPAC small molecule screen exhibiting more than 50 % protein binding determined with negative control DMSO (0 %) and positive control
piroxicam (100 %). Three drug classes are depicted: a anionic P2 receptors antagonists, b modulators of phosphorylation, and c prostaglandin synthesis (COX) inhibitors
kinase inhibitors [27]. The protein binding in this group ranges between 53 and 100 %. Group C, Fig. 8, represents FDA-approved COX inhibitors used as pain medication. The majority of COX inhibitors have a hydrophobic and a hydrophilic part (carboxylate) and exhibit high in vivo plasma protein binding. The majority of COX inhibitors were identified with the Red Mega 500 assay with HSA binding ranging from 55 to 100 %. All fluorescence-based assays suffer from false positive hits due to intrinsic fluorescence properties of small molecule. For the LOPAC small molecule screen, we determined the
fluorescence intensity of all compounds at the concentration of 100 μM using an excitation and emission wavelength of 514 and 532 nm, respectively. The results of this screen are depicted in Fig. 9. Only a very limited number of compounds exhibited significant fluorescence intensities at the wavelengths use (Fig. 9a). Using a cutoff of 1σ of the mean fluorescence intensity, eight compounds were identified and their chemical structures are depicted in Fig. 9b. Interestingly, only four compounds depicted in black of the total eight compounds were identified with the Red Mega 500 protein binding assay
a
b 25000
Fluorescence Intensity (514/532 nm)
Fig. 9 a Fluorescence intensities (514/532 nm) of all LOPAC compounds (white data points correspond to DMSO; b structures of compounds with high fluorescence intensity (FI) at 514/532 nm
20000 15000 10000 5000 1000 800 600 400 200 0
Compounds
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(Fig. 8). The molecules included Cy5, which saturated the signal, and three tyrosine kinase inhibitors. The other four compounds exhibited a protein binding of less than 50 % in the Red Mega 500 protein binding assay.
Discussion The direct measurement of drug−plasma protein binding using dialysis, chromatography, CD, and ITC enables the determination of % plasma binding and if repeated with different concentrations of small molecule, the determination of a plasma binding constant (KD). However, no information is gained in respect to small molecule occupancy of discreet plasma protein binding sites. For HSA, three main small molecule binding sites have been identified by x-ray crystallography [28]. Importantly, drug molecules as well as fluorescent molecules can bind to a single site of HSA or a combination of HSA binding sites [29]. The binding site of Red Mega 500 is not known, but for the drugs investigated, a good correlation between in vivo plasma binding and inhibition of the HSA−Red Mega 500 interaction was observed. The assay buffer conditions have a significant influence on the measured fluorescence intensity. Especially, the addition of glycerol and surfactant NP-40 increased the quality of the assay by stabilizing HSA and solubilizing hydrophobic assay compounds. The dose–response analysis of piroxicam, diethylstilbestrol, and naproxen identified two classes of HSA binders. Diethylstilbestrol exhibits medium plasmid binding capacity and binds to an unknown HSA binding site. In contrast, piroxicam and naproxen are high plasma protein binder and bind to the IIA and IIIA HSA sites. In regard to potency, we observed that piroxicam interacts stronger with HSA than naproxen (IC50 =4.2 vs. 24.6 μM). Diethylstilbestrol also has a strong interaction with HSA (IC50 =4.4 μM) but binds HSA differently than naproxen because of the weaker efficacy. It can be hypothesized that diethylstilbestrol interacts with a more limited number of HSA binding sites than naproxen leaving some specific HSA−Red Mega 500 interactions undisrupted. This in turn might cause the higher but saturable fluorescence intensity. Efficacy differences due to different HSA binding sites were also reported by Yasgar et al. [13] A large efficacy was observed for naproxen, while a small efficacy was measured for phenylbutazone, which binds predominately to the IIA site instead of all three HSA binding sites. The HTS screen identified 97 compounds with more than 50 % protein binding, determined by the ability to disrupt the interaction between HSA and Red Mega 500 in comparison with naproxen. Although not all compounds of the LOPAC library have been investigated by other plasma protein binding methods, a significantly higher percentage of high protein binders than the 7.7 % determined by the Red Mega 500 assay
can be assumed. The physiological concentration of HSA is 600 μM, and high protein concentrations are usually used to determine direct plasma protein binding. However, for competition plasma binding assays, a minimal protein concentration should be applied to enable the specific competition between fluorescent probe and molecules. Thus, it is very likely that more high protein binders will be identified with lower HSA concentrations, which unfortunately will reduce the Red Mega 500 assay performance due to a smaller fluorescence intensity signal window. The 97 compounds identified by the Red Mega 500 assay bear different scaffolds and functional groups complicating the identification of a relationship between plasma protein binding and compound structure. However, we found that more than half of the hit compounds belong to certain drug classes that include COX inhibitors, kinase inhibitors, anionic P2 receptors antagonists, and glutamate receptor modulators. Thus, structurally diverse compounds that were developed to interact with the same drug target are likely to have similar plasma protein binding. In order to compare % protein binding from a direct and a competition assay, the probe must have the same HSA binding mode than the compound investigated. As a result, we observe different degrees of inhibition (efficacy) for the Red Mega 500 assay depending on the similarity of the binding modes of compound and probe (Fig. 5). Therefore, the lower the fluorescence intensity for a particular compound in Fig. 6, the more similar is the HSA binding mode to Red Mega 500. Our future approach to improve the general correlation between % protein binding from a direct and a competition assay is to multiplex fluorescent probes to bind most of the small molecule HSA binding sites. This will allow the identification of selective inhibitors binding to a particular HSA site and those inhibiting the HSA binding of multiple fluorescent probes. Nevertheless, most of the hit compounds identified by Red Mega 500 were confirmed as high protein binders in comparison with their literature plasma protein binding. Further advances of the developed assay are short incubation times and relative low concentrations of HSA and fluorescent probe. The longer wavelength detection using Red Mega 500 limited the number of molecules interfering with the assay, which was 0.63 % for the LOPAC screening library. Overall, the novel HTS protein binding assay allowed the detection of high protein binders, especially those targeting kinases, COX enzymes, and P2 receptors. We will continue our investigations to develop multiplexed protein binding assays and include the application of blood serum and plasma. Acknowledgments The work was supported by the University of Wisconsin–Milwaukee, the University of Wisconsin-Milwaukee Research Growth Initiative (RGI), National Institute of Drug Abuse
A fluorescence-based serum protein binding assay DA031090, the University of Wisconsin-Milwaukee Research Foundation, the Lynde and Harry Bradley Foundation, and the Richard and Ethel Herzfeld Foundation. Further support was enabled by the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research and the Intramural Research Program of the NHGRI, NIH. We also thank Dr. Frick to enable the use of the ITC instrument.
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13. Yasgar A, Furdas SD, Maloney DJ, Jadhav A, Jung M, Simeonov A (2012) High-throughput 1,536-well fluorescence polarization assays for a1-acid glycoprotein and human serum albumin binding. PLoS ONE 7(9):1–12 14. Seidler J, McGovern SL, Doman TN, Shoichet BK (2003) Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem 46(21):4477–4486 15. Zhang J-H, Chung TDY, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73 16. Wenzel M, Czerney P, Schweder B, Lehmann F (2006) Protein Identifying and Quantifying Method. WO 2006/079334A2 17. Sarkar N, Das K, Nath DN, Bhattacharyya K (1994) Twisted charge transfer process of Nile Red in homogeneous solution and in faujasite seolite. Langmuir 10(326–329):326 18. Dayer P, Leemann T, Marmy A, Rosenthaler J (1985) Interindividual variation of beta-adrenoceptor blocking drugs, plasma concentration and effect: influence of genetic status on behaviour of atenolol, bopindolol and metoprolol. Eur J Clin Pharmacol 28(2):149–153 19. Patel L, Johnson A, Turner P (1984) Nadolol binding to human serum proteins. J Pharm Pharmacol 36(6):414–415 20. Wan H, Rehngren M (2006) High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry. J Chromatogr A 1102(1–2):125– 134 21. Gladziwa U, Klotz U (1993) Pharmacokinetics and pharmacodynamics of H2-receptor antagonists in patients with renal insufficiency. Clin Pharmacokinet 24(4):319–332 22. MacGowan AP (2003) Pharmacokinetic and pharmacodynamic profile of linezolid in healthy volunteers and patients with gram-positive infections. J Antimicrob Chemother 51(S2):ii17–ii25 23. Zlotos G, Bucker A, Kinzig-Schippers M, Sorgel F, Holzgrabe U (1998) Plasma protein binding of gyrase inhibitors. J Pharm Sci 87(2):215–220 24. Anderson B, Peyster Ad, Gad S, Hakkinen PJ, Kamrin M, Locey B, Mehendale H, Pope C, Shugart L (eds) (2005) Encyclopedia of Toxicology. 2nd edn. Elsevier Inc 25. Andersson T, Weidolf L (2008) Stereoselective disposition of proton pump inhibitors. Clin Drug Investig 28(5):263–279 26. Bree F, Urien S, Nguyen P, Riant P, Albengres E, Tillement JP (1990) A re-evaluation of the HSA-piroxicam interaction. Eur J Drug Metab Pharmacokinet 15(4):303–307 27. Levitzki A, Mishani E (2006) Tyrphostins and other tyrosine kinase inhibitors. Annu Rev Biochem 75:93–109 28. Ghuman J, Zunszain PA, Petitpas I, Bhattacharya AA, Otagiri M, Curry S (2005) Structural basis of the drug-binding specificity of human serum albumin. J Mol Biol 353(1):38–52 29. Zsila F (2013) Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model. Mol Pharm 10(5):1668–1682
RESEARCH PAPER
A fluorescence-based high throughput assay for the determination of small molecule−human serum albumin protein binding Megan M. McCallum & Alan J. Pawlak & William R. Shadrick & Anton Simeonov & Ajit Jadhav & Adam Yasgar & David J. Maloney & Leggy A. Arnold
Received: 4 October 2013 / Revised: 26 November 2013 / Accepted: 6 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the library of pharmacological active compounds (LOPAC) small molecule library of 1,280 compounds identifying known high protein binders. The small molecule competition of HSA−Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3 and 24 μM. The compound affinity toward HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding.
Keywords Drug–protein binding . High throughput screening . Red Mega 500 . Human serum albumin M. M. McCallum : A. J. Pawlak : W. R. Shadrick : L. A. Arnold (*) Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, 3210 N. Cramer St, Milwaukee, WI 53211, USA e-mail: [email protected] A. Simeonov : A. Jadhav : A. Yasgar : D. J. Maloney NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892-3370, USA
Introduction Drug−plasma protein binding is a major determinant of drug distribution and elimination. Thus, the pharmacological effect of a drug is directly related to its extent of plasma protein binding. Only the unbound drug is capable of passing through membranes, interact with membrane proteins, and bind to freely circulating proteins. Thus, non-specific plasma protein binding of small molecules decreases the free drug concentration available to the site of action. The interactions between drug and plasma protein include ionic, hydrogen bond, and Van der Waals interactions. Although highly reversible, drug−plasma protein binding can be regarded as temporary storage of small molecules with a significant influence on pharmacokinetics, pharmacodynamics, and drug metabolism. Human serum albumin (HSA) is one of the most abundant blood proteins and carrier for many neutral and weak acidic metabolites and drugs [1]. The lack of specificity toward particular ligands, multiple HSA binding sites, and allosteric effects complicate the studies of plasma protein binding. Nevertheless, several preclinical in vitro methods to determine drug−plasma protein binding have been developed [2]. Separative methods to determine plasma protein binding such as equilibrium dialysis are commonly performed in pharmaceutical industry. This process is labor intensive, costly, time consuming, and difficult to automate. The application of 96-well dialysis blocks improves the throughput of equilibrium dialysis, but long incubation times are still required to reach equilibrium. In addition, small molecule binding to the apparatus can greatly affect the results [3]. Ultra-filtration methods have also been employed for the determination of plasma protein binding. It is a relatively fast and simple method that has been shown to have a good correlation to other methods. However, non-specific binding to the filtration device has been a major issue for this technique [4]. In effort to
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increase throughput of plasma protein binding techniques, LCMS methods in conjunction with immobilized HSA columns [5], capillary electrophoresis, or silica beads with immobilized HSA have been applied [6]. The main disadvantage of separative methods is the disturbance of the drug−protein equilibrium by the separation of the free drug. In addition, some methods assume that immobilized albumin retains its full binding characteristics, which is also relevant for surface plasmon resonance-based protein binding assays [7]. Non-seperative methods include calorimetric methods for plasma protein binding such as isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) [8]. Although the direct compound−HSA equilibrium constants and heat of binding can be determined, there is a lack of automation and throughput affiliated with these methods. Higher throughput can be achieved with spectroscopic methods such as circular dichroism (CD) and fluorescence. Fluorescent molecules with high protein affinity have been used as responsive probes to quantify and analyze proteins [9]. Usually, these probes, such as 1-anilinonaphthalene-8-sulfonate (ANS), are very sensitive to their environment so that the presence of proteins will cause a blue-shift of their emission spectrum [10]. The fluorescence change is due to ionic, hydrogen bond, and Van der Waals interactions between the fluorophore and the macromolecule altering the rates of non-radiative decay. ANS and its dimeric form, 4,4′-bis-1-anilinonaphthalene-8-sulfonate (Bis-ANS) have been most frequently used for protein characterization. The anilinonaphthalene analog Prodran has been applied to characterize the interaction between warfarin and HSA [11]. A fluorescence polarization method to determine small molecule−plasma protein binding was developed using danslyamide and dansylsarcosine [12]. In contrast to fluorescence intensity, fluorescence polarization (FP) is dependent on the fluorophore movement, which in turn is size-dependent. Thus, ratios of fluorescent molecule and fluorescent molecule in complex with plasma protein can be distinguished by FP. Recently, a high throughput FP plasma protein binding assay was introduced by Yasgar et al. using dansyl sarcosine and dipyridamole to determine the small molecule binding to α1-acid glucoprotein and HSA, respectively [13]. The assay was carried out in a 1,536well plate format but suffered like all fluorescence-based small molecule assays from false positives and false negatives hits due to small molecule auto-fluorescence and fluorescence quenching especially at short excitation wavelengths. Other potential problems of fluorescence-based assays can occur through compound aggregation in the absence of detergent [14]. Herein, we report a high throughput method to quantify the HSA protein binding of small molecules using fluorescence intensity detection with a novel fluorophore Red Mega 500. The assay employs low concentrations of probe and HSA and tolerates the presence of detergent NP-40 to suppress compound aggregation. The inhibition of HSA−Red Mega 500
binding by known high protein binders is concentrationdependent and similar to the direct HSA binding determined by isothermal titration calorimetry. The high throughput capability of this assay was demonstrated by determining the ability of 1,280 pharmacological compounds to inhibit the interaction between HSA and Red Mega 500.
Materials and methods Materials All materials were used as received with no further purification. The following small molecules were used as standards: diethylstilbestrol (Spectrum Chemical Mfg. Corp.), caffeine (Alfa Aesar), piroxicam (MP-Biomedicals), metoprolol tartarate (LKT Laboratories), naproxen (MP-Biomedicals), atenolol (MP-Biomedicals), ranitidine hydrochloride (Alfa Aesar), lansoprazole (Sigma Aldrich), omeprazole (Sigma Aldrich), nadolol (Sigma Aldrich), linezolid (Sigma Aldrich), antipyrine (Sigma Aldrich), and ofloxacin (Sigma Aldrich). Each of the small molecules was dissolved in DMSO (Acros, Spectroscopic Grade 99.9+ %) to make a 10 mM solution. Solutions From lyophilized powder, fatty acid free, globulin free, and ≥99 % human serum albumin (HSA) (Sigma Aldrich) were made in buffer. HSA solutions were stored for no longer than 1 week at 4 °C. Phosphate buffered saline (PBS) was prepared in 1 L batches using 18 MΩ water with 3.23 mM K2HPO4 7H2O (J.T. Baker), 7.84 mM KH2PO4 (J.T. Baker), 5 mM KCl (Fisher), 150 mM NaCl (Fisher), and adjusted to pH 7.2 with HCl (Mallinckrodt) and NaOH (Fisher). Nonylphenyl polyethylene glycol (NP-40) 0.01 % (v/v) surfactant (Boston BioProducts) and glycerol (Fisher) were used as buffer additives. In the study of binding dependence on buffer composition, a 200 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) (Acros) at pH 7.2 was used. Fluorescent molecule Red Mega 500 (Sigma) was dissolved to 10 mM in DMSO upon receiving and stored at −20 °C until used. The absorbance readings were completed in a 384-well UV plate (Greiner Bio-One, 781801). The Red Mega 500 assay was performed in a 384-well, flat bottom, black assay plate (Corning, 3573). Instrumentation All of the absorbance and fluorescence readings were performed with a Tecan Infinite M1000 plate reader. A total of 100 nL transfers were carried out with the Tecan Freedon EVO liquid handling system using a slotted stainless steel pin tool (V & P Scientific). A MicroFlo Select instrument
A fluorescence-based serum protein binding assay
(BioTek) was used for the addition of solutions to the assay plate. TA Instruments low volume nano isothermal titration calorimeter (ITC) was used for the determination of binding constants of standard molecules to HSA. The analysis of ITC data were completed using a Thermo NanoAnalyze software. High throughput protein binding assay In the preparation of the ‘compound plate’, 15 μL of the 10 mM solution of small molecules in DMSO were dispensed in a 384-well polystyrene plate filling columns 1–20. A second 384-well polystyrene plate had column 21 filled with 15 μL of control compounds naproxen and piroxicam (10 mM) and 22–24 filled with 15 μl DMSO to determine the vehicle control. A total of 19.6 μL assay buffer containing PBS, 0.20 mg/mL HSA (3 μM) and 500 nM Red Mega 500, 10 % glycerol, and 0.01 % NP-40 and was dispensed in columns 1–23. A 500 nM solution of Red Mega 500 in assay buffer was used as further control, and 20 μL was dispensed into column 24 of the assay plate. With the Tecan liquid handling system, 200 nL from the compound plate and 200 nL from the control plate were transferred into each assay plate. The assay plate was then centrifuged for 2 min at 2,500 rpm and put on the plate shaker for agitation for about 5 min followed by fluorescence detection. An excitation and emission wavelength of 514 and 532 nm with a band width of 5 nm was used for Red Mega 500 with 100 flashes, 20 μs integration time, optimized gain, and z-position. The Z' values for the assay were calculated using DMSO as negative control (0 % binding) and high protein binder piroxicam as the positive control (100 % binding) [15].
Results In order to determine the extent of small molecule HSA binding, the fluorescent molecule Red Mega 500 was chosen because of the drug-like structure, high excitation and emission wavelength, large fluorescence intensity in the presence of proteins, and low background fluorescence in pure water Fig. 1 a Structure of Red Mega 500 with wavelengths of maximal fluorescence excitation and emission in PBS with 0.5 mg/mL HSA. b Absorbance and fluorescence spectra of Red Mega 500 (500 nM) in PBS with 0.5 mg/mL HSA
[16]. Red Mega 500 is a charged molecule that contains a conjugated coumarin moiety, which demonstrates the ability to form ionic and Van der Waals interactions with proteins (Fig. 1a). The fluorescence properties of Red Mega 500 are governed by a twisted intramolecular charge transfer (TICT) in which an electron transfers from the diethylamino group to the electronwithdrawing aromatic system similar to that of fluorescent compound Nile-Red [17]. In polar solvents, the TICT state is non-radiative causing the low quantum yield in buffer. In apolar environments with HSA, the TICT process is thermodynamically unfavorable, which results in a significant increase in fluorescence lifetime and quantum yield. The absorbance and fluorescence spectra of Red Mega 500 were determined and depicted in Fig. 1b. The maximal absorbance wavelength is 509 nm, and the maximal fluorescence occurs at an emission wavelengths of 532 nm when exited at 510 nm. Because of the small Stokes shift, a narrow bandwidth (BW) of 5 nm was used for excitation and emission. The optimal setting for this assay with a maximal fluorescence intensity signal window between Red Mega 500 in the presence and absence of HSA was 514 nm (5 nm BW) for the excitation wavelength and 532 nm (5 nm BW) for the emission wavelength. The relationship between concentrations of Red Mega 500 and HSA was determined using a serial dilution of Red Mega 500 (1, 3, 9, 27, 81, 243, 729, and 2,187 nM) and 0, 0.2, 0.5 and 1.0 mg/mL of HSA in PBS (Fig. 2). A linear increase in fluorescence intensity was observed for Red Mega 500 concentrations up to 729 nM for each HSA concentration. The fluorescence intensity at a constant Red Mega 500 concentration saturated at an HSA concentration of 0.5 mg/mL. In order to minimize the presence of unbound HSA, a concentration of 0.2 mg/mL was selected. With respect to the fluorescence intensity, we observed that the background fluorescence was less than 5 % when a Red Mega 500 concentration of at least 500 nM was used in the presence and absence of 0.2 mg/mL HSA. Based on these findings, a concentration of 500 nM Red Mega 500 and 0.2 mg/mL (3 μM) HSA was used for all consecutive experiments.
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Fig. 2 Fluorescence intensity of varying concentrations of Red Mega 500 and HSA in PBS
In order to optimize the assay buffer, Red Mega 500 and HSA were analyzed in the presence and absence of high protein binder naproxen (100 μM). Several buffer additives were investigated, and the results are summarized in Fig. 3. For the assay, the gain of the TECAN M1000 reader was optimized to give 48,300 fluorescence intensity units for 500 nM Mega Red 500 and 0.2 mg/mL HSA in PBS (10 mM phosphate, 150 mM NaCl, pH 7.2). In the presence of naproxen (100 μM), the fluorescence intensity dropped to 30,302 units caused by the competition between naproxen and Red Mega 500 for HSA binding. The change of the ionic strength of the buffer from 150 mM to 500 mM NaCl resulted in a very limited competition between Red Mega 500 and naproxen, possibly due to a stronger interaction between HSA and Red Mega 500 under these conditions. To obtain further insight on the effect of ionized groups, the pH of the buffer was changed to 8. A small increase in HSA−naproxen binding was observed. The change to a buffer with zwitterionic HEPES at 20 mM and pH 7.2 showed no improvement of naproxen−HSA binding. Increasing amounts of DMSO in order to enhance the solubility of naproxen was investigated. Fig. 3 Inhibition of Red Mega 500−HSA binding in the presence and absence of naproxen (100 μM) using different buffer and additives
No significant fluorescence intensity changes were observed for concentrations of up to 5 % by volume. The addition of glycerol, typically 10 % by volume, is sometimes added to protein buffers to enhance the solubility and stability proteins especially upon freezing. NP-40 and other surfactants have been shown to increase the solubility of hydrophobic molecules and circumvent aggregation [14]. The HSA−Red Mega 500 binding in the presence of naproxen was determined at varying percentages of glycerol, 5, 10, and 20 by volume, with and without DMSO, and with 0.01 or 0.001 % NP-40. The buffer with 5 % glycerol did not change the degree of naproxen binding. However, higher amounts of glycerol (10 and 20 %) in concert with 0.01 % NP-40 showed a drastic increase of HSA−naproxen binding. About 10 % glycerol gave the best results. Using 0.001 % NP-40 or no surfactant with 10 % glycerol showed a decreased naproxen binding. Finally, the addition of 2 % DMSO to the buffer with 10 % glycerol showed an increase of BSA−naproxen binding. The optimized assay buffer giving a signal window of more 30,000 fluorescence intensity units consists of PBS at pH 7.2, 150 mM NaCl, 10 % glycerol, 0.01 % NP-40, and 1 % DMSO, which was added together with the test compound naproxen. Next, we assembled a set of 13 drug compound with different protein binding constants reported in the literature. This include the low plasma protein binder atenolol [18], metoprolol [18], and nadolol [19] of the β receptor antagonist family (beta-blocker), the analgesic antipyrine [20], the histamine H2-receptor antagonist ranitidine [21], the antibiotics linezolid [22], and ofloxacin [23], and caffeine. The literature plasma binding constants of these molecules range between 4 and 31 % (Fig. 4). Diethylstilbestrol [24] is a medium plasma protein binder, whereas proton pump inhibitors omeprazole [25] and lansoprazole [25] have a high protein binding, probably due to the reactivity toward nucleophilic residues such as cysteine moieties. The COX inhibitors naproxen and piroxicam have high plasma protein binding [26]. Figure 4 compares the
A fluorescence-based serum protein binding assay Fig. 4 Average drug literature values for in vivo plasma protein binding in comparison with fluorescence intensity values for 100 μM compound in the presence of 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA
Compound Antipyrine Atenolol Metoprolol Ranitidine Nadolol Ofloxacin Caffeine Linezolid Diethylstilbestrol Omeprazole Lansoprazole Naproxen Piroxicam
Plasma Fluorescence Binding (%) Intensity 4.0 ± 2.0 10.0 ± 5 11.0 ± 1 15.0 ± 3 15.5 ± 11.5 19.0 ± 11.0 30.0 ± 5 31.0 ± 1.0 65.0 ± 15 95.0 95.5 ± 1.5 99.7 ± 0.1 99.0 ± 0.3
average in vivo plasma binding of these compounds with the reduced fluorescence intensity determined in the presence of HSA and Red Mega 500. A good inverse correlation between both values was observed, which is graphically presented in the scatter plot of Fig. 4. Further investigations to determine the specific disruption of the HSA−Red Mega 500 complex by drug compounds included the dose–response analysis. Therefore, compounds were serial diluted and added to 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA. The results for piroxicam, diethylstilbestrol, and naproxen are presented in Fig. 5. The inhibition constants determined for piroxicam and diethylstilbestrol were similar with IC50 values of 4.2± Fig. 5 Dose–response analysis of piroxicam, diethylstilbestrol, and naproxen. a, b, c Quantification of dose-dependent inhibition of HSA−Red Mega 500 binding in the presence of piroxicam, diethylstilbestrol, and naproxen using 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA by fluorescence intensity; d determination of naproxen−HSA binding by isothermal titration calorimetry (ITC)
47797 ± 1743 48374 ± 1267 48498 ± 2419 48057 ± 872 48167 ± 2125 46833 ± 2301 46573 ± 2028 48009 ± 911 37610 ±2803 25162 ± 970 23081 ± 658 15879 ± 482 12011 ± 347
1.1 μM and 4.4±1.9 μM, respectively. Interestingly, the efficacy of inhibition is significantly different between the medium plasma protein binder diethylstilbestrol and the high plasma protein binder piroxicam. The high plasma protein binder naproxen showed an IC50 value of 24.6±8.3 μM, and the efficacy was comparable with piroxicam. In addition, we determined the KD value of the HSA−naproxen interaction using isothermal titration calorimetry (ITC), which was 58.2± 5 μM. A significant heat of 221±9 KJ/mol was generated upon binding. The Library of Pharmacologically Active Compounds1280 (LOPAC, Sigma) was used to determine the HTS quality of the assay and the ability to identify compounds that inhibit the interaction between HSA and Red Mega 500. Each
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Fig. 6 HTS small molecule assay using the LOPAC-1280 compounds screening collection at a concentration of 100 μM in the presence of 500 nM Red Mega 500 in PBS with 10 % glycerol, 0.01 % NP-40, and 0.2 mg/mL HSA. DMSO was used as negative control and piroxicam and naproxen as positive control compounds
LOPAC compound was measured in triplicate at a single concentration of 100 μM, and the results are depicted in Fig. 6. The majority of the compounds exhibited a fluorescence intensity ranging between 44,000 and 56,000 units. All negative control DMSO values fell in between this range. For the positive control compounds piroxicam and naproxen fluorescence, intensities between 16,000 and 24,000 were observed for all assay plates. The average Z' factor for controls DMSO and piroxicam was 0.69. The normalization of all compound data to DMSO (0 % HSA binding) and piroxicam (100 % HSA binding) resulted in the identification of 97 compounds with an HSA binding of more than 50 % as indicated as the horizontal line in Fig. 6. Of these hit compounds, 92 % was determined to be moderately to highly lipophilic (>1
calculated logP(o/w)). In comparison, 73 % of all compounds of the LOPAC library have a calculated logP(o/w) of greater than 1. A composition map of the LOPAC compounds in respect to drug classes is depicted in Fig. 7a. The pie diagrams in Fig. 7 reflect the number of compounds that interact with a particular drug targets. For the LOPAC library (Fig. 7a), the largest groups of compounds are modulators of serotonin receptors, kinases, glutamate receptors, acetylcholine receptors, and adrenoceptors. In comparison, the pie diagram for the 97 hit compounds is given in Fig. 7b. The largest groups of HSA binders are COX inhibitors (prostaglandin synthesis), kinases inhibitors (phosphorylation), anionic P2 receptors antagonists and glutamate receptor modulators. Three for the four groups (COX, kinases and P2 receptors modulators) are significantly enlarge in Fig. 7b in comparison with 7A due numerous Red Mega 500 assay hit compounds with high HSA binding. Usually, compounds interacting with the same biological target have a common pharmacophore thus their chemical structure is related. Figure 8 presents the chemical structure of hit compounds from three classes (COX, kinases, and P2 receptors modulators) identified by presenting assay compounds in a pie diagram (Fig. 7). The pharmacophore of the anionic P2 receptor antagonists is a linear core structure capped with anionic carboxylate or sulfate groups (Fig. 8a). All members of this drug class inhibited the interaction between Red Mega 500 and HSA as least as good as piroxicam. The group of kinase inhibitors is structurally diverse due to the different enzyme inhibition modes that include direct ATP inhibition, allosteric modulation, reversible binding, and irreversible inhibition (Fig. 8b). The largest groups within B are tyrphostins and other tyrosine
Fig. 7 a Composition of LOPAC 1280 library in regard of drug classes; b Red Mega 500−HSA inhibitors in regard to drug classes
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Fig. 8 Structures of compounds identified with the LOPAC small molecule screen exhibiting more than 50 % protein binding determined with negative control DMSO (0 %) and positive control
piroxicam (100 %). Three drug classes are depicted: a anionic P2 receptors antagonists, b modulators of phosphorylation, and c prostaglandin synthesis (COX) inhibitors
kinase inhibitors [27]. The protein binding in this group ranges between 53 and 100 %. Group C, Fig. 8, represents FDA-approved COX inhibitors used as pain medication. The majority of COX inhibitors have a hydrophobic and a hydrophilic part (carboxylate) and exhibit high in vivo plasma protein binding. The majority of COX inhibitors were identified with the Red Mega 500 assay with HSA binding ranging from 55 to 100 %. All fluorescence-based assays suffer from false positive hits due to intrinsic fluorescence properties of small molecule. For the LOPAC small molecule screen, we determined the
fluorescence intensity of all compounds at the concentration of 100 μM using an excitation and emission wavelength of 514 and 532 nm, respectively. The results of this screen are depicted in Fig. 9. Only a very limited number of compounds exhibited significant fluorescence intensities at the wavelengths use (Fig. 9a). Using a cutoff of 1σ of the mean fluorescence intensity, eight compounds were identified and their chemical structures are depicted in Fig. 9b. Interestingly, only four compounds depicted in black of the total eight compounds were identified with the Red Mega 500 protein binding assay
a
b 25000
Fluorescence Intensity (514/532 nm)
Fig. 9 a Fluorescence intensities (514/532 nm) of all LOPAC compounds (white data points correspond to DMSO; b structures of compounds with high fluorescence intensity (FI) at 514/532 nm
20000 15000 10000 5000 1000 800 600 400 200 0
Compounds
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(Fig. 8). The molecules included Cy5, which saturated the signal, and three tyrosine kinase inhibitors. The other four compounds exhibited a protein binding of less than 50 % in the Red Mega 500 protein binding assay.
Discussion The direct measurement of drug−plasma protein binding using dialysis, chromatography, CD, and ITC enables the determination of % plasma binding and if repeated with different concentrations of small molecule, the determination of a plasma binding constant (KD). However, no information is gained in respect to small molecule occupancy of discreet plasma protein binding sites. For HSA, three main small molecule binding sites have been identified by x-ray crystallography [28]. Importantly, drug molecules as well as fluorescent molecules can bind to a single site of HSA or a combination of HSA binding sites [29]. The binding site of Red Mega 500 is not known, but for the drugs investigated, a good correlation between in vivo plasma binding and inhibition of the HSA−Red Mega 500 interaction was observed. The assay buffer conditions have a significant influence on the measured fluorescence intensity. Especially, the addition of glycerol and surfactant NP-40 increased the quality of the assay by stabilizing HSA and solubilizing hydrophobic assay compounds. The dose–response analysis of piroxicam, diethylstilbestrol, and naproxen identified two classes of HSA binders. Diethylstilbestrol exhibits medium plasmid binding capacity and binds to an unknown HSA binding site. In contrast, piroxicam and naproxen are high plasma protein binder and bind to the IIA and IIIA HSA sites. In regard to potency, we observed that piroxicam interacts stronger with HSA than naproxen (IC50 =4.2 vs. 24.6 μM). Diethylstilbestrol also has a strong interaction with HSA (IC50 =4.4 μM) but binds HSA differently than naproxen because of the weaker efficacy. It can be hypothesized that diethylstilbestrol interacts with a more limited number of HSA binding sites than naproxen leaving some specific HSA−Red Mega 500 interactions undisrupted. This in turn might cause the higher but saturable fluorescence intensity. Efficacy differences due to different HSA binding sites were also reported by Yasgar et al. [13] A large efficacy was observed for naproxen, while a small efficacy was measured for phenylbutazone, which binds predominately to the IIA site instead of all three HSA binding sites. The HTS screen identified 97 compounds with more than 50 % protein binding, determined by the ability to disrupt the interaction between HSA and Red Mega 500 in comparison with naproxen. Although not all compounds of the LOPAC library have been investigated by other plasma protein binding methods, a significantly higher percentage of high protein binders than the 7.7 % determined by the Red Mega 500 assay
can be assumed. The physiological concentration of HSA is 600 μM, and high protein concentrations are usually used to determine direct plasma protein binding. However, for competition plasma binding assays, a minimal protein concentration should be applied to enable the specific competition between fluorescent probe and molecules. Thus, it is very likely that more high protein binders will be identified with lower HSA concentrations, which unfortunately will reduce the Red Mega 500 assay performance due to a smaller fluorescence intensity signal window. The 97 compounds identified by the Red Mega 500 assay bear different scaffolds and functional groups complicating the identification of a relationship between plasma protein binding and compound structure. However, we found that more than half of the hit compounds belong to certain drug classes that include COX inhibitors, kinase inhibitors, anionic P2 receptors antagonists, and glutamate receptor modulators. Thus, structurally diverse compounds that were developed to interact with the same drug target are likely to have similar plasma protein binding. In order to compare % protein binding from a direct and a competition assay, the probe must have the same HSA binding mode than the compound investigated. As a result, we observe different degrees of inhibition (efficacy) for the Red Mega 500 assay depending on the similarity of the binding modes of compound and probe (Fig. 5). Therefore, the lower the fluorescence intensity for a particular compound in Fig. 6, the more similar is the HSA binding mode to Red Mega 500. Our future approach to improve the general correlation between % protein binding from a direct and a competition assay is to multiplex fluorescent probes to bind most of the small molecule HSA binding sites. This will allow the identification of selective inhibitors binding to a particular HSA site and those inhibiting the HSA binding of multiple fluorescent probes. Nevertheless, most of the hit compounds identified by Red Mega 500 were confirmed as high protein binders in comparison with their literature plasma protein binding. Further advances of the developed assay are short incubation times and relative low concentrations of HSA and fluorescent probe. The longer wavelength detection using Red Mega 500 limited the number of molecules interfering with the assay, which was 0.63 % for the LOPAC screening library. Overall, the novel HTS protein binding assay allowed the detection of high protein binders, especially those targeting kinases, COX enzymes, and P2 receptors. We will continue our investigations to develop multiplexed protein binding assays and include the application of blood serum and plasma. Acknowledgments The work was supported by the University of Wisconsin–Milwaukee, the University of Wisconsin-Milwaukee Research Growth Initiative (RGI), National Institute of Drug Abuse
A fluorescence-based serum protein binding assay DA031090, the University of Wisconsin-Milwaukee Research Foundation, the Lynde and Harry Bradley Foundation, and the Richard and Ethel Herzfeld Foundation. Further support was enabled by the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research and the Intramural Research Program of the NHGRI, NIH. We also thank Dr. Frick to enable the use of the ITC instrument.
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