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Evaluation of the binding of polyhedral borane anions to representative proteins
Journal of Inorganic Biochemistry 124 (2013) 11–14
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Evaluation of the binding of polyhedral borane anions to representative proteins R. Corey Waller, Rachell E. Booth, Debra A. Feakes ⁎ Department of Chemistry and Biochemistry, Texas State University-San Marcos, 601 University Drive, San Marcos, TX 78666, United States
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Article history: Received 10 October 2012 Received in revised form 13 March 2013 Accepted 13 March 2013 Available online 21 March 2013 Keywords: BNCT Boron Polyhedral borane anion SDS-PAGE
a b s t r a c t The ability of three polyhedral borane anions, [B20H18] 2−, [B20H17SH] 4−, and [B20H19]3−, to bind to proteins was evaluated by measuring the total boron content using inductively coupled plasma-atomic emission spectroscopy after purification by gel electrophoresis. Results were correlated to the known chemical reactivity of the compounds as well as the reported murine biodistributions of the liposomally encapsulated sodium salts of each of the polyhedral borane anions. Qualitative reactions were performed with the [B20H18] 2− anion to determine the potential reactivity with simple molecular building blocks. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Boron neutron capture therapy (BNCT) is a binary cancer therapy proposed for the treatment of glioblastoma multiforme, a particularly lethal brain tumor, and metastatic melanoma [1–3]. The treatment, first proposed by Locher in 1936 [4], is initiated by the irradiation of boron-10 atoms, which have been selectively delivered to the tumor, by thermal neutrons. The irradiation process results in the formation of an unstable boron-11 atom which subsequently undergoes fission, forming an alpha particle, a lithium-7 particle, and a substantial amount of energy [5]. The distance traveled by the alphaparticles in tissue is approximately 8–10 μm, the approximate diameter of an average eukaryotic cell. Therefore, the damage induced by the alpha-particles is localized to the tumor cells which contain high concentrations of the boron-10 isotope. The tumor boron concentration required for successful BNCT has been estimated to be between 15 and 30 μg of boron/gram of tumor [6]. To achieve these concentrations, researchers have utilized several approaches including, but not limited to, the preparation of boroncontaining derivatives of compounds known to localize in tumors [7–14], the synthesis of compounds which have the potential to be incorporated into the metabolic cycles of rapidly proliferating cancer cells [15–21], and the preparation of boron-containing compounds which have the potential to target specific biological molecules such as proteins, receptors, and growth factors [19,22,23]. In addition, ⁎ Corresponding author at: Department of Chemistry and Biochemistry, Texas State University-San Marcos, 601 University Drive, San Marcos, TX 78666, United States. Fax: +1 512 245 2374. E-mail address: [email protected] (D.A. Feakes). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.03.007
researchers have investigated the utilization of tumor selective delivery vehicles for boron-containing compounds which have little to no inherent tumor specificity [24–33]. Unilamellar liposomes, encapsulating aqueous solutions of watersoluble polyhedral borane compounds, have been shown to deliver their contents selectively to the tumor in murine biodistribution experiments [24,25,27,28,30,32]. Although essentially any water-soluble polyhedral borane compound can be delivered to the tumor in this manner, investigations have shown that the retention of the compound by the tumor is dependent on the reactivity of the anion [24,25,27]. Polyhedral borane ions which have the potential to react with intracellular and extracellular proteins, such as the [B20H18] 2− ion and the [B20H17SH]4− ion, are retained by the tumor over the time course experiment. The retention of the [B20H18]2− ion within the tumor has been attributed to the susceptibility of the electron-deficient, threecenter, two-electron bonds to nucleophilic attack [24]. Two possible mechanisms exist for the retention of the [B20H17SH]4− ion in the tumor mass [27]. The first mechanism is based on the potential of the thiol substituent to react with intracellular and extracellular protein thiol moieties to bind the compound in the tumor through the formation of disulfide bonds. The second mechanism is based on the potential of the [B20H17SH]4− ion to oxidize to the more reactive species, [B20H17SH]2−, which is again susceptible to nucleophilic attack by a wide variety of protein substituents. Unreactive anions, such as the [B20H19]3− ion, may exhibit a high initial tumor boron concentration, but eventually clear from the tumor, resulting in tumor boron concentrations below the therapeutic level [24]. Although numerous compounds have been investigated in murine biodistribution experiments, no studies have been reported regarding the localization of the polyhedral borane anions once delivered to the
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R.C. Waller et al. / Journal of Inorganic Biochemistry 124 (2013) 11–14
tumor by the liposomes and, as a result, no specific proteins have been identified as targets for reactivity. In an earlier investigation, our laboratory reported the MALDI-MS results of the products obtained from the reaction of three polyhedral borane anions, [B20H18] 2−, [B20H17OH] 4−, and [B20H17SH] 4−, with either bovine serum albumin (BSA) or human serum albumin (HSA) [34]. Although each of the products exhibited a shift in the mass spectrum consistent with an interaction between the polyhedral borane anion and the albumin, no direct measurement of boron concentration was obtained and the nature of the interaction, covalent or electrostatic, could not be ascertained from the mass spectrum. Analysis of the products of the reaction between the polyhedral borane anions and HSA using native polyacrylamide gel electrophoresis (native-PAGE) resulted in a variation in the migration of HSA for only the [B20H17SH]4− ion, consistent with the presence of a covalent bond. Analysis using Ellman's test for free thiols [35] confirmed the presence of the disulfide bond with the single free cysteine residue. While the existence of covalent bond formation between the [B20H17SH]4− ion and albumin was established in the earlier investigation, the proteins of greatest interest for application in BNCT are those which occur intracellularly and which have the potential to retain the polyhedral borane anion within the cell as a result of covalent binding. In order to further the understanding of the reactivity of this class of compounds, as well as their potential application in BNCT, delineation of the cellular interactions as well as the interactions of the polyhedral borane anions with a variety of representative intracellular proteins is needed. To determine the potential molecular interactions exhibited by polyhedral borane anions in a cellular setting, an investigation of the reactivity of Na2[B20H18] with simple molecules, as well as the reactivity of Na2[B20H18], Na4[B20H17SH], and Na3[B20H19] with common proteins, was completed. Structural building block molecules, such as aspartic acid, choline, cysteine, glutamic acid, and tyrosine were used to determine the reactivity of Na2[B20H18] to common nucleophiles found in cellular systems. Although the products of these reactions were not isolated in their pure form, the reactivity was monitored by 1 H and 11B NMR spectroscopy. Reaction of Na4[B20H17SH] with reduced glutathione as a representative small biomolecule was reported earlier [34]. SDS-PAGE was used to separate the products of the reaction between Na2[B20H18], Na4[B20H17SH], and Na3[B20H19] and BSA, G-actin, cytochrome-c, β-galactosidase, and α-mannosidase. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to measure the total boron content of each of the gel purified proteins to verify the binding of the polyhedral borane anions to the proteins.
ICP-AES [38] at the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID). 2.2. Investigation of the interactions with molecular building blocks The general reaction procedure was the same for each of the amino acids and choline. An aqueous solution of Na2[B20H18] and the test compound were combined in a 1:3 M ratio and the pH of the solution adjusted to 7.4–7.6, depending on the particular reactant utilized. The reaction vessel was placed in a 37 °C water bath and the reaction was monitored by 11B NMR spectroscopy at 24-hour intervals. When desired, products were isolated by precipitation of the resulting anion as the potassium salt, using a saturated solution of potassium acetate in absolute ethanol, or as the methyltriphenylphosphonium salt, using a saturated solution of methyltriphenylphosphonium bromide in distilled water. The solids were filtered, dried in vacuo, and characterized spectroscopically. The control reactions, which contained only Na2[B20H18] and distilled water, were performed at the temperature and pH of the individual reaction mixtures. 2.3. Investigation of the interactions with proteins by reducing SDS-PAGE The protein (100 μg) was dissolved in 10 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) buffered saline (pH 7.4) to produce a final volume of 500 μL. The protein solution was incubated with Na2[B20H18], Na4[B20H17SH], or Na3[B20H19] equal to 100× the molar amount of the protein at 37 °C for 36 h. Reaction samples (~20 μg) were mixed with 1× SDS sample buffer (100 mM Tris–HCl, 10% glycerol, 2% SDS, 0.5 mL bromophenol blue, and 25 μL β-mercaptoethanol and 475 μL stock solution), heated at 95 °C for 8 min, and loaded into a 10% Tris–Gly SDS-PAGE, and separated for 1 h at 250 V. The proteins were visualized with G-250 coomassie blue stain, destained with a methanol/acetic acid mixture, and photographed. All protein controls were incubated and prepared using the same protocols as described. 2.4. Investigation of the interactions with proteins by SDS-PAGE in combination with ICP-AES analysis Reactions and SDS-PAGE analysis were performed as described in Section 2.3. The resulting proteins were excised from the gel and sent to the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID) to obtain the total boron analysis by ICP-AES
2. Materials and methods
Decaborane, B10H14, was obtained from Alfa Aesar (Ward Hill, MA) and sublimed prior to use. CAUTION: Decaborane is a highly toxic, impact sensitive compound which forms explosive mixtures, especially with halogenated materials. A careful examination of the MSDS is recommended before usage. Na2[B20H18] and Na4[B20H17SH] were prepared using published methods [27,36,37]. Na3[B20H19] was obtained as a gift from Professor M. Frederick Hawthorne (presently at the University of Missouri-Columbia). The amino acids and proteins were obtained from either Sigma Chemicals (St. Louis, MO) or Sigma-Aldrich Chemicals (Milwaukee, WI) and used without further purification. Molecular weight (mass) standards were obtained from BioRad (Hercules, CA) and contained myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). The 1H and 11B NMR nuclear magnetic resonance (NMR) spectra were obtained with a Varian INOVA instrument operating at 400 MHz and 128 MHz, respectively. Proton chemical shifts were referenced to residual solvent protons. Boron chemical shifts were externally referenced to BF3·Et2O in C6D6; peaks upfield of the reference are designated as negative. Total boron analysis was performed by
µg of boron/g of protein
60000
2.1. Chemicals and analysis
40000
20000
0
BSA
Actin
Cytochrome-C
Galactosidase
Mannosidase Fig. 1. The total boron content in each protein, in μg of boron/g of protein, after reaction of the [B20H18]2− anion with the protein and separation by SDS-PAGE. Five proteins were analyzed in each case; no bar is observed when the value is zero.
R.C. Waller et al. / Journal of Inorganic Biochemistry 124 (2013) 11–14
µg of boron/g of protein
40000
30000
20000
10000
0
BSA
Actin
Cytochrome-C
Galactosidase
Mannosidase Fig. 2. The total boron content in each protein, in μg of boron/g of protein, after reaction of the [B20H17SH]4− anion with the protein and separation by SDS-PAGE. Five proteins were analyzed in each case; no bar is observed when the value is zero.
[38]. The results were obtained as parts per million boron in the sample tube. The values reported in Figs. 1 and 2 are calculated as μg of boron per gram of protein and represent the average of triplicate analysis. Control sections were taken and analyzed from empty lanes of the gel as well as the portion of the gel above the first band seen. 3. Results and discussion Water-soluble polyhedral borane anions, encapsulated in unilamellar liposomes, have been investigated as potential agents for application in BNCT [24,25,27,28,30,32]. Although the unilamellar liposomes provide the tumor selectivity necessary for the therapy, the observed retention of some polyhedral borane anions has been attributed to the potential reactivity of the compounds with the reactive substituents of intracellular proteins. Compounds lacking suitable reactivity are cleared from the tumor mass during the time course experiment [24]. The [B20H18]2− anion and its substituted derivatives, of the form [B20H17X]2−, are characterized by two electron-deficient, three-center, two-electron bonds which are susceptible to nucleophilic attack [37]. The reaction of [B20H18]2− with simple nucleophiles, such as [OH]− [39], [OR]− [39], [NH2]− [25], and [NHR]− [40], has been reported. Retention of the [B20H18]2− ion has been attributed to the formation of covalent bonds with nucleophilic protein substituents [24]. The reduced, substituted derivatives of [B20H18]2−, of the form [B20H17X]4−, have the potential to oxidize to the more reactive [B20H17X] 2 − derivatives. Retention of [B20H17NH3] 3 − [25] and [B20H17SH]4− [27] has been attributed to the in vivo oxidation of these compounds followed by nucleophilic attack on the oxidized derivatives. The thiol derivative, [B20H17SH]4−, also has the potential to form intracellular disulfide bonds. The reactivity of three polyhedral borane anions, [B20H18] 2−, [B20H17SH] 4−, and [B20H19] 3−, was investigated. The first two were selected based on the potential reactivity with intracellular protein substituents. The [B20H19] 3− anion was selected for investigation because the anion is known to be essentially unreactive [41]. The in vivo murine biodistribution of the liposomally encapsulated [B20H19] 3− supports the unreactive nature of the anion [24]. Although the initial tumor boron concentration was quite high based on the low injected dose, rapid clearance of the boron from the tumor was observed over the remainder of the time course experiment. The susceptibility of the electron-deficient, three-center, twoelectron bonds in [B20H18] 2− was investigated qualitatively by the reaction of [B20H18] 2− with amino acids and choline, which were each
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selected for the presence of representative organic functional groups (\OH and \SH) with the potential to react as nucleophiles. Reactions were monitored by 11B NMR spectroscopy. The starting material, [B20H18]2−, exhibits a characteristic seven peak spectrum. The highest downfield peak is a doublet centered at 29.1 ppm. Reductive substitution of the [B20H18] 2− ion results in a dramatic alteration of the 11B NMR spectrum, characterized by the disappearance of the highest downfield peak and the formation of apical boron–hydrogen signals and a substituted boron signal in the +5 to −15 ppm region of the spectrum. Representative 1H and 11B NMR spectra can be viewed in the supplemental information (Supplemental Figs. S1–S4). Reaction of the [B20H18]2− anion with the sodium salts of L-tyrosine and L-cysteine and the bromide salt of choline was observed while reaction of the [B20H18]2− anion with aspartic acid and glutamic acid was not observed. The control reactions at the same temperature and pH of the reaction mixtures exhibited no change in the 11B NMR spectrum of the reaction mixture. The results indicate that the sodium salts of L-tyrosine and L-cysteine and the bromide salt of choline are sufficiently nucleophilic to react with the [B20H18]2− anion while aspartic acid and glutamic acid are not, consistent with the known reactivity of the hydroxyl group of a carboxylic acid. In addition, reactivity with amino substituents from the backbone or side chain was not evident, which was expected at physiological pH where the protonated amino groups are less reactive. The results of the reaction between the [B20H18] 2− ion and the reactive functional groups of L-tyrosine, L-cysteine, and choline suggested that the polyhedral borane anion may also react with the reactive substituents of proteins. Thus, various proteins including α-mannosidase, β-galactosidase, bovine serum albumin (BSA), cytochrome-c, and G-actin, were selected for investigation. The proteins were selected based on availability as well as to provide diversity in their cellular localization, thiol content, and amino acid composition. α-Mannosidase (GeneBank P02769) is localized in the lysosome and has 11 cysteine residues; β-galactosidase (Q58D55) is also localized in the lysosome and has 7 cysteine residues; BSA (CAA76847) is secreted outside of the cell and has 35 cysteine residues; cytochrome-c (NP_001039526) is localized in the membrane mitochondrion and has 2 cysteine residues; and, G-actin (NP_001028790) is localized in the cytoplasm and has 6 cysteine residues. Reactions between the selected proteins and each of the polyhedral borane anions were performed at 37 °C and unreacted and reacted proteins were separated by reducing SDS-PAGE. The increase in molecular weight due to polyhedral borane binding was difficult to quantify as a result of relatively small increases in molecular weight on the gels (representative gels can be viewed in the supplemental information, Supplemental Fig. S5). As a result, a more quantitative investigation of the reaction of each of the three selected polyhedral borane anions and the chosen proteins was performed. SDS-PAGE was utilized in conjunction with the quantitative measurement of total boron in the gel slices by ICP-AES. Results of the ICP-AES analysis indicate that BSA, β-galactosidase and cytochrome-c had slight reactivity with [B20H18]2− while α-mannosidase exhibited significantly higher reactivity and actin had no detectable reactivity (Fig. 1). All of the proteins except for actin exhibited reactivity with [B20H17SH]4− (Fig. 2). The relatively unreactive [B20H19] 3− ion exhibited no detectable reactivity with actin and cytochrome-c and only negligible reactivity with α-mannosidase. Some reactivity between the [B20H19]3− was observed with BSA and β-galactosidase. These results are consistent with both the known chemical reactivity of the polyhedral borane anions as well as the reported murine biodistribution of the liposomally encapsulated compounds [24,27]. The [B20H18] 2− anion has the potential to undergo nucleophilic attack and is retained to a large degree in the tumor in murine biodistribution experiments [24]. The total boron analysis (Fig. 1) indicates that the compound reacts with four of the five proteins investigated and exhibits significant reactivity with α-mannosidase. This is likely due, in part, to the greater size of α-mannosidase resulting in a
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greater number of hydroxyl substituents present in α-mannosidase (163 residues contain hydroxyl groups based on the number of serine, threonine, and tyrosine) compared to the other model proteins (13 residues in cytochrome-c, 87 residues in BSA, and 103 residues in β-galactosidase) as well as the relative number of those residues that are surface exposed as seen in x-ray crystal structures from the Protein Data Bank (PDB files 1O7D, 2B4Z, and 3THC, respectively). In comparison, the murine biodistribution of the liposomally encapsulated sodium salt of the [B20H17SH]4− ion exhibits not only retention, but accretion, of tumor boron concentration over the entire time course experiment [27]. Therefore, this anion should exhibit a higher degree of reactivity to the proteins that have accessible free thiols. BSA, β-galactosidase, and α-mannosidase contain free thiols and were reactive with the [B20H17SH]4− anion (Fig. 2) whereas cytochrome-c had significantly less reactivity and actin had no detectible reactivity. The latter of these proteins contain thiol containing residues, but they are likely involved in disulfide bonds. For each of the proteins containing free thiols, except for the α-mannosidase reaction, the amount of boron meets or exceeds that of the reactions with the [B20H18] 2− ion (Fig. 1). The [B20H19] 3− anion was the least reactive of the three polyhedral borane anions investigated and the murine biodistribution of the liposomally encapsulated sodium salt of [B20H19] 3− exhibited relatively high boron concentration in the tumor early in the time course experiment, but overall clearance of tumor boron concentration throughout the remainder of the experiment [24]. The ICP-AES analysis indicated little to no reaction with three of the proteins (αmannosidase, cytochrome-c, and G-actin) investigated and only moderate reactivity with the other two (β-galactosidase and BSA). Similar reactivity may account for the high tumor boron concentrations early in the in vivo biodistribution experiment [24]. The [B20H19] 3− anion, like the other anions, has the potential to form strong electrostatic interactions with the proteins. The anion also has the potential to oxidize to form a more reactive species, the [B20H19]− anion, though this reaction is unlikely under the existing reaction conditions. 4. Conclusions Evaluation and correlation of the potential reactivity of polyhedral borane anions with both extracellular and intracellular proteins have been achieved by ICP-AES analysis after separation by reducing SDS-PAGE. The thiol derivative, [B20H17SH] 4−, was generally more reactive with the proteins investigated which is consistent with both the anticipated reactivity of the polyhedral borane anion and the reported uptake and retention obtained in the murine biodistribution experiment [27]. The results of the current investigation, in combination with cellular fractionation studies, should assist in the elucidation of the subcellular pathway of the Na4[B20H17SH], once delivered to the tumor by the liposomes. Additionally, the correlations observed between the results of the current study and the in vivo biodistribution experiments provide evidence for the potential to evaluate the relative retention of polyhedral borane anions within the tumor using the methods developed and reported here and a series of model proteins prior to extensive and expensive murine biodistribution experiments. Abbreviations BNCT boron neutron capture therapy BSA bovine serum albumin HSA human serum albumin ICP-AES inductively coupled plasma-atomic emission spectroscopy Native-PAGE native polyacrylamide gel electrophoresis Acknowledgments This research was supported by awards from the Robert A. Welch Foundation (AI1292), Research Corporation (CC3913), and Texas State
University-San Marcos. The authors wish to thank Dr. William Bauer of the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID) for the total boron analysis by ICP-AES and Professor M. Frederick Hawthorne for his generous gift of Na3[B20H19]. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jinorgbio.2013.03.007.
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Evaluation of the binding of polyhedral borane anions to representative proteins R. Corey Waller, Rachell E. Booth, Debra A. Feakes ⁎ Department of Chemistry and Biochemistry, Texas State University-San Marcos, 601 University Drive, San Marcos, TX 78666, United States
a r t i c l e
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Article history: Received 10 October 2012 Received in revised form 13 March 2013 Accepted 13 March 2013 Available online 21 March 2013 Keywords: BNCT Boron Polyhedral borane anion SDS-PAGE
a b s t r a c t The ability of three polyhedral borane anions, [B20H18] 2−, [B20H17SH] 4−, and [B20H19]3−, to bind to proteins was evaluated by measuring the total boron content using inductively coupled plasma-atomic emission spectroscopy after purification by gel electrophoresis. Results were correlated to the known chemical reactivity of the compounds as well as the reported murine biodistributions of the liposomally encapsulated sodium salts of each of the polyhedral borane anions. Qualitative reactions were performed with the [B20H18] 2− anion to determine the potential reactivity with simple molecular building blocks. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Boron neutron capture therapy (BNCT) is a binary cancer therapy proposed for the treatment of glioblastoma multiforme, a particularly lethal brain tumor, and metastatic melanoma [1–3]. The treatment, first proposed by Locher in 1936 [4], is initiated by the irradiation of boron-10 atoms, which have been selectively delivered to the tumor, by thermal neutrons. The irradiation process results in the formation of an unstable boron-11 atom which subsequently undergoes fission, forming an alpha particle, a lithium-7 particle, and a substantial amount of energy [5]. The distance traveled by the alphaparticles in tissue is approximately 8–10 μm, the approximate diameter of an average eukaryotic cell. Therefore, the damage induced by the alpha-particles is localized to the tumor cells which contain high concentrations of the boron-10 isotope. The tumor boron concentration required for successful BNCT has been estimated to be between 15 and 30 μg of boron/gram of tumor [6]. To achieve these concentrations, researchers have utilized several approaches including, but not limited to, the preparation of boroncontaining derivatives of compounds known to localize in tumors [7–14], the synthesis of compounds which have the potential to be incorporated into the metabolic cycles of rapidly proliferating cancer cells [15–21], and the preparation of boron-containing compounds which have the potential to target specific biological molecules such as proteins, receptors, and growth factors [19,22,23]. In addition, ⁎ Corresponding author at: Department of Chemistry and Biochemistry, Texas State University-San Marcos, 601 University Drive, San Marcos, TX 78666, United States. Fax: +1 512 245 2374. E-mail address: [email protected] (D.A. Feakes). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.03.007
researchers have investigated the utilization of tumor selective delivery vehicles for boron-containing compounds which have little to no inherent tumor specificity [24–33]. Unilamellar liposomes, encapsulating aqueous solutions of watersoluble polyhedral borane compounds, have been shown to deliver their contents selectively to the tumor in murine biodistribution experiments [24,25,27,28,30,32]. Although essentially any water-soluble polyhedral borane compound can be delivered to the tumor in this manner, investigations have shown that the retention of the compound by the tumor is dependent on the reactivity of the anion [24,25,27]. Polyhedral borane ions which have the potential to react with intracellular and extracellular proteins, such as the [B20H18] 2− ion and the [B20H17SH]4− ion, are retained by the tumor over the time course experiment. The retention of the [B20H18]2− ion within the tumor has been attributed to the susceptibility of the electron-deficient, threecenter, two-electron bonds to nucleophilic attack [24]. Two possible mechanisms exist for the retention of the [B20H17SH]4− ion in the tumor mass [27]. The first mechanism is based on the potential of the thiol substituent to react with intracellular and extracellular protein thiol moieties to bind the compound in the tumor through the formation of disulfide bonds. The second mechanism is based on the potential of the [B20H17SH]4− ion to oxidize to the more reactive species, [B20H17SH]2−, which is again susceptible to nucleophilic attack by a wide variety of protein substituents. Unreactive anions, such as the [B20H19]3− ion, may exhibit a high initial tumor boron concentration, but eventually clear from the tumor, resulting in tumor boron concentrations below the therapeutic level [24]. Although numerous compounds have been investigated in murine biodistribution experiments, no studies have been reported regarding the localization of the polyhedral borane anions once delivered to the
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tumor by the liposomes and, as a result, no specific proteins have been identified as targets for reactivity. In an earlier investigation, our laboratory reported the MALDI-MS results of the products obtained from the reaction of three polyhedral borane anions, [B20H18] 2−, [B20H17OH] 4−, and [B20H17SH] 4−, with either bovine serum albumin (BSA) or human serum albumin (HSA) [34]. Although each of the products exhibited a shift in the mass spectrum consistent with an interaction between the polyhedral borane anion and the albumin, no direct measurement of boron concentration was obtained and the nature of the interaction, covalent or electrostatic, could not be ascertained from the mass spectrum. Analysis of the products of the reaction between the polyhedral borane anions and HSA using native polyacrylamide gel electrophoresis (native-PAGE) resulted in a variation in the migration of HSA for only the [B20H17SH]4− ion, consistent with the presence of a covalent bond. Analysis using Ellman's test for free thiols [35] confirmed the presence of the disulfide bond with the single free cysteine residue. While the existence of covalent bond formation between the [B20H17SH]4− ion and albumin was established in the earlier investigation, the proteins of greatest interest for application in BNCT are those which occur intracellularly and which have the potential to retain the polyhedral borane anion within the cell as a result of covalent binding. In order to further the understanding of the reactivity of this class of compounds, as well as their potential application in BNCT, delineation of the cellular interactions as well as the interactions of the polyhedral borane anions with a variety of representative intracellular proteins is needed. To determine the potential molecular interactions exhibited by polyhedral borane anions in a cellular setting, an investigation of the reactivity of Na2[B20H18] with simple molecules, as well as the reactivity of Na2[B20H18], Na4[B20H17SH], and Na3[B20H19] with common proteins, was completed. Structural building block molecules, such as aspartic acid, choline, cysteine, glutamic acid, and tyrosine were used to determine the reactivity of Na2[B20H18] to common nucleophiles found in cellular systems. Although the products of these reactions were not isolated in their pure form, the reactivity was monitored by 1 H and 11B NMR spectroscopy. Reaction of Na4[B20H17SH] with reduced glutathione as a representative small biomolecule was reported earlier [34]. SDS-PAGE was used to separate the products of the reaction between Na2[B20H18], Na4[B20H17SH], and Na3[B20H19] and BSA, G-actin, cytochrome-c, β-galactosidase, and α-mannosidase. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to measure the total boron content of each of the gel purified proteins to verify the binding of the polyhedral borane anions to the proteins.
ICP-AES [38] at the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID). 2.2. Investigation of the interactions with molecular building blocks The general reaction procedure was the same for each of the amino acids and choline. An aqueous solution of Na2[B20H18] and the test compound were combined in a 1:3 M ratio and the pH of the solution adjusted to 7.4–7.6, depending on the particular reactant utilized. The reaction vessel was placed in a 37 °C water bath and the reaction was monitored by 11B NMR spectroscopy at 24-hour intervals. When desired, products were isolated by precipitation of the resulting anion as the potassium salt, using a saturated solution of potassium acetate in absolute ethanol, or as the methyltriphenylphosphonium salt, using a saturated solution of methyltriphenylphosphonium bromide in distilled water. The solids were filtered, dried in vacuo, and characterized spectroscopically. The control reactions, which contained only Na2[B20H18] and distilled water, were performed at the temperature and pH of the individual reaction mixtures. 2.3. Investigation of the interactions with proteins by reducing SDS-PAGE The protein (100 μg) was dissolved in 10 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) buffered saline (pH 7.4) to produce a final volume of 500 μL. The protein solution was incubated with Na2[B20H18], Na4[B20H17SH], or Na3[B20H19] equal to 100× the molar amount of the protein at 37 °C for 36 h. Reaction samples (~20 μg) were mixed with 1× SDS sample buffer (100 mM Tris–HCl, 10% glycerol, 2% SDS, 0.5 mL bromophenol blue, and 25 μL β-mercaptoethanol and 475 μL stock solution), heated at 95 °C for 8 min, and loaded into a 10% Tris–Gly SDS-PAGE, and separated for 1 h at 250 V. The proteins were visualized with G-250 coomassie blue stain, destained with a methanol/acetic acid mixture, and photographed. All protein controls were incubated and prepared using the same protocols as described. 2.4. Investigation of the interactions with proteins by SDS-PAGE in combination with ICP-AES analysis Reactions and SDS-PAGE analysis were performed as described in Section 2.3. The resulting proteins were excised from the gel and sent to the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID) to obtain the total boron analysis by ICP-AES
2. Materials and methods
Decaborane, B10H14, was obtained from Alfa Aesar (Ward Hill, MA) and sublimed prior to use. CAUTION: Decaborane is a highly toxic, impact sensitive compound which forms explosive mixtures, especially with halogenated materials. A careful examination of the MSDS is recommended before usage. Na2[B20H18] and Na4[B20H17SH] were prepared using published methods [27,36,37]. Na3[B20H19] was obtained as a gift from Professor M. Frederick Hawthorne (presently at the University of Missouri-Columbia). The amino acids and proteins were obtained from either Sigma Chemicals (St. Louis, MO) or Sigma-Aldrich Chemicals (Milwaukee, WI) and used without further purification. Molecular weight (mass) standards were obtained from BioRad (Hercules, CA) and contained myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). The 1H and 11B NMR nuclear magnetic resonance (NMR) spectra were obtained with a Varian INOVA instrument operating at 400 MHz and 128 MHz, respectively. Proton chemical shifts were referenced to residual solvent protons. Boron chemical shifts were externally referenced to BF3·Et2O in C6D6; peaks upfield of the reference are designated as negative. Total boron analysis was performed by
µg of boron/g of protein
60000
2.1. Chemicals and analysis
40000
20000
0
BSA
Actin
Cytochrome-C
Galactosidase
Mannosidase Fig. 1. The total boron content in each protein, in μg of boron/g of protein, after reaction of the [B20H18]2− anion with the protein and separation by SDS-PAGE. Five proteins were analyzed in each case; no bar is observed when the value is zero.
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µg of boron/g of protein
40000
30000
20000
10000
0
BSA
Actin
Cytochrome-C
Galactosidase
Mannosidase Fig. 2. The total boron content in each protein, in μg of boron/g of protein, after reaction of the [B20H17SH]4− anion with the protein and separation by SDS-PAGE. Five proteins were analyzed in each case; no bar is observed when the value is zero.
[38]. The results were obtained as parts per million boron in the sample tube. The values reported in Figs. 1 and 2 are calculated as μg of boron per gram of protein and represent the average of triplicate analysis. Control sections were taken and analyzed from empty lanes of the gel as well as the portion of the gel above the first band seen. 3. Results and discussion Water-soluble polyhedral borane anions, encapsulated in unilamellar liposomes, have been investigated as potential agents for application in BNCT [24,25,27,28,30,32]. Although the unilamellar liposomes provide the tumor selectivity necessary for the therapy, the observed retention of some polyhedral borane anions has been attributed to the potential reactivity of the compounds with the reactive substituents of intracellular proteins. Compounds lacking suitable reactivity are cleared from the tumor mass during the time course experiment [24]. The [B20H18]2− anion and its substituted derivatives, of the form [B20H17X]2−, are characterized by two electron-deficient, three-center, two-electron bonds which are susceptible to nucleophilic attack [37]. The reaction of [B20H18]2− with simple nucleophiles, such as [OH]− [39], [OR]− [39], [NH2]− [25], and [NHR]− [40], has been reported. Retention of the [B20H18]2− ion has been attributed to the formation of covalent bonds with nucleophilic protein substituents [24]. The reduced, substituted derivatives of [B20H18]2−, of the form [B20H17X]4−, have the potential to oxidize to the more reactive [B20H17X] 2 − derivatives. Retention of [B20H17NH3] 3 − [25] and [B20H17SH]4− [27] has been attributed to the in vivo oxidation of these compounds followed by nucleophilic attack on the oxidized derivatives. The thiol derivative, [B20H17SH]4−, also has the potential to form intracellular disulfide bonds. The reactivity of three polyhedral borane anions, [B20H18] 2−, [B20H17SH] 4−, and [B20H19] 3−, was investigated. The first two were selected based on the potential reactivity with intracellular protein substituents. The [B20H19] 3− anion was selected for investigation because the anion is known to be essentially unreactive [41]. The in vivo murine biodistribution of the liposomally encapsulated [B20H19] 3− supports the unreactive nature of the anion [24]. Although the initial tumor boron concentration was quite high based on the low injected dose, rapid clearance of the boron from the tumor was observed over the remainder of the time course experiment. The susceptibility of the electron-deficient, three-center, twoelectron bonds in [B20H18] 2− was investigated qualitatively by the reaction of [B20H18] 2− with amino acids and choline, which were each
13
selected for the presence of representative organic functional groups (\OH and \SH) with the potential to react as nucleophiles. Reactions were monitored by 11B NMR spectroscopy. The starting material, [B20H18]2−, exhibits a characteristic seven peak spectrum. The highest downfield peak is a doublet centered at 29.1 ppm. Reductive substitution of the [B20H18] 2− ion results in a dramatic alteration of the 11B NMR spectrum, characterized by the disappearance of the highest downfield peak and the formation of apical boron–hydrogen signals and a substituted boron signal in the +5 to −15 ppm region of the spectrum. Representative 1H and 11B NMR spectra can be viewed in the supplemental information (Supplemental Figs. S1–S4). Reaction of the [B20H18]2− anion with the sodium salts of L-tyrosine and L-cysteine and the bromide salt of choline was observed while reaction of the [B20H18]2− anion with aspartic acid and glutamic acid was not observed. The control reactions at the same temperature and pH of the reaction mixtures exhibited no change in the 11B NMR spectrum of the reaction mixture. The results indicate that the sodium salts of L-tyrosine and L-cysteine and the bromide salt of choline are sufficiently nucleophilic to react with the [B20H18]2− anion while aspartic acid and glutamic acid are not, consistent with the known reactivity of the hydroxyl group of a carboxylic acid. In addition, reactivity with amino substituents from the backbone or side chain was not evident, which was expected at physiological pH where the protonated amino groups are less reactive. The results of the reaction between the [B20H18] 2− ion and the reactive functional groups of L-tyrosine, L-cysteine, and choline suggested that the polyhedral borane anion may also react with the reactive substituents of proteins. Thus, various proteins including α-mannosidase, β-galactosidase, bovine serum albumin (BSA), cytochrome-c, and G-actin, were selected for investigation. The proteins were selected based on availability as well as to provide diversity in their cellular localization, thiol content, and amino acid composition. α-Mannosidase (GeneBank P02769) is localized in the lysosome and has 11 cysteine residues; β-galactosidase (Q58D55) is also localized in the lysosome and has 7 cysteine residues; BSA (CAA76847) is secreted outside of the cell and has 35 cysteine residues; cytochrome-c (NP_001039526) is localized in the membrane mitochondrion and has 2 cysteine residues; and, G-actin (NP_001028790) is localized in the cytoplasm and has 6 cysteine residues. Reactions between the selected proteins and each of the polyhedral borane anions were performed at 37 °C and unreacted and reacted proteins were separated by reducing SDS-PAGE. The increase in molecular weight due to polyhedral borane binding was difficult to quantify as a result of relatively small increases in molecular weight on the gels (representative gels can be viewed in the supplemental information, Supplemental Fig. S5). As a result, a more quantitative investigation of the reaction of each of the three selected polyhedral borane anions and the chosen proteins was performed. SDS-PAGE was utilized in conjunction with the quantitative measurement of total boron in the gel slices by ICP-AES. Results of the ICP-AES analysis indicate that BSA, β-galactosidase and cytochrome-c had slight reactivity with [B20H18]2− while α-mannosidase exhibited significantly higher reactivity and actin had no detectable reactivity (Fig. 1). All of the proteins except for actin exhibited reactivity with [B20H17SH]4− (Fig. 2). The relatively unreactive [B20H19] 3− ion exhibited no detectable reactivity with actin and cytochrome-c and only negligible reactivity with α-mannosidase. Some reactivity between the [B20H19]3− was observed with BSA and β-galactosidase. These results are consistent with both the known chemical reactivity of the polyhedral borane anions as well as the reported murine biodistribution of the liposomally encapsulated compounds [24,27]. The [B20H18] 2− anion has the potential to undergo nucleophilic attack and is retained to a large degree in the tumor in murine biodistribution experiments [24]. The total boron analysis (Fig. 1) indicates that the compound reacts with four of the five proteins investigated and exhibits significant reactivity with α-mannosidase. This is likely due, in part, to the greater size of α-mannosidase resulting in a
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greater number of hydroxyl substituents present in α-mannosidase (163 residues contain hydroxyl groups based on the number of serine, threonine, and tyrosine) compared to the other model proteins (13 residues in cytochrome-c, 87 residues in BSA, and 103 residues in β-galactosidase) as well as the relative number of those residues that are surface exposed as seen in x-ray crystal structures from the Protein Data Bank (PDB files 1O7D, 2B4Z, and 3THC, respectively). In comparison, the murine biodistribution of the liposomally encapsulated sodium salt of the [B20H17SH]4− ion exhibits not only retention, but accretion, of tumor boron concentration over the entire time course experiment [27]. Therefore, this anion should exhibit a higher degree of reactivity to the proteins that have accessible free thiols. BSA, β-galactosidase, and α-mannosidase contain free thiols and were reactive with the [B20H17SH]4− anion (Fig. 2) whereas cytochrome-c had significantly less reactivity and actin had no detectible reactivity. The latter of these proteins contain thiol containing residues, but they are likely involved in disulfide bonds. For each of the proteins containing free thiols, except for the α-mannosidase reaction, the amount of boron meets or exceeds that of the reactions with the [B20H18] 2− ion (Fig. 1). The [B20H19] 3− anion was the least reactive of the three polyhedral borane anions investigated and the murine biodistribution of the liposomally encapsulated sodium salt of [B20H19] 3− exhibited relatively high boron concentration in the tumor early in the time course experiment, but overall clearance of tumor boron concentration throughout the remainder of the experiment [24]. The ICP-AES analysis indicated little to no reaction with three of the proteins (αmannosidase, cytochrome-c, and G-actin) investigated and only moderate reactivity with the other two (β-galactosidase and BSA). Similar reactivity may account for the high tumor boron concentrations early in the in vivo biodistribution experiment [24]. The [B20H19] 3− anion, like the other anions, has the potential to form strong electrostatic interactions with the proteins. The anion also has the potential to oxidize to form a more reactive species, the [B20H19]− anion, though this reaction is unlikely under the existing reaction conditions. 4. Conclusions Evaluation and correlation of the potential reactivity of polyhedral borane anions with both extracellular and intracellular proteins have been achieved by ICP-AES analysis after separation by reducing SDS-PAGE. The thiol derivative, [B20H17SH] 4−, was generally more reactive with the proteins investigated which is consistent with both the anticipated reactivity of the polyhedral borane anion and the reported uptake and retention obtained in the murine biodistribution experiment [27]. The results of the current investigation, in combination with cellular fractionation studies, should assist in the elucidation of the subcellular pathway of the Na4[B20H17SH], once delivered to the tumor by the liposomes. Additionally, the correlations observed between the results of the current study and the in vivo biodistribution experiments provide evidence for the potential to evaluate the relative retention of polyhedral borane anions within the tumor using the methods developed and reported here and a series of model proteins prior to extensive and expensive murine biodistribution experiments. Abbreviations BNCT boron neutron capture therapy BSA bovine serum albumin HSA human serum albumin ICP-AES inductively coupled plasma-atomic emission spectroscopy Native-PAGE native polyacrylamide gel electrophoresis Acknowledgments This research was supported by awards from the Robert A. Welch Foundation (AI1292), Research Corporation (CC3913), and Texas State
University-San Marcos. The authors wish to thank Dr. William Bauer of the Idaho National Engineering and Environmental Laboratories (INEEL, Idaho Falls, ID) for the total boron analysis by ICP-AES and Professor M. Frederick Hawthorne for his generous gift of Na3[B20H19]. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jinorgbio.2013.03.007.
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