Exploiting Blood Transport Proteins as Carborane Supramolecular Vehicles for Boron Neutron Capture Therapy

Carboranes are promising agents for applications in boron neutron capture therapy (BNCT), but their hydrophobicity prevents their use in physiological environments. Here, by using reverse docking and molecular dynamics (MD) simulations, we identified blood transport proteins as candidate carriers of carboranes. Hemoglobin showed a higher binding affinity for carboranes than transthyretin and human serum albumin (HSA), which are well-known carborane-binding proteins. Myoglobin, ceruloplasmin, sex hormone-binding protein, lactoferrin, plasma retinol-binding protein, thyroxine-binding globulin, corticosteroid-binding globulin and afamin have a binding affinity comparable to transthyretin/HSA. The carborane@protein complexes are stable in water and characterized by favorable binding energy. The driving force in the carborane binding is represented by the formation of hydrophobic interactions with aliphatic amino acids and BH-π and CH-π interactions with aromatic amino acids. Dihydrogen bonds, classical hydrogen bonds and surfactant-like interactions also assist the binding. These results (i) identify the plasma proteins responsible for binding carborane upon their intravenous administration, and (ii) suggest an innovative formulation for carboranes based on the formation of a carborane@protein complex prior to the administration.


Introduction
Boron neutron capture therapy (BNCT) is a non-invasive binary approach for the treatment of difficult-to-cure tumors such as glioblastoma multiforme or head and neck cancers [1][2][3][4][5][6]. The effectiveness of BNCT is based on the adequate and selective accumulation of 10 B in the tumor tissue (approximately 10 9 atoms/cell), followed by irradiation with a thermal neutron beam. The fission reaction that occurs upon the neutron beam irradiation on the boron atom ( 10 B[n,α] 7 Li) produces a high linear energy transfer (LET) alpha particle ( 4 He), a recoiled lithium nucleus ( 7 Li) and γ radiation (0.48 MeV). The alpha particles, which have a LET in the range of 50-230 keV/µm, also possess a very short travel distance, causing their energy to be released within the diameter of the cell (Bragg's peak). Thus, BNCT is potentially highly selective, since only the cells that have accumulated enough boron are destroyed by the radiation. Low uptake and toxicity in healthy cells and persistence in the malignant tissues are the requirements for effective BNCT agents. The first boron-containing compounds employed in BNCT were based on boric acids and their derivatives (first-generation compounds). In the 1980s, concomitantly with the improvement of neutron beam sources, BPA (boronphenylalanine) and BSH (sodium borocaptate) were extensively employed in BNCT, and were authorized for clinical trials [7].
Proteins, acting as Trojan horses [56,62,63], can also promote the accumulation of boron in the tumor tissue by taking advantage of the enhanced permeability and retention Scheme 1. Representation of the possible intermolecular interactions that can be generated between carborane and the protein-binding pockets. Proteins, acting as Trojan horses [56,62,63], can also promote the accumulation of boron in the tumor tissue by taking advantage of the enhanced permeability and retention (EPR) effect and the tumor-targeting activity of some specific proteins [64][65][66].
Among the human proteome, blood transport proteins are the natural carriers of endogenous and exogenous hydrophobic molecules in the bloodstream [67]. Blood transport proteins have well-defined binding pockets for the transport of non-soluble compounds [67]. Human serum albumin (HSA), the most abundant protein in blood, and α-fetoprotein (AFP) and afamin are responsible for the binding of fatty acids, exogenous drugs and hormones [67]; vitamin D-binding protein (DBP), thyroxine-binding globulin (TBG), transthyretin (TTR), corticosteroid-binding globulin (CBG), sex hormone-binding globulin (SHBG) and plasma retinol-binding protein (RBP) carry hormones, steroids and vitamins in plasma [67]. Other proteins, such as hemoglobin (Hb), myoglobin (Mb), hemopexin (HPX) and haptoglobin (Hp), are heme-binding proteins and they are generally responsible for the transport of oxygen and carbon dioxide [67], while cerulosplasmin (CP), serotransferrin (TF), lactotransferrin (LF) and ferritin (FT) are involved in iron/copper transport and storage [67].
Carboranes are highly hydrophobic [36,37], and their transport in blood as pristine moieties is hardly possible. The notion of using plasma proteins as potential carriers of hydrophobic species is rather straightforward. In addition, the different chemical moieties present on the protein surface can be chemically functionalized with (i) targeting tags able to improve both the cell selectivity and promote the selective uptake of the carborane@protein hybrid in cancer therapy and (ii) imaging tags to create innovative, protein-based theranostic platforms [68].
Some of the blood transport plasma proteins can also be uptaken by cancer cells via active targeting: cancer cells can selectively accumulate human serum albumin and transferrin because of the high expression levels of albumin-binding proteins (gp60 and SPARC) and transferrin receptors. BNCT was clinically approved for head and neck cancers; it is well known that many head and neck cancers overexpress SPARC and transferrin receptors [69,70]. So, the use of HSA and transferrin as carborane carriers can be potentially exploited for the development of boron neutron capture targeted therapy.
HSA and transferrin are also effective carriers for delivery across the blood-brain barrier, and the dispersion of carborane by these proteins can be an opportunity for the application of BNCT to brain cancers [71].
Therefore, in this work, a reverse docking approach was used to investigate the interaction of blood plasma with carborane. Molecular dynamics (MD) simulations, followed by molecular mechanics/generalized Born surface area (MM-GBSA) analysis, quantitatively evaluated the affinity of carborane to blood transport proteins. This approach allows for (i) the identification of the most promising proteins to act as carriers for carborane in blood, (ii) the characterization of the carborane-binding pocket(s) and (iii) the determination of the nature of the non-covalent interactions between the amino acids of the protein and the ligand.

Blood Transport Protein Structural Database
Blood transport proteins were identified following the classification of Schaller [67]. When the 3D structures of the proteins are experimentally available, they were downloaded from the Protein Data Bank [72]. When multiple PDBs were available, a representative structure was identified, using as selecting criteria the completeness of the sequence, the absence of mutations and the highest resolution. These structures, reported in Table 1, were cleaned of their co-crystallized ligands, ions and water molecules. When the 3D structure was not experimentally available, as in the case of hemopexin, the computed structure model (CSM) was used [73].

Docking and Refinement
The ortho-carborane structure was used as a representative of the closo-carboranes, due to its higher stability compared to meta-and para-carboranes. Docking calculations were carried out using the ortho-carborane structure and the blood transport proteins structural database, using the PatchDock algorithm [77], which is a valid tool for generating poses for rigid and spherical ligand molecules such as fullerenes [78][79][80][81][82][83][84][85][86], carbon nanotubes [87][88][89][90] and carboranes [55]. All the carborane@protein complexes obtained as poses of the docking procedure were then refined by minimization and MM-GBSA calculations. The first 50 poses, sorted by binding affinity, were further refined by 1 ns MD simulation followed by MM-GBSA calculations. For the complex of each blood transport protein, characterized by the highest affinity, 100 ns of MD simulations were carried out. In the case of human serum albumin (HSA), a refinement of 100 ns was carried out for all the possible binding pockets.

MD Simulations
The FF14SB force field [91], as implemented in Amber, was used to describe the proteins, while an ad hoc force field was used for the ortho-carborane, combining the GAFF force field with the parameters developed by Sarosi et al. [92]. In all simulations, the carborane@protein complexes were solvated by explicit water molecules (TIP3P model) and Na + and Cl − counterions were added to neutralize the total charge of the system. Periodic boundary condition (PBC) and the particle mesh Ewald summations, with a cut-off radius of 10.0 Å, were used during all the simulations. RMSD values of carborane@protein complexes (see Figures S1-S15) were calculated using CPPTRAJ [93].

Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) Calculations
From each MD trajectory, 1000 frames were extracted by CPPTRAJ [93] and employed as input for the MM-GBSA analysis [94]. The MM-GBSA analysis was carried out using the MM-PBSA.py module, considering an infinite cut-off in the calculation of the electrostatic and van der Waals (vdW) interactions. The generalized Born (GB) model developed by Hawkins and coworkers (GB HCT ) was used to compute the polar solvation term [95,96], whereas the non-polar solvation term was determined using solvent-accessible, surfacearea-dependent terms. A fingerprint analysis, based on the decomposition of the total binding energy on a per-residue basis, offers the possibility to identify the protein residues more responsible for the binding of the ligand.

Identification of Blood Transport Proteins as Carriers for Carborane by Virtual Screening
A protocol of reverse docking was used to identify the propensity of the blood transport proteins to bind carborane. Figure 1 shows the strength of the interaction between blood transport proteins and carboranes, as calculated by the MD simulation followed by MM-GBSA analysis. The binding between carborane derivatives and HSA [18,49,[97][98][99][100] and transthyretin [101] has already been experimentally demonstrated. [95,96], whereas the non-polar solvation term was determined using solvent-accessib surface-area-dependent terms. A fingerprint analysis, based on the decomposition of total binding energy on a per-residue basis, offers the possibility to identify the prot residues more responsible for the binding of the ligand.
The interactions of the carborane moieties with HSA were ascribed to the formation of hydrophobic interactions between the lipophilic cage of the carborane and the non-polar binding pockets of HSA.
Nevertheless, to our knowledge, the structural characterization of the carborane@HSA complex has never been reported. Following the proposed docking approach, we identified the most probable binding sites for carborane in HSA. The carborane occupies all the binding pockets that are specific for fatty acids (FA1-7), and the cleft site ( Figure 2). The highly hydrophobic nature of the carborane explains its tendency to bind in the HSA pockets where hydrophobic molecules, such as warfarine (Sudlow's 1), ibuprofen (FA6), iodipamide (cleft), oxyphenbutazone (FA5) and diazepam (FA3,4), usually bind to HSA. Two additional binding pockets for the carborane were also identified. The binding affinity computed for the carborane in each of the nine pockets is reported in Figure 2B. polar binding pockets of HSA.
Nevertheless, to our knowledge, the structural characterization of the ca borane@HSA complex has never been reported. Following the proposed docking ap proach, we identified the most probable binding sites for carborane in HSA. The carboran occupies all the binding pockets that are specific for fatty acids (FA1-7), and the cleft si ( Figure 2). The highly hydrophobic nature of the carborane explains its tendency to bin in the HSA pockets where hydrophobic molecules, such as warfarine (Sudlow's 1), ibu profen (FA6), iodipamide (cleft), oxyphenbutazone (FA5) and diazepam (FA3,4), usual bind to HSA. Two additional binding pockets for the carborane were also identified. Th binding affinity computed for the carborane in each of the nine pockets is reported i Figure 2B. After 100 ns MD simulations, the three most favorable binding pockets were foun to be FA5 (−23.5 kcal mol −1 ), Sudlow's I (−21.1 kcal mol −1 ) and the cleft region (−20.9 kc mol −1 ). Our results suggest that FA5, located in domain III of HSA, is the principal site fo carborane binding in HSA. Carborane interacts with Phe554 (−1.3 kcal mol −1 ) and Phe55 (−1.3 kcal mol −1 ) by BH-π and CH-π interactions ( Figure 3A). Leu529 (−0.9 kcal mol −1 ) an Ala528 (−0.8 kcal mol −1 ) give non-polar interactions with the carborane, while Lys525 (−0 kcal mol −1 ) is characterized by a surfactant-like interaction: the aliphatic side-chain of Ly binds the hydrophobic surface of the carborane, while its hydrophilic head interacts wit water.

Carborane@transthyretin
Transthyretin (TTR) is a homotetramer, responsible for carrying the thyroid hormone thyroxine (T4) [67]. TTR also acts as a carrier of retinol (vitamin A) through its association with retinol-binding protein (RBP) [67]. TTR is a 55 kDa protein and it comprises four subunits of 127 amino acids each [67]. The interface between two dimers creates the two binding pockets for T4. Less than 1% of TTR's T4-binding sites are occupied in blood. The docking and the MD simulations reveal that carborane occupies the T4-binding site (Figure 4A), as also suggested by experimental studies [101]. In this binding pocket ( Figure  4B,C), aliphatic amino acids such as the two Leu110 (−1.3 kcal mol −1 ) and the two Ala108

Carborane@transthyretin
Transthyretin (TTR) is a homotetramer, responsible for carrying the thyroid hormone thyroxine (T 4 ) [67]. TTR also acts as a carrier of retinol (vitamin A) through its association with retinol-binding protein (RBP) [67]. TTR is a 55 kDa protein and it comprises four subunits of 127 amino acids each [67]. The interface between two dimers creates the two binding pockets for T 4 . Less than 1% of TTR's T 4 -binding sites are occupied in blood. The docking and the MD simulations reveal that carborane occupies the T 4 -binding site ( Figure 4A), as also suggested by experimental studies [101]. In this binding pocket ( Figure 4B,C), aliphatic amino acids such as the two Leu110 (−1.3 kcal mol −1 ) and the two Ala108 (−0.9 kcal mol −1 ) give strong hydrophobic interactions with the carborane. The hydroxyl side-chain of Thr119 (−0.9 kcal mol −1 ) is involved in dihydrogen bonds with the hydridic B-H groups of the cage.

Carborane@hemoglobin
Hemoglobin (Hb) is a metalloprotein responsible for the transport of oxygen to the tissues. Hb (64 kDa) comprises four subunits, namely α and β chains of 141 and 146 residues, respectively, that pack into a tetramer α2β2 [67]. The four subunits interact via noncovalent interactions, and each chain contains a heme prosthetic group (i.e., iron protoporphyrin IX) which is responsible for the binding of molecular oxygen.
Carboranes occupy the heme-binding pockets of Hb ( Figure 5A). This is not surprising since the heme-binding pockets in proteins are usually characterized by the presence of hydrophobic/aromatic residues responsible for the binding of the hydrophobic portions of the heme group.

Carborane@hemoglobin
Hemoglobin (Hb) is a metalloprotein responsible for the transport of oxygen to the tissues. Hb (64 kDa) comprises four subunits, namely α and β chains of 141 and 146 residues, respectively, that pack into a tetramer α 2 β 2 [67]. The four subunits interact via non-covalent interactions, and each chain contains a heme prosthetic group (i.e., iron protoporphyrin IX) which is responsible for the binding of molecular oxygen.
Carboranes occupy the heme-binding pockets of Hb ( Figure 5A). This is not surprising since the heme-binding pockets in proteins are usually characterized by the presence of hydrophobic/aromatic residues responsible for the binding of the hydrophobic portions of the heme group.

Carborane@myoglobin
Myoglobin (Mb) is formed by a single polypeptide chain, folded into a globular shape. Mb shares almost identical secondary and tertiary structures with the α and β subunits of Hb and consequently binds only one heme group [67]. The fingerprint analysis ( Figure 5B,C) suggests that Phe98, Leu100 and Leu125 are the residues that mostly contribute to the stabilization of the carborane inside the heme-binding pocket: Phe98 (−1.4 kcal mol −1 ) by BH-π interactions, and Leu100 (−1.2 kcal mol −1 ) and Leu125 (−1.2 kcal mol −1 ) by hydrophobic contacts. Two polar amino acids, i.e., Ser131 (−1.2 kcal mol −1 ) and Ser101 (−1.1 kcal mol −1 ), are also important for the binding, engaging dihydrogen bonds with the carborane cage.

Carborane@myoglobin
Myoglobin (Mb) is formed by a single polypeptide chain, folded into a globular shape. Mb shares almost identical secondary and tertiary structures with the α and β subunits of Hb and consequently binds only one heme group [67].
Mb is responsible for oxygen transport and storage in muscles [67]. In analogy to Hb, our in silico approach suggested that carborane occupies the binding pocket of the heme group of Mb ( Figure 6A). His93 (−1.5 kcal mol −1 ) and His97 (−0.9 kcal mol −1 ) interact with the carborane cage by BH-π and CH-π interactions. Other three non-polar residues are responsible for the formation of the carborane@myoglobin complex via hydrophobic interactions, namely Ile99 (−1.1 kcal mol −1 ), Leu104 (−1.0 kcal mol −1 ) and Ile107 (−0.9 kcal mol −1 ). The fingerprint analysis and the close-up of the most interacting residues are reported in Figure 6B,C, respectively.
Removing the heme group [50], it is possible to use myoglobin as a drug delivery system/supramolecular host for carborane.

Carborane@myoglobin
Myoglobin (Mb) is formed by a single polypeptide chain, folded into a globular shape. Mb shares almost identical secondary and tertiary structures with the α and β subunits of Hb and consequently binds only one heme group [67].
Mb is responsible for oxygen transport and storage in muscles [67]. In analogy to Hb, our in silico approach suggested that carborane occupies the binding pocket of the heme group of Mb ( Figure 6A). His93 (−1.5 kcal mol −1 ) and His97 (−0.9 kcal mol −1 ) interact with the carborane cage by BH-π and CH-π interactions. Other three non-polar residues are responsible for the formation of the carborane@myoglobin complex via hydrophobic interactions, namely Ile99 (−1.1 kcal mol −1 ), Leu104 (−1.0 kcal mol −1 ) and Ile107 (−0.9 kcal mol −1 ). The fingerprint analysis and the close-up of the most interacting residues are reported in Figure 6B,C, respectively.
Removing the heme group [50], it is possible to use myoglobin as a drug delivery system/supramolecular host for carborane.

Carborane@cerulosplamin
Ceruloplasmin (CP) is the most important copper-carrying protein in the blood and plays an important role in iron metabolism [67]. Ceruloplasmin is also able to prevent oxidative damage to biological systems (proteins, DNA and lipids) because of its antioxidant activity. CP has a molecular weight of~132 kDa and is composed by a single peptide chain; in physiological conditions, it is a trimer [67]. Carborane binds CP at the interface of the three domains ( Figure 7A), and the formation of the complex is stabilized by the formation of a set of BH-π and CH-π interactions with aromatic residues composing the binding pocket ( Figure 7B,C), namely Phe303 (−1.5 kcal mol −1 ), Tyr992 (−1.1 kcal mol −1 ), Phe997 (−1.1 kcal mol −1 ) and Tyr986 (−0.8 kcal mol −1 ). The hydroxyl group of Thr301 (−0.7 kcal mol −1 ), instead, generates a dihydrogen bond. oxidative damage to biological systems (proteins, DNA and lipids) because of its antioxidant activity. CP has a molecular weight of ~132 kDa and is composed by a single peptide chain; in physiological conditions, it is a trimer [67]. Carborane binds CP at the interface of the three domains ( Figure 7A), and the formation of the complex is stabilized by the formation of a set of BH-π and CH-π interactions with aromatic residues composing the binding pocket ( Figure 7B,C), namely Phe303 (−1.5 kcal mol −1 ), Tyr992 (−1.1 kcal mol −1 ), Phe997 (−1.1 kcal mol −1 ) and Tyr986 (−0.8 kcal mol −1 ). The hydroxyl group of Thr301 (−0.7 kcal mol −1 ), instead, generates a dihydrogen bond.

Carborane@sex Hormone-Binding Globulin
Sex hormone-binding globulin (SHBG) is a homodimeric glycoprotein responsible for carrying testosterone, dihydrotestosterone androgens and estradiol in the bloodstream [67]. It is produced by the liver. SHBG regulates the level of these hormones, keeping their concentration stable and preventing them from being broken down too quickly. Testosterone generally binds at the N-terminal domain and estrogen at the C-terminal portion of SHBG. The location of carborane in the protein is depicted in Figure 8A, indicating that the carborane is located in the binding pocket of dihydrosterone. At this binding site (Figure 8B,C), the carborane interacts with Phe67 (−1.8 kcal mol −1 ) by BH-π and CH-π interactions and with the hydrophobic side-chains of Val105 (−0.9 kcal mol −1 ), Met107 (−1.1 kcal mol −1 ), Met139 (−0.8 kcal mol −1 ) and Leu171 (−0.8 kcal mol −1 ).

Carborane@sex Hormone-Binding Globulin
Sex hormone-binding globulin (SHBG) is a homodimeric glycoprotein responsible for carrying testosterone, dihydrotestosterone androgens and estradiol in the bloodstream [67]. It is produced by the liver. SHBG regulates the level of these hormones, keeping their concentration stable and preventing them from being broken down too quickly. Testosterone generally binds at the N-terminal domain and estrogen at the C-terminal portion of SHBG. The location of carborane in the protein is depicted in Figure 8A, indicating that the carborane is located in the binding pocket of dihydrosterone. At this binding site ( Figure 8B,C), the carborane interacts with Phe67 (−1.8 kcal mol −1 ) by BH-π and CH-π interactions and with the hydrophobic side-chains of Val105 (−0.9 kcal mol −1 ), Met107

Carborane@lactotransferrin
Transferrins (LT) are glycoproteins found in mammal fluids, such as blood (seotransferrin) and milk (lactotransferrin) [67]. Even if it is not a heme protein, transferrin is responsible for the binding of two iron atoms, their transport in the body and the regulation of the iron metabolism [67]. The residues involved in Fe(III) binding are also responsible

Carborane@lactotransferrin
Transferrins (LT) are glycoproteins found in mammal fluids, such as blood (seotransferrin) and milk (lactotransferrin) [67]. Even if it is not a heme protein, transferrin is responsible for the binding of two iron atoms, their transport in the body and the regulation of the iron metabolism [67]. The residues involved in Fe(III) binding are also responsible for the recognition of the carborane cage ( Figure 9A). In particular, as depicted in Figure 9B,C, the aromatic imidazole of Hie597 (−0.8 kcal mol −1 ) and the phenol ring of Tyr528 (−0.8 kcal mol −1 ) give BH-π and CH-π interactions with the aromatic cage of the carborane. Arg465 (−0.5 kcal mol −1 ) interacts with its guanidinium group via cation-π and BH-π interactions, while two asparagine residues, Asn640 (−0.7 kcal mol −1 ) and Asn644 (−0.6 kcal mol −1 ), are involved in the dihydrogen bond with the carborane cage.

Carborane@plasma Retinol-Binding Protein
Plasma retinol-binding protein (RBP) is a single-chain protein of ~180 amino acids. RBP carries retinol (vitamin A) in blood, binding it in a hydrophobic pocket near the center of the protein [67]. Vitamin A is crucial for vision, immune function and cellular growth and differentiation; therefore, adequate levels of RBP are essential for the correct delivery of vitamin A in the body. Carborane occupies the retinol-binding pocket in RBP ( Figure  10A); here, two methionine residues, Met88 (−1.1 kcal mol −1 ) and Met73 (−0.9 kcal mol −1 ), engage hydrophobic interactions with the carborane, while three aromatic amino acids, Tyr90 (−0.9 kcal mol −1 ), Phe135 (−0.7 kcal mol −1 ) and Phe36 (−0.7 kcal mol −1 ), give BH-π and CH-π interactions with the aromatic cage of the carborane (Figure 10B,C).

Carborane@plasma Retinol-Binding Protein
Plasma retinol-binding protein (RBP) is a single-chain protein of~180 amino acids. RBP carries retinol (vitamin A) in blood, binding it in a hydrophobic pocket near the center of the protein [67]. Vitamin A is crucial for vision, immune function and cellular growth and differentiation; therefore, adequate levels of RBP are essential for the correct delivery of vitamin A in the body. Carborane occupies the retinol-binding pocket in RBP ( Figure 10A); here, two methionine residues, Met88 (−1.1 kcal mol −1 ) and Met73 (−0.9 kcal mol −1 ), engage hydrophobic interactions with the carborane, while three aromatic amino acids, Tyr90 (−0.9 kcal mol −1 ), Phe135 (−0.7 kcal mol −1 ) and Phe36 (−0.7 kcal mol −1 ), give BH-π and CH-π interactions with the aromatic cage of the carborane (Figure 10B,C).
Plasma retinol-binding protein (RBP) is a single-chain protein of ~180 amino acids. RBP carries retinol (vitamin A) in blood, binding it in a hydrophobic pocket near the center of the protein [67]. Vitamin A is crucial for vision, immune function and cellular growth and differentiation; therefore, adequate levels of RBP are essential for the correct delivery of vitamin A in the body. Carborane occupies the retinol-binding pocket in RBP ( Figure  10A); here, two methionine residues, Met88 (−1.1 kcal mol −1 ) and Met73 (−0.9 kcal mol −1 ), engage hydrophobic interactions with the carborane, while three aromatic amino acids, Tyr90 (−0.9 kcal mol −1 ), Phe135 (−0.7 kcal mol −1 ) and Phe36 (−0.7 kcal mol −1 ), give BH-π and CH-π interactions with the aromatic cage of the carborane (Figure 10B,C).

Carborane@thyroxine-Binding Globulin
Thyroxine-binding globulin (TBG) regulates the binding of thyroxine (T 4 ) and triiodothyronine (T 3 ) [67], together with transthyretin and serum albumin. TBG has only one binding site for the thyroid hormones and has a molecular mass of~54 kDa [67]. Carborane binds TBG in the T 4 -binding pocket ( Figure 11A) superimposing to one of the aromatic groups of the endogenous ligand. At this binding site ( Figure 11B,C), Leu289 (−1.2 kcal mol −1 ) gives hydrophobic interactions with the carborane. Two charged residues, Lys290 (−1.2 kcal mol −1 ) and Arg401 (−1.0 kcal mol −1 ), interact with the carborane cage with their aliphatic side chains via surfactant-like interactions. The aromatic Tyr40, instead, arranges its phenol group to give sandwich-like BH-π and CH-π interactions (−0.8 kcal mol −1 ) with the carborane, while the hydroxyl portion of Ser43 (−0.7 kcal mol −1 ) engages a dihydrogen bond with the hydridic B-H groups of the ligand.

Carborane@thyroxine-Binding Globulin
Thyroxine-binding globulin (TBG) regulates the binding of thyroxine (T4) and triiodothyronine (T3) [67], together with transthyretin and serum albumin. TBG has only one binding site for the thyroid hormones and has a molecular mass of ~54 kDa [67]. Carborane binds TBG in the T4-binding pocket ( Figure 11A) superimposing to one of the aromatic groups of the endogenous ligand. At this binding site ( Figure 11B,C), Leu289 (−1.2 kcal mol −1 ) gives hydrophobic interactions with the carborane. Two charged residues, Lys290 (−1.2 kcal mol −1 ) and Arg401 (−1.0 kcal mol −1 ), interact with the carborane cage with their aliphatic side chains via surfactant-like interactions. The aromatic Tyr40, instead, arranges its phenol group to give sandwich-like BH-π and CH-π interactions (−0.8 kcal mol −1 ) with the carborane, while the hydroxyl portion of Ser43 (−0.7 kcal mol −1 ) engages a dihydrogen bond with the hydridic B-H groups of the ligand.

Carborane@corticosteroid-Binding Globulin
Glucocorticoids and hormones, such as cortisol and progesterone, are mainly carried in the blood by corticosteroid-binding globulin (CBG), a member of the SERPIN (serine protease inhibitor) family [67]. CBG is a single-chain protein of 383 amino acids with a weight of ~52 kDa. The carborane binds in a pocket ( Figure 12A

Carborane@corticosteroid-Binding Globulin
Glucocorticoids and hormones, such as cortisol and progesterone, are mainly carried in the blood by corticosteroid-binding globulin (CBG), a member of the SERPIN (serine protease inhibitor) family [67]. CBG is a single-chain protein of 383 amino acids with a weight of~52 kDa. The carborane binds in a pocket ( Figure 12A-C) characterized by BH-π and CH-π interactions with Phe94 (−1.2 kcal mol −1 ) and hydrophobic interactions with Leu93 and Leu374 (−0.9 kcal mol −1 ). Very interestingly, here, a new type of interaction appears, i.e., a hydrogen bond with Asp367 (−0.9 kcal mol −1 ). In fact, the negatively charged carboxylate group of Asp389 interacts with the acidic C-H groups of the carborane cage.

Analysis of the Nature of the Non-Covalent Interactions between the Amino Acids and the Carborane in the Protein-Binding Pockets
The analysis of the most interacting amino acids (the five most interacting amino acids were selected for every protein considered) allows the identification of the most recurrent amino acids, responsible for the interaction of a protein with the carborane cage (Figure 14). The driving force in the recognition of the carborane in the protein-binding pockets

Analysis of the Nature of the Non-Covalent Interactions between the Amino Acids and the Carborane in the Protein-Binding Pockets
The analysis of the most interacting amino acids (the five most interacting amino acids were selected for every protein considered) allows the identification of the most recurrent amino acids, responsible for the interaction of a protein with the carborane cage ( Figure 14).

Analysis of the Nature of the Non-Covalent Interactions between the Amino Acids and the Carborane in the Protein-Binding Pockets
The analysis of the most interacting amino acids (the five most interacting amino acids were selected for every protein considered) allows the identification of the most recurrent amino acids, responsible for the interaction of a protein with the carborane cage (Figure 14). The driving force in the recognition of the carborane in the protein-binding pockets is represented by: The driving force in the recognition of the carborane in the protein-binding pockets is represented by: (i) Hydrophobic interactions of the hydrophobic cage of the carborane with the aliphatic side chains of amino acids such as leucine (18%), methionine (9%), isoleucine (7%), valine (5%) and alanine (5%); (ii) BH-π and CH-π interactions of the hydridic B-H groups and acidic C-H groups of the carborane with aromatic amino acids such as phenylalanine (18%), tyrosine (9%) and histidine (5%).
Other important contributions arise from the: (iii) Formation of dihydrogen bonds with serine (5%), threonine (4%) and asparagine (4%), where serine and threonine use their hydroxylic moieties, while asparagine uses its amidic N-H to form the dihydrogen bonds with the hydridic B-H groups of the carborane; (iv) Surfactant-like interactions with lysine (4%) and arginine (4%) which wrap the hydrophobic cage of the carborane with their aliphatic chains, while their hydrophilic moieties interact with water; (v) Formation of classical hydrogen bonds with Asp (2.5%) where the carborane interacts with its acidic C-H groups with the carboxylate group of the amino acid.

Conclusions
A virtual screening approach allowed the identification of blood transport proteins as candidate carriers of carboranes. Human serum albumin (HSA) and transthyretin have already demonstrated their ability to bind carborane derivatives, but the structural characterization of their complexes has never been reported. Here, we identified the most probable binding sites for carborane in HSA, i.e., the FA5-binding pocket, Sudlow's I site and the cleft region. In transthyretin, as suggested also by experimental data, the carborane occupies the T4-binding site. Hemoglobin showed a higher binding affinity for carboranes than transthyretin and human serum albumin (HSA). Myoglobin, ceruloplasmin, sex hormone-binding protein, lactoferrin, plasma retinol-binding protein, thyroxine-binding globulin, corticosteroid-binding globulin and afamin have a binding affinity comparable to HSA/transthyretin. The "blind identification" of HSA and transthyretin as carboranebinding proteins (second and third position in the ranking) vouches for the accuracy of the model and validates its predictions.
Carborane shows the tendency to bind in the protein pockets where hydrophobic molecules, such as fatty acids, heme, thyroxine, androgens and retinol, are usually recognized. The driving force for the carborane binding is represented by the formation of hydrophobic interactions with aliphatic amino acids (i.e., leucine, methionine) and BH-π and CH-π interactions with aromatic amino acids (i.e., phenylalanine, tyrosine). Dihydrogen bonds, classical hydrogen bonds and surfactant-like interactions also assist the binding.
The blood transport proteins identified by the reverse docking protocol are potentially responsible to bind carborane upon their intravenous administration. These proteins can be also exploited, ex vivo, to develop boron neutron capture targeted therapy, following two different strategies: (i) the chemical conjugation of targeting (i.e., folate) and imaging (i.e., TRITC) tags on the surface of the protein to both improve the cell selectivity and promote the uptake of the carborane@protein hybrids in cancer cells, generating innovative protein-based theranostic platforms, and (ii) the selection of proteins that can be selectively uptaken by cancer cells via active targeting, such as albumin or transferrin, because their receptors are overexpressed in many cancer cells.
In addition, the dispersion of carboranes with specific proteins able to pass the BBB represents an opportunity for the development of a drug delivery system to target brain cancers with BNCT. Future works will be carried out to experimentally test the possibility to use the identified blood transport proteins as carborane carriers for BCNT.