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Keywords:

  • X-ray;
  • crystallography;
  • drug design;
  • fragment screening;
  • purple acid phosphatase;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with Ki values in the 30–60 μm range. Ligand efficiency values are 0.39–0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3–2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

Purple acid phosphatases (PAP) are metalloenzymes that hydrolyse phosphate esters and anhydrides, generally at low pH values (1). The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II/III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP). In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.

A number of biological roles for PAP have been proposed. These include (i) the transport of iron from the mother to the developing foetus during gestation (14); (ii) bone resorption in osteoclasts (evidence for this activity is derived from the effect of PAP on osteoclasts that were cultured on cortical bone slices (15) and from the observation that the addition of anti-PAP antibodies to osteoclasts reduces in vitro resorption of bone (16)); (iii) an involvement in the inflammatory response of antigen presenting cells (17); and (iv) catalysis of Fenton’s reaction, thus promoting the generation of reactive oxygen species (ROS). ROS can be targeted to destroy collagen and other proteins in resorbing osteoclasts. Such a mechanism of action may be important in tissue remodelling (18). In plants, it is speculated that PAP may play a role in mobilizing organic phosphates in the soil for the germinating embryo (19). There is also speculation that PAP in plants may be important in peroxidations, especially when the plant experiences oxygen starvation (20).

In mice, where the gene encoding for PAP is selectively disrupted, an increase in bone mineralization reflecting mild osteopetrosis is observed (21), while in transgenic mice with increased PAP activity in osteoclasts, a decrease in trabecular bone density is recorded, consistent with the onset of osteoporosis (22). This data, taken together with the abundance of PAP in osteoclasts, provide substantial evidence to implicate PAP as a target for the development of chemotherapeutic treatments for osteoporosis. There is also the possibility that potent PAP inhibitors will find additional clinical applications in the treatment of conditions that are also associated with elevated levels of this enzyme in sera, for example, AIDS encephalopathy (23), Guacher’s disease (24), hairy cell leucemia (25), Alzheimer’s disease (26) and bone metastases (27,28).

A number of non-specific inhibitors of PAP have been identified, including fluoride (29), and several tetrahedral inorganic oxyanions, including phosphate, arsenate, vanadate, tungstate and molybdate (4,30). Another class of PAP inhibitors are modified phosphotyrosine-containing tripeptides. These molecules have IC50 values in the mid-micromolar range (31). More recently, α-alkoxynaphthylmethylphosphonic acids and acyl derivatives of α-aminonaphthylmethyl phosphonic acid have also been shown to be PAP inhibitors with Ki and IC50 values in the low micromolar range (32,33). These molecules were designed as the derivatives of 1-naphthylmethylphosphonic acid, a PAP inhibitor previously reported by Schwender et al. (34).

A recent advance in drug discovery is the use of fragment-based screening (FBS) to discover new enzyme inhibitor leads (35). The advantage of this technique is that small fragment molecules (MW < ∼ 200 Da) may bind to the active site of an enzyme with high ligand efficiency. These molecules can then be elaborated to produce more potent and specific inhibitors. Here, we use this approach to identify new PAP inhibitor leads. Initially we used a spectrophotometric assay to screen a 500-compound fragment library to find potential hits. Fragments that showed the most potent inhibition were subjected to more detailed kinetic analysis, molecular modelling and X-ray crystallographic studies to identify their mode of binding.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Enzyme preparation and purification

Purple acid phosphatases from red kidney bean (rkbPAP) was purified following a previously published protocol (6). Briefly, red kidney beans (Phaseolus vulgaris) were ground in a Waring blender and suspended in 0.5 m sodium chloride. The suspension was filtered through a muslin cloth, followed by ethanol fractionation and ammonium sulphate precipitation. Purification was achieved by ion-exchange chromatography using a CM-cellulose column followed by gel filtration on a Sephadex S-300 column. The resulting preparation was concentrated to 23.8 mg/mL using a Millipore Amicon centrifugal concentrator and stored at 4 °C in 0.5 m sodium chloride. Pig PAP was extracted from the uterine fluid of a pregnant sow and purified by ion-exchange chromatography using CM-cellulose followed by gel filtration on a Sephadex G-75 (36). Purified pig PAP was concentrated to 8.1 mg/mL and stored at −20 °C in 100 mm acetate buffer at pH 4.9. Protein concentrations were determined by measuring the absorbance at 280 nm using extinction coefficients of 1.41 for a 1 mg/mL solution (28.6 μm) of pig PAP and 2.1 for a 1 mg/mL solution (9.1 μm) of rkbPAP. SDS-PAGE analysis showed that the enzymes were >95% pure.

Enzyme kinetics

Inhibition assays for both pig PAP and rkbPAP were performed in 96-well 400-μL multi-titre plates using a UV/Vis multiplate spectrophotometer. Prior to its use in kinetic assays, pig PAP was fully reduced with 0.77 mmβ-mercaptoethanol for 10 min at 37 °C. Kinetic measurements were taken at pH 4.9 (0.1 m sodium acetate buffer with 25% DMSO) at 25 °C using para-nitrophenol phosphate (pNPP) as substrate at concentrations of 1, 3, 5, 7.5, 10 and 12.5 mm. The rate of product formation was measured at λ = 405 nm (ε = 343/M/cm). The concentration of enzyme used was 20.9 nm for pig PAP and 7.4 nm for rkbPAP, while the concentration of the inhibitors ranged from 50 to 300 μm. The data were analysed by nonlinear regression using the general inhibition equation (eqn 1) (37) and the program WinCurveFit (Kevin Raner software).

  • image(1)

In this equation, Kic and Kiuc represent the equilibrium dissociation constants for competitive and uncompetitive inhibitor binding, respectively, while v, Vmax, Km, [S] and [I] represent the rate of product formation, the maximum rate of product formation, the Michaelis constant, the substrate concentration and the inhibitor concentration, respectively.

Fragment-based screening

The 500-fragment compound library was purchased from ™. Each compound was dissolved in DMSO to a concentration of 200 mm and then diluted with the assay buffer to 1 mm, followed by the addition of enzyme. Inhibition was measured as the reduction in PAP activity towards pNPP in the presence of fragment compound. The most promising leads were subsequently tested in inhibition assays as described in the preceding paragraph.

Crystallization and inhibitor soaking

Crystallization of rkbPAP was achieved using previously determined conditions (38). Once crystals had reached a size of ∼0.1 mm in all three dimensions, an equivalent volume of cryoprotectant containing the inhibitor was added to the hanging drop. This solution consisted of 0.1 m sodium citrate pH 5.0, 0.1 m lithium chloride, 25% polyethylene glycol 3350, 20% isopropyl alcohol, 10% glycerol and 4 mm of inhibitor. This was introduced ∼4 days before the data collection. For cryoprotection, a crystal from the above preparation was soaked in cryoprotectant (containing the inhibitor) for 10 s before placing it in the cryostream (100 K).

Data collection and structure determination

X-ray data collection was undertaken on the MX2 beamline at the Australian Synchrotron, Victoria, Australia using Blu-Ice (39). The program XDS (40) was used to index and integrate the data, and Scala in CCP4 was used to scale and merge the data (41). Although the same conditions as described previously (38) were used for crystallization, a new space group (P3121) and lattice parameters were obtained. Hence, molecular replacement, using PHASER (42), was undertaken to solve the phase problem. Coordinates for the starting model were obtained from the protein data bank (PDB code 4KBP) (43). The fitting of two dimers in the asymmetric unit yielded a Z-score of 72. After refinement of the atomic coordinates, occupancies and individual B factors followed by the placement of solvent molecules and fitting of carbohydrates and ligands, the final Rfree was in the range of 0.21–0.23 for all three structures (Table 1). Model refinements were undertaken using PHENIX (44) and Refmac5 (45). Model building was undertaken using Coot (46). Figures were produced using PyMOL unless otherwise stated. Coordinates and structure factors for the three complexes have been deposited in the Protein Data Bank with access codes 4DHL, 4DSY and 4DT2. A list of contacts between the inhibitor and the enzyme is provided in Table S1.

Table 1.   Three inhibitors identified by fragment-based screening and their docking score Thumbnail image of

Docking studies

Docking studies were undertaken with Molegro Virtual Docker (MVD) (47) using the MolDock SE algorithm. The ligand search space was confined to an 8 Å sphere originating from the metal centre of PAP. For rkbPAP, the receptor coordinates used were those of the crystal structure of the corresponding rkbPAP-fragment complex with the inhibitor omitted. For human PAP, the coordinates used were those of recombinant human PAP in complex with phosphate (PDB code 1WAR) (8) with the phosphate anion omitted from the active site. Water molecules were removed from all coordinate files prior to docking.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Purple acid phosphatases has been identified as an attractive target for the development of chemotherapeutics to treat osteoporosis (16,21,22). The purpose of this study was to identify new inhibitor leads by screening a fragment library of compounds – the philosophy behind this approach is to discover new leads (i.e., small molecules) that can then be elaborated to generate potent and specific PAP inhibitors. Although the desired target for such inhibitors is human PAP, the enzymes from pig or red kidney bean (which have similar active sites) were used here for initial testing because of their ready availability.

Fragment-based screening

A number of inhibitors of PAPs have been discovered but most of these do not possess drug-like properties and so are not suitable for further modifications that may convert them into chemotherapeutic agents. We therefore embarked upon on a FBS approach using a library of small molecules that possess drug-like properties to discover novel compounds as inhibitor leads of PAP. The initial screening was based on a plate assay where all 500 compounds were tested for inhibitory activity at a concentration of 1 mm against pig PAP. Three compounds, CC27209, CC24201 and MO07123 (Table 1; Figure S1), inhibit pig PAP by >70% at that concentration. Subsequently, the modes of inhibition and magnitudes of inhibition constants were determined (Table 2). The three compounds are all competitive inhibitors of pig PAP with Ki values in the 30–60 μm range. The three compounds also inhibit rkbPAP in a similar manner (Table 2; Figure S1), an observation that suggests that despite some structural variations between the two enzymes (discussed in detail later), the overall interactions between inhibitor and enzyme are largely conserved. This interpretation is also in agreement with previous studies using phosphonate derivatives as inhibitors for pig and rkbPAP – in each case, the observed modes and magnitudes of inhibition were similar in the two enzymes (32,33).

Table 2.   Kinetic data for Maybridge compounds against reduced pig purple acid phosphatases (PAP) and red kidney bean PAP at pH 4.9
  Pig PAPRed kidney bean PAP
K icm) K iucm)Ligand efficiencya (competitive) K icm) K iucm)Ligand efficiencya (competitive)
  1. NSE, no significant effect.

  2. aLigand efficiency is calculated as the binding free energy (in kcal/mol) divided by the number of heavy atoms.

CC2420153 ± 19223 ± 1810.3937 ± 16NSE0.40
CC2720959 ± 21NSE0.4443 ± 14359 ± 3360.46
MO0712342 ± 14NSE0.4033 ± 10NSE0.41

Structure and sequence comparisons of plant and animal PAPs

An obvious caveat in the preceding section is the use of pig and rkbPAP instead of the human enzyme. As pointed out earlier, the rationale for this approach is that the sufficient amounts of pure human PAP required for screening a large number of compounds cannot be obtained easily (48). Furthermore, as it is an aim of this study (as a necessary step towards visualizing enzyme–inhibitor interactions) to obtain crystal structures of a PAP with a bound fragment, it is not practical to use human PAP. In contrast, pig and rkbPAP can be obtained easily in large quantities, and in particular, plant PAPs have been shown to crystallize readily, thus facilitating the screening and visualizing process (38,49). Although pig PAP is available in large quantities and crystallization using previously identified conditions (9) has been successful, it has not been possible to obtain crystals of this enzyme where access to the active site is not blocked by a phosphate anion, and attempts to soak the fragments into pre-grown pig PAP crystals to replace the phosphate anion have been unsuccessful. In an attempt to further substantiate the use of both pig and rkbPAP as valid models for human PAP, we carried out a detailed sequence and structure comparison, focusing on the vicinity of the active site of these three enzymes. A partial structure-based sequence alignment of plant, human and pig PAP shows that the metal-binding ligands, as expected from previous studies (9,15,50,51), are completely conserved across the three species and that the amino acids that make up the surface of the active site are identical for the human and pig enzymes, with only three differences in rkbPAP (Figure 1). The two differences closest to the metal centre occur at positions R170 and Y365 (numbering according to the rkbPAP sequence), both of which are occupied by phenylalanine side chains in the animal enzymes (i.e., F244 and F56 in pig PAP). The most significant difference between the animal and plant enzymes lies in the deletion of a short loop in the sequence of rkbPAP relative to the animal PAPs (Figure 1). In animal PAPs, this region of the polypeptide chain forms a surface-exposed loop that consists of 21 amino acids (i.e., N144-X145–163-T164 in pig PAP). For the human enzyme, this loop has been shown to be susceptible to proteolytic cleavage, resulting in an increase in activity of up to tenfold (including increased efficiency towards ATP as a substrate depending on the protease used for cleavage) (15,47,52). In rkbPAP, the repression loop is replaced by a single amino acid R258, which comes from the adjoining subunit of the dimer.

image

Figure 1.  Structure-based sequence alignment for residues in the active site region of plant and animal purple acid phosphatases (PAPs). Residues that have a red arrow coordinate directly to the metal ions. Residues that are located on the surface of the active site are highlighted in purple. The residues in the green box form an extended loop in human and pig PAPs. When the peptide bond residues A161 and R162 are proteolytically cleaved (green arrow) in human PAP, the enzyme is ten times more active. In plant PAP, this loop is missing, but R258 from the other subunit of the dimer reaches across to occupy this space.

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The residues that contribute to the exposed surface of the active site and are located within the repression loop in the animal enzymes are N142, S143, D144 and Q149 (numbering according to the human PAP sequence). Thus, the active sites of the two animal enzymes are virtually indistinguishable. When comparing the plant and animal enzymes, the positions of the metals and the residues that are close to the metal centres are conserved, but some differences do exist at the periphery. It is therefore postulated that because of the small size of the fragments and similarity of the active site environment surrounding the metal centre, the binding modes of the fragments would be conserved between rkbPAP and animal PAPs. The structural similarities, the ready availability of rkbPAP and the fact that rkbPAP crystallizes easily make this enzyme suitable for crystallographic studies of inhibitor binding.

Crystallographic investigations into the fragment binding modes

The structures of the three fragments in complex with rkbPAP were determined by X-ray crystallography to 2.3 Å for CC24201 and MO07123 and to 2.7 Å resolution for CC27209 (Table 3). The asymmetric unit in all of the structures consists of two dimers of rkbPAP. The alignment of the dimeric structure reported here onto the first reported crystal structure of rkbPAP (PDB ID: 4KBP) gave an RMSD value of 0.38 Å after superimposition of all Cα atoms, demonstrating that the overall fold is conserved across all of the structures.

Table 3.   Data collection and refinement statistics for the red kidney bean purple acid phosphatases – fragment complexes
 CC24201CC27209MO07123
  1. b R work = Σ|Fo|−|Fc|/Σ|Fo|.Rwork is calculated using 95% of the total reflections, and cRfree using the remaining 5% of the reflections. aRmerge = ΣhklΣi |Ii(hkl)−I(hkl)|/ΣhklΣi Ii(hkl) Values in () are for the highest resolution shell.

Data collection
 Temperature (K)100100100
 Resolution range (Å)19.86–2.3019.90–2.7019.89–2.30
 Total number of observations677 862420 125618 281
 Total number unique126 11577 045115 253
 Completeness (%)99.8 (99.8)99.7 (100)94.8 (96.3)
 Rmergea0.11 (0.58)0.11 (0.58)0.15 (0.55)
 Rmeas (all I+ & I−)0.13 (0.65)0.12 (0.64)0.17 (0.63)
 Rpim (all I+ & I−)0.05 (0.28)0.05 (0.27)0.07 (0.28)
 Mean I/σI14.0 (3.5)13.6 (2.9)8.4 (2.6)
 Multiplicity5.4 (5.1)5.5 (5.5)5.4 (4.5)
 Unit cell lengths (Å) = 127.71 = 299.76 = 126.76 = 298.95 = 126.10 = 297.98
 Space group P31 21 P31 21 P31 21
Refinement
 Total number of atoms16 28815 25316 359
 Number of water molecules14244321609
 Wilson B-factor (Å2)27.4645.4325.46
 Rworkb0.1580.1720.155
 Rfreec0.2060.2270.205
 RMS bonds (Å)0.0150.0170.014
 RMS angles (°)1.11.41.1
Ramachandran statistics (%)
 Favoured95.296.195.2
 Allowed4.103.804.40
 Outlier0.500.100.40

For the CC24201 complex, the inhibitor was modelled into the difference electron density (σ > 4.5) in one of the subunits (A). However, it was not possible to unequivocally fit it or any other ligand to the electron density in subunits B, C and D (the correlation of the model to 2Fo-Fc electron density was poor). For the MO07123 complex, the inhibitor was modelled in three active sites and covered by electron density (σ > 3.7) in subunits A, B and C, but for subunit D, there was only electron density for the thiazole ring and carboxylate group of the inhibitor. Variation in the binding mode of this inhibitor in the different active sites was observed, with one binding mode being more similar to that of CC24201 (i.e., the phenyl ring forms π-cation interactions with the side-chain imidazole ring of H295) and the other binding mode being more consistent with docking predictions (this is illustrated in Figure 2A). For CC27209, the inhibitor was modelled in all four active sites (σ > 5.0), but also with variation between the individual sites, differing particularly in its magnitude of interaction with R258 and in the spatial arrangement of the functional groups of the inhibitor (this variability is illustrated in Figure 2B). The binding of CC24201 in active site A, MO07123 in active site B and CC27209 in active site B is described later.

image

Figure 2.  Superimposition of subunit B (cyan) and subunit A (yellow) of the complex formed between rkbPAP and (A) MO07123 (B) CC27209. For CC27209, the major difference in inhibitor binding is observed as a stronger interaction with R258 (yellow) and a change of the conformation of the inhibitor. For MO07123, the major difference in inhibitor binding is driven by the interaction of the phenyl ring with the side chains that line the active site. One fragment (yellow) forms π-cation interactions through the phenyl ring with a nitrogen atom in the H295 side-chain imidazole ring, whereas another (cyan) forms π-cation interactions through the phenyl ring with the R258 side-chain guanidino group.

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CC24201 coordinates to the metal centre through its carboxylate group with one oxygen bridging between the metals (2.7 Å from Fe and 2.9 Å from Zn) and the other oxygen forming a hydrogen bond with the N201 side-chain amine (2.3 Å) and an electrostatic interaction with a nitrogen (3.1 Å) in the side-chain imidazole of H202 (Figure 3). This oxygen interacts only weakly with Zn (3.3 Å). The pyridine nitrogen forms a hydrogen bond (2.4 Å) with the side-chain hydroxyl of Y365 and with a water molecule (3.1 Å), and the phenyl ring forms a π-cation interaction with a nitrogen in the side-chain imidazole group of H295.

image

Figure 3.  (A) Fo-Fc electron density map (σ = 4.0) and stick representation showing the complex formed between the active site of rkbPAP (green) and CC24201 (cyan); a water molecule (red sphere) is observed bridging the inhibitor to rkbPAP. (B) Surface and stick representation showing the fit of CC24201 (cyan) in the active site of rkbPAP (yellow). (C) Surface and stick representation of the predicted binding mode of CC24201 (green) to red kidney bean purple acid phosphatases (PAP) (yellow) based on MVD docking simulations. (D) Surface and stick representation of the predicted binding mode of CC24201 (green) to human PAP (cyan) based on MVD docking simulations.

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CC27209 is bound to the metal centre with the aromatic ring being approximately co-planar to the metals and the closest heavy atom to the metal centre being a carbon from the phenyl ring (2.6 Å from Fe and 2.7 Å from Zn) (Figure 4). The hydroxyl oxygen of the inhibitor forms a hydrogen bond with the hydroxyl oxygen in the Y365 side-chain (2.5 Å), and the oxygen in the 5-membered ring is hydrogen bonded to a water molecule (2.8 Å) that bridges the inhibitor to a nitrogen atom (2.8 Å) in the side-chain imidazole of H295. The unexpected observation here is that the aromatic carbons form close contacts with the metal centre, whereas the hydroxyl group on the inhibitor faces away from the metal centre and interacts with the side-chain hydroxyl of Y365.

image

Figure 4.  (A) Fo-Fc electron density map (σ = 5.0) and stick representation showing the unusual complex formed between the active site of rkbPAP (green) and CC27209 (cyan); two water molecules (red spheres) are observed bridging the inhibitor to rkbPAP. (B) Surface and stick representation showing the fit of CC27209 (cyan) in the active site of rkbPAP (yellow). (C) Surface and stick representation of the predicted binding mode of CC27209 (green) to rkbPAP (yellow) based on MVD docking simulations. (D) Surface and stick representation of the predicted binding mode of CC27209 (green) to human purple acid phosphatases (cyan) based on MVD docking simulations.

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For MO07123, the carboxylate group binds to the metal centre in a bidentate manner, with one oxygen coordinating to Fe (1.9 Å) and to Zn (3.1 Å) (Figure 5). The second oxygen of the carboxylate group binds to Zn (2.0 Å), while forming a hydrogen bond (2.9 Å) with the N201 side-chain amide. The thiazole ring of MO07123 forms π-cation interactions with a nitrogen atom in the H202 side-chain imidazole and the phenyl ring forms close contacts with the side-chain guanidino group of R258.

image

Figure 5.  (A) Fo-Fc electron density map (σ = 3.7) and stick representation showing the complex formed between the active site of rkbPAP (green) and MO07123 (cyan); a water molecule (red sphere) is observed bridging the inhibitor to rkbPAP. (B) Surface and stick representation showing the fit of MO07123 (cyan) in the active site of rkbPAP (yellow). (C) Surface and stick representation of the predicted binding mode of MO07123 (green) to rkbPAP (yellow) based on MVD docking simulations. (D) Surface and stick representation of the predicted binding mode of MO07123 (green) to human purple acid phosphatases (cyan) based on MVD docking simulations.

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Docking studies using MVD (47) were undertaken to model the binding of the three Maybridge inhibitors to rkbPAP. It was rationalized that if the program could successfully predict the binding modes of the inhibitors to rkbPAP, then it should be useful for predictions of the binding of the inhibitors to human PAP. For the CC24201-rkbPAP complex, MVD successfully predicted the key elements of molecular recognition (i.e., the interaction of the carboxylate group with the metal centre, the interaction of the pyridine nitrogen with Y365 and the interaction of the phenyl ring with R258) but the predicted orientation of the inhibitor was slightly different to that observed in the crystal structure (RMSD = 1.56 Å for all atoms; Figure 3). For the CC27209-rkbPAP complex, MVD predicted a binding orientation similar to that of CC27209 observed in subunit A (RMSD = 0.91 Å for all atoms). This result is illustrated in Figure 4. For the rkbPAP-MO07123 complex, MVD predicted a binding orientation similar to that of MO07123 observed in subunit B (RMSD = 0.64 Å for all atoms; Figure 5).

Subsequent docking studies were undertaken to predict the binding of the three inhibitors to human PAP (Figures 3–5). Analysis shows that they can all bind to the binuclear centre in a similar fashion to that observed in the rkbPAP but that there is an ∼180° rotation when their orientations are compared to how they bind to rkbPAP. For MO07123 and CC24201, the carboxylate group is predicted to coordinate directly to the metal centre and interact with the conserved N89 and H90 (N201 and H202 in rkbPAP), whilst the thiazole sulphur or pyridine nitrogen are oriented towards a pocket in the interface between the conserved metal ligating residues N89 and H221 (N201 and H325 in rkbPAP) and the mammalian repression loop (N142, S143 and D144, spatially equivalent to R258 in rkbPAP), whereby the sulphur or nitrogen are held in place by electrostatic interactions with the side chains of these residues. The phenyl ring of both inhibitors is observed forming hydrophobic interactions with F54 (spatially equivalent to R170 in rkbPAP). For CC27209, the phenyl group is held close to the metal centre and forms π-cation interactions with H90, whilst the 5-membered ring and the dimethyl tail form hydrophobic interactions with H90. The hydroxyl group is oriented towards the same pocket discussed earlier, where it is held in place by hydrogen bonds and electrostatic interactions.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Three novel inhibitors of PAPs have been discovered by FBS. Such molecules would not have been identified by traditional methods of designing inhibitors based around the structures of the substrates, products or transition states for the enzyme. In addition, the crystal structures presented here are the first structures of any PAP with drug-like compounds (i.e., they obey Lipinski’s rule of five) bound to the active site. MO07123 and CC24201 coordinate to the binuclear metal centre of rkbPAP through their carboxylate oxygen atoms. CC27209 shows an unusual binding mode to rkbPAP, whereby the closest aromatic carbons in the phenyl ring are positioned within 3 Å of the metal centre. A ligand database search of the Protein Data Bank (PDB) and the CCDC (Cambridge Crystallographic Data Centre) via Relibase concluded that this type of ligand binding has not been observed previously in any other crystal structures of metalloenzymes.

The three compounds show markedly improved ligand efficiency for binding to pig PAP (0.39–0.44 kcal/mol per heavy atom) when compared with previously designed PAP inhibitors, including the phosphotyrosine analogues (31) and the alpha-alkoxynaphthylmethylphosphonic acids (32) (the best of these has a ligand efficiency of 0.28 kcal/mol per heavy atom). Thus, there is great potential to elaborate their structures further while still maintaining favourable drug-like properties. The crystallographic and molecular modelling studies presented here now make possible the design and development of more potent and selective PAP inhibitors that can be tested for their therapeutic value.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

The authors acknowledge funding from the Australian Research Council (DP0986292). Preliminary X-ray data were measured at the University of Queensland Remote Operation Crystallization and X-ray diffraction facility (UQROCX). The final X-ray data were obtained at the Australian Synchrotron. The assistance of Tom Caradoc-Davies and Alan Riboldi-Tunnicliffe during data collection was greatly appreciated.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
  8. Supporting Information

Figure S1. Non-linear regression curve fitting for the inhibition of (a) rkbPAP and (b) pigPAP.

Table S1. Contacts (Å) between inhibitors and rkbPAP in the crystal structures.

FilenameFormatSizeDescription
CBDD_12001_sm_FigS1-TableS1.docx246KSupporting info item

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