Identification of novel PTPRQ phosphatase inhibitors based on the virtual screening with docking simulations
© Park et al.; licensee BioMed Central Ltd. 2013
Received: 20 May 2013
Accepted: 23 August 2013
Published: 28 August 2013
Protein tyrosine phosphatase receptor type Q (PTPRQ) is an unusual PTP that has intrinsic dephosphorylating activity for various phosphatidyl inositides instead of phospho-tyrosine substrates. Although PTPRQ was known to be involved in the pathogenesis of obesity, no small-molecule inhibitor has been reported so far. Here we report six novel PTPRQ inhibitors identified with computer-aided drug design protocol involving the virtual screening with docking simulations and enzyme inhibition assay. These inhibitors exhibit moderate potencies against PTPRQ with the associated IC50 values ranging from 29 to 86 μM. Because the newly discovered inhibitors were also computationally screened for having desirable physicochemical properties as a drug candidate, they deserve consideration for further development by structure-activity relationship studies to optimize the antiobestic activities. Structural features relevant to the stabilization of the inhibitors in the active site of PTPRQ are addressed in detail.
Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of the phosphorylated tyrosine residues of protein substrates, which is a hallmark of cellular signal transduction. Because these dephosphorylation activities of PTPs have been implicated in a variety of cellular processes, abnormal PTP activities may cause various diseases including cancer, diabetes, and immune deficiencies . Total 38 members of PTP family (21 receptor-type PTPs and 17 nonreceptor-type PTPs) are known to have specificity for the phosphorylated tyrosine substrates . Most PTPs share a highly conserved catalytic module that plays a crucial role in the enzymatic action for dephosphorylation reaction. This catalytic core comprises a PTP loop (Cys-Ser-Xaa-Gly-Xaa-Gly-Arg-Thr/Ser), WPD loop, and Q loop . The invariant cysteine is located at the bottom of the PTP loop to act as a nucleophile to attack the substrate phosphorous atom, while the side-chain guanidinium ion of the conserved Arg residue in the PTP loop stabilizes the negative charge on the oxygen atoms of the substrate accumulated during the hydrolysis reaction. The conserved Gln and Asp residues in the Q and WPD loops also participate in the hydrolysis reaction of the substrate through the role of a general acid/base catalyst .
PTP receptor type Q (PTPRQ) is a member of the receptor type PTP family that contains 18 extracellular fibronectin domains and one cytoplasmic catalytic domain. Although the primary sequence of the catalytic domain of PTPRQ (PTPRQ-C) shows a high degree of similarity to those of the known PTPs, PTPRQ displays an unusual catalytic behavior. For example, it has intrinsic dephosphorylating activity for various phosphatidyl inositides (PIs) but not for phospho-tyrosine substrates . Furthermore, PTPRQ negatively regulates the proliferation and survival of cells by lowering the level of phosphoinositol phosphates (PIPs) . This characteristic dephosphorylating activity can be attributed in a large part to the difference in amino-acid sequence of the WPD loop in which the conserved aspartate is replaced with a glutamate. This hypothesis was supported by the experimental finding that the reverse mutation of glutamate to aspartate in the WPE motif caused PTPRQ to gain catalytic activity toward pTyr while losing the activity with respect to PI substrates . Four catalytically active members of the classical PTP family (PTPRQ, PTPRU, PTPD1 and HDPTP) in human genome possess the WPE motif instead of the WPD one, the structures of which have not characterized yet except for PTPRQ. PTPRQ is also homologous to a few PTPs such as phosphatase and tensin homolog (PTEN) and myotubularin phosphatases, which have the catalytic capability to dephosphorylate PIs . A line of experimental evidence showed that the loss of PTPRQ gene could lead to the hearing impairment associated with vestibular dysfunction [6–8]. It was also demonstrated that the overexpression of PTPRQ caused the differentiation of mesenchymal stem cells (MSCs) into adipocytes, which leads to the pathogenesis of obesity . This indicates that PTPRQ can serve as an effective target for development of new antiobestic drugs.
Very recently, X-ray crystal structure of human PTPRQ has been reported in complex with the sulfate ion bound in the active site as a surrogate for the phosphate group of substrates . In this structure, PTPRQ adopts an open conformation in which the residues of WPE loop stay distant from the active site. It has a flatter active site than other PTPs to accommodate the PIP substrates that are larger than the phosphorylated tyrosine. The presence of structural information about the nature of the interactions between PTPRQ and small-molecule ligands can make it a plausible task to design the potent inhibitors that may develop into an antiobestic drug. Nonetheless, the discovery of PTPRQ inhibitors has lagged behind the biological and structural studies. To the best of our knowledge, no small-molecule PTPRQ inhibitor has been reported so far in the literature at least. In this paper, we report the novel classes of PTPRQ inhibitors identified through the structure-based drug design protocol involving the virtual screening with docking simulations and in vitro enzyme assay. Computer-aided drug design has not always been successful due to the inaccuracy in the scoring function, which leads to a weak correlation between the computational predictions and experimental results for binding affinities . Therefore, we implement an accurate solvation free energy function into the scoring function to enhance the accuracy in calculating the binding free energies between PTPRQ and the putative inhibitors. This modification of the scoring function seems to improve the potential for designing the new inhibitors with high activity . It will be shown that docking simulations with the improved binding free energy function can be a useful tool for enriching the chemical library with molecules that are likely to have desired biological activities, as well as for elucidating the activities of the identified inhibitors.
3D atomic coordinates in the X-ray crystal structure of human PTPRQ in complex with the sulfate ion as a substrate analogue (PDB code: 4ikc) were selected as the receptor model in the virtual screening. After removing the crystallographic water molecules, hydrogen atoms were added to each protein atom. A special attention was paid to assign the protonation states of the ionizable Asp, Glu, His, and Lys residues in the original X-ray structure of PTPRQ. The side chains of Asp and Glu residues were assumed to be neutral if one of their carboxylate oxygens pointed toward a hydrogen-bond accepting group including the backbone aminocarbonyl oxygen at a distance within 3.5 Å, a generally accepted distance limit for a hydrogen bond of moderate strength . Similarly, the lysine side chains were assumed to be protonated unless the NZ atom was in proximity of a hydrogen-bond donating group. The same procedure was also applied to determine the protonation states of ND and NE atoms in His residues.
The docking library for PTPRQ comprising about 260,000 synthetic and natural compounds was constructed from the latest version of the chemical database distributed by Interbioscreen (http://www.ibscreen.com) containing approximately 500,000 synthetic and natural compounds. Prior to the virtual screening with docking simulations, they were filtrated on the basis of Lipinski’s “Rule of Five” to adopt only the compounds with the physicochemical properties of potential drug candidates  and without reactive functional group(s). To remove the structural redundancies in the chemical library, structurally similar compounds with a Tanimoto coefficient exceeding 0.85 were clustered into a single representative molecule. Molecular similarities were measured using the fingerprints of each molecule, generated using the Daylight software as an ASCII string of 1’s and 0’s. In this way, a docking library consisting of 260,000 compounds was constructed. All compounds included in the docking library were then processed with the CORINA program to generate their 3D atomic coordinates, followed by the assignment of Gasteiger-Marsilli atomic charges . We used the AutoDock program  in the virtual screening of PTPRQ inhibitors because the outperformance of its scoring function over those of the others had been shown in several target proteins . AMBER force field parameters were assigned for calculating the van der Waals interactions and the internal energy of a ligand as implemented in the original AutoDock program. Docking simulations with AutoDock were then carried out in the active site of PTPRQ to score and rank the compounds in the docking library according to their calculated binding affinities.
Here W vdW , W hbond , W elec , W tor , and W sol are the weighting factors of van der Waals, hydrogen bond, electrostatic interactions, torsional term, and desolvation energy of the inhibitors, respectively. r ij represents the interatomic distance, and A ij , B ij , C ij , and D ij are related to the depths of the potential energy well and the equilibrium separations between the two atoms. The hydrogen bond term has an additional weighting factor, E(t), representing the angle-dependent directionality. Cubic equation approach was applied to obtain the dielectric constant required in computing the interatomic electrostatic interactions between PTPRQ and a ligand molecule . In the entropic term, N tor is the number of rotatable bonds in the ligand. In the desolvation term, S i and V i are the solvation parameter and the fragmental volume of atom i, respectively, while O i max stands for the maximum atomic occupancy. The self-solvation parameter P i represents the extent of the stabilization of the solute atom i due to the intramolecular interactions with the rest of solute atoms. Inclusion of this self-solvation effect in the scoring function is necessary because the calculated molecular solvation free energies were shown to be inaccurate in the absence of the self-solvation term . To calculate the contribution of molecular solvation free energy term in Eq. (1), we used the atomic parameters developed by Choi and coworkers . This modification of the solvation free energy term is expected to increase the accuracy in virtual screening because the underestimation of ligand solvation often leads to the overestimation of the binding affinity of a ligand with many polar atoms .
The catalytic domain of PTPRQ (PTPRQ-C, residues 2661–2948) was subcloned into pET28a and overexpressed using Escherichia coli BL21 (DE3) strain. Cells were grown at 291 K after induction with 0.1 mM IPTG for 20 hours. His-tagged PTPRQ-C was purified by nickel-affinity chromatography. 150 compounds selected from the precedent virtual screening were evaluated for their in vitro inhibitory activity against the recombinant human PTPRQ. Initial inhibitor screening was performed by monitoring the extent of hydrolysis of p-Nitrophenyl Phosphate (pNPP) with a spectrofluorometric assay. The purified PTPRQ-C (1.5 μM), pNPP (5 mM), and a candidate inhibitor were incubated in the reaction mixture containing 50 mM Bis-Tris (pH 6.0), 2 mM dithiothreitol for 60 minutes. This enzymatic reaction was stopped with the addition of sodium hydroxide (0.5 M). The phosphatase activities were then checked by the absorbance changes due to the hydrolysis of the substrate at 405 nm. IC50 values of the inhibitors were determined from direct regression curve analysis.
Six PTPRQ inhibitors identified under the above reaction conditions were further investigated using PI(3,4,5)P3 as the substrate (Cayman Chemical). The enzymatic activity of PTPRQ was measured in 80 μL reaction mixture containing 50 mM Tris–HCl (pH 6.0), 10 mM dithiothreitol, 300 μM PI(3,4,5)P3, 100 μM inhibitor, and 1.5 μM of the purified catalytic domain of PTPRQ. The mixture was incubated for 60 minutes at 310 K, and the enzymatic reaction was stopped by the addition of 20 μL of malachite green/ammonium molybdate reagent (Bioassay systems). The absorbance was measured at 650 nm using a plate reader.
Results and discussion
IC 50 values (in μM) of 1–6 against PTPRQ
As can be seen in Table 1, the potencies of the six PTPRQ inhibitors are moderate with the IC50 values ranging from 29.9 to 85.7 μM. These modest inhibitory activities can be understood because PTPRQ has a flat and shallow active site, which makes it difficult for the inhibitors to be fully accommodated . To improve the inhibitory activities, therefore, some chemical groups should be added to 1–6 in such a way that the resulting derivatives can be stabilized not only in the active site but also in other peripheral binding pockets. Despite the modest inhibitor potencies, 1–6 deserve consideration for further development by structure-activity relationship (SAR) studies to optimize the antiobestic activities because they are structurally diverse and were computationally screened for having desirable physicochemical properties as a drug candidate.
Comparison of the inhibitory activities of 1–6 for PTPRQ and PTPRO
% inhibition at 100 μM for PTPRQ
% inhibition at 100 μM for PTPRO
71.1 ± 0.8
94.4 ± 0.5
57.7 ± 0.4
94.2 ± 1.5
50.9 ± 2.2
48.7 ± 2.1
82.6 ± 0.2
95.0 ± 3.5
62.5 ± 1.6
95.0 ± 0.8
57.0 ± 2.1
81.2 ± 0.9
In summary, we have identified six novel inhibitors of PTPRQ by applying a computer-aided drug design protocol involving the structure-based virtual screening with docking simulations under consideration of the effects of ligand solvation in the scoring function. These inhibitors are expected to have desirable physicochemical properties as a drug candidate and reveal a moderate activity with IC50 values ranging from 29.9 to 85.7 μM. Therefore, each of the newly discovered inhibitors deserves consideration for further development by SAR studies to optimize the antiobestic activities. The results of binding mode analysis with docking simulations indicate that the inhibitors can be stabilized in active site by the simultaneous establishment of multiple hydrogen bonds and van der Waals contacts.
This work was supported by the grants from National Research Foundation of Korea (NRF; 2011–0030027) and World Class Institute (WCI) Program of NRF (WCI 2009–002) funded by the Korean Government Ministry of Education Science and Technology (MEST).
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