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Abstract

VH1-like phosphatase Z (VHZ) has proved to be a promising target for the development of therapeutics for the treatment of human cancers. Here, we report the first example for a successful application of structure-based virtual screening to identify the novel small-molecule inhibitors of VHZ. These inhibitors revealed high potencies with the associated IC₅₀ values ranging from 3 to 20 µM and were also screened for having desirable physicochemical properties as a drug candidate. Therefore, they deserve consideration for further development by structure-activity relationship studies to optimize inhibitory and anticancer activities. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of VHZ are discussed in detail.

Keywords

cheminformatics computational chemistry enzyme assays medicinal chemistry

Introduction

Protein tyrosine phosphatases (PTPs) are often involved in mitogenic signal transduction for cell growth and differentiation. VH1-like phosphatase Z (VHZ), which is also called dual-specificity protein phosphatase 23 (DUSP23), catalyzes the dephosphorylation reactions of phosphotyrosyl and phosphoseryl/threonyl residues in specific protein substrates.¹ VHZ is one of the smallest phosphatases with 150 amino acids and contains the minimal set of secondary structure elements conserved in all known phosphatases.² Recently, it was demonstrated that the activation and the knockdown of VHZ would, respectively, have an effect of enhancing and retarding the progression of the cell cycle associated with carcinogenesis from the G1 to S phase.³ Furthermore, VHZ was shown to be overexpressed in multiple human primary cancers, including breast cancer.³ These series of experimental observations indicated that VHZ should be a promising target for the development of therapeutics for human cancers.

VHZ can dephosphorylate p44-ERK1 (MAPK3)⁴ and enhance the activation of c-Jun N-terminal kinase (JNK) and p38 (MAPK14) stimulated by several osmotic stress, and it functions as an enhancer of osmotic stress.⁵ X-ray crystal structures of human VHZ were reported in complex with the substrate analogue, D-malate.⁶ The presence of structural information about the nature of the interactions between the active-site residues of VHZ and the small-molecule ligand can make it a plausible task to design the potent inhibitors that may develop into an anticancer medicine. Indeed, the usefulness of such structural information has been well appreciated in designing the potent and selective small-molecule inhibitors of various PTPs.⁷ Nonetheless, the discovery of VHZ inhibitors has lagged behind the biochemical and structural studies. To the best of our knowledge, no small-molecule VHZ inhibitor has been reported so far in the literature or in patents.

In the present study, we aim to identify the potent VHZ inhibitors by means of a structure-based drug design protocol involving virtual screening with docking simulations and an in vitro enzyme assay. The characteristic feature that discriminates our virtual screening approach from the others lies in the implementation of an accurate solvation model in calculating the binding free energy between VHZ and its putative inhibitors, which would have an effect of increasing the hit rate in the enzyme assay.⁸ We find in this study that the docking simulations with the improved binding free energy function can be a useful computational tool for elucidating the activities of the identified inhibitors, as well as for enriching the chemical library with molecules that are likely to have desired biological activities.

Materials and Methods

The atomic coordinates in the X-ray crystal structure of human VHZ⁶ (PDB entry: 2IMG) were selected as the receptor model in the virtual screening with docking simulations. The docking library for VHZ, comprising about 240 000 compounds, was constructed from the latest version of the chemical database distributed by InterBioScreen (Moscow, Russia) containing approximately 477 000 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 candidates⁹ and without reactive functional group(s). All of the compounds included in the docking library were then processed with the CORINA program (Molecular Networks, Erlangen, Germany) to generate their 3D atomic coordinates, which was followed by the assignment of Gasteiger-Marsilli atomic charges. We used the AutoDock program (AutoDock, La Jolla, CA)¹⁰ in the virtual screening of VHZ inhibitors because the outperformance of its scoring function over those of the others had been shown in several target proteins.¹¹ Docking simulations with AutoDock were carried out in the active site of VHZ to score and rank the compounds in the docking library according to their calculated binding affinities.

In the actual docking simulation of the compounds in the docking library, we used the empirical AutoDock scoring function improved by the implementation of a new solvation model for a compound. The modified scoring function has the following form:

\begin{array}{l} Δ G_{b i n d} = W_{v d W} \sum_{i = 1} \sum_{j = 1} (\frac{A_{i j}}{r_{i j}^{12}} - \frac{B_{i j}}{r_{i j}^{6}}) + W_{h b o n d} \sum_{i = 1} \sum_{j = 1} E (t) (\frac{C_{i j}}{r_{i j}^{12}} - \frac{D_{i j}}{r_{i j}^{10}}) \\ + W_{e l e c} \sum_{i = 1} \sum_{j = 1} \frac{q_{i} q_{j}}{ε (r_{i j}) r_{i j}} + W_{t o r} N_{t o r} + W_{s o l} \sum_{i = 1} S_{i} (O_{i}^{\max} - \sum_{j > i} V_{j} e^{- \frac{r_{i j}^{2}}{2 σ^{2}}}), \end{array}

where 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 a putative inhibitor, 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. 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,¹² respectively, whereas O^max _i stands for the maximum atomic occupancy. In the calculation of the molecular solvation free energy term in equation (1), we used the atomic parameters developed by Kang et al.¹³ 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.⁸ Indeed, the superiority of this modified scoring function to the previous one was well appreciated in recent studies for virtual screening of kinase and phosphatase inhibitors.^14,15

The gene for residues 1 to 150 of VHZ was cloned into Escherichia coli expression vector pET28a. Cells containing the vector were induced with 0.2 mM IPTG and grown further at 18 °C for 16 h. Cell pellets were resuspended in the lysis buffer containing 50 mM Tris-HCl pH (7.5), 0.5 M NaCl, 1% PMSF, and 5% glycerol. The resuspension was then lysed by sonication on ice. The His-tagged VHZ protein was purified by an Ni-NTA agarose column and dialyzed against 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 50 mM NaCl, and 5% glycerol. In total, 142 compounds selected from the virtual screening were evaluated for their in vitro inhibitory activity against the purified VHZ. These enzyme assays were performed by monitoring the extent of hydrolysis of 6,8-difluoro-4-methyl-umbelliferyl phosphate (DiFMUP) with the spectrofluorometric assay. The purified VHZ (100 nM), DiFMUP (10 µM), and a candidate inhibitor were incubated in the reaction mixture containing 20 mM Tris-HCl (pH 8.0), 0.01% Trition X-100, and 5 mM dithiothreitol (DTT) for 20 min. The resulting fluorescence was measured at 460 nm by using the PerkinElmer 2030 instrument (PerkinElmer, Waltham, MA). To determine the IC₅₀ values of the inhibitors, their inhibitory activities were measured in duplicate at the concentrations of 0.0, 0.1, 0.5, 1.0, 2.0, 5.0, 10, 20, and 50 µM to obtain the dose-response curve fits. IC₅₀ values of the inhibitors were then determined from direct regression analysis using the four-parameter sigmoidal curve as implemented in the SigmaPlot program (Systat Software, Chicago, IL).

Results and Discussion

Of the 240 000 compounds screened with docking simulations, 150 compounds were selected as virtual hits. In total, 142 were available from the compound supplier and were tested for inhibitory activity against VHZ by an in vitro enzyme assay. As a result, we identified seven compounds that inhibited the catalytic activity of VHZ by more than 50% at the concentration of 20 µM, which were selected to determine the IC₅₀ values. The chemical structures and the inhibitory activities of the newly identified inhibitors are shown in Figure 1 and Table 1 , respectively. The dose-response behaviors of the inhibitors measured to obtain their IC₅₀ values are presented in the supplementary data. We note that compounds 1–7 exhibit high inhibitory activities, five of which are found to inhibit the enzymatic activity of VHZ at a micromolar level. These high inhibitory activities are surprising because the identification of a potent PTP inhibitor has been very difficult because the flat and shallow active site prevents the inhibitors from being fully accommodated in the active site. Therefore, the inhibitors found in this study seem to be capable of establishing strong multiple hydrogen bonds in the active site of VHZ. Benzoate, benzene-1,2-diol, 5-imino-5,6-dihydro-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole, nitrobenzene, and 2-hydroxybenzoate moieties in 1, 2 and 5, 3, 4, 6, and 7 are respectively expected to serve as an effective surrogate for the phosphotyrosine group in substrates of VHZ. To the best of our knowledge, compounds 1–7 have not been reported as a phosphatase inhibitor so far, and no additional biological activity was found at least in the two most public chemical databases, ChEMBL and PubChem. Besides the high potencies, these inhibitors were also screened for having desirable physicochemical properties as a drug candidate. Therefore, they deserve consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory and anticancer activities.

Figure 1.

Chemical structures of the newly identified VHZ phosphatase inhibitors.

Table 1.

IC₅₀ Values (in µM) against Various Dual-Specificity Protein Phosphatases and the Fluorescence Interference (FI) in Enzyme Assay for 1–7

Compound	VHZ	MKP2	PRL-1	PRL-2	FI, %
1	3.5	>100	>100	32.6	2.1
2	3.7	15.0	60.3	57.7	4.5
3	4.0	21.0	37.2	27.6	14.2
4	4.6	2.29	21.0	>100	5.5
5	7.7	22.3	4.7	3.7	21.4
6	19.6	>100	>100	57.0	0.3
7	20.6	55.4	>100	>100	16.4

To confirm the inhibitory activity of a phosphatase inhibitor, one must disprove the possibility that the inhibitor would fluorescently interfere with the assay signal. This validation study started with the incubation of an enzymatic reaction mixture for 20 min without the treatment of an inhibitor to obtain the maximum fluorescence signal. At this point, the enzymatic reaction was terminated by the treatment of vanadate, which was followed by the addition of each inhibitor in the reaction mixture to determine its effect of fluorescence interference. The extent of the decrease in the fluorescence signal was then measured at the inhibitor concentration of 10 µM after being incubated for 5 min. As can be seen in the last column of Table 1, the fluorescence signals appear to be reduced by only 0.3% to 21.4% due to the interference effects from the inhibitors. These small interference effects indicate that the major contribution to the decrease in fluorescence signal in enzyme assays should come from the inhibition of enzymatic activity rather than from the role of the fluorescence quencher played by the inhibitors.

Because selectivity has been one of the most important issues in the development of phosphatase inhibitors, we determined the inhibitory activities of 1–7 for some other phosphatases, including MKP2, PRL-1, and PRL-3. As can be seen in Table 1 , most of the identified VHZ inhibitors reveal lower potencies for the three DUSP phosphatases than for VHZ, although 4 and 5 also appear to be potent inhibitors of MKP2 and the two PRL isoforms, respectively. In particular, 1, 6, and 7 are found to be almost inactive against two of the three other DUSPs. Thus, the inhibitors 1–7 seem to bind in the active site of VHZ in a stronger fashion than in those of the other structurally similar DUSPs. The selectivities of the identified inhibitors also indicate that they would impair the enzymatic activity of VHZ through the nonbond interactions in the active site instead of making a covalent bond with the side-chain thiolate ion of the catalytic cysteine residue contained in common in most DUSPs.

To obtain structural insight into the inhibitory mechanisms of the identified VHZ inhibitors, we investigated their binding modes in the active site in a comparative fashion. The active site of VHZ comprises a distinctive HCXXGXXRS(T) signature motif, which is known as the PTP loop and is characteristic of cysteine-dependent PTPs. The PTP loop includes the conserved catalytic Cys95 that plays the role of nucleophile to attack the phosphorous atom in the catalytic dephosphorylation of pTyr/Thr/Ser substrates. The lowest-energy conformations of 1–7 in the active site gorge of VHZ are shown in the supplementary data. The results of these docking simulations are self-consistent in the sense that the functional groups of similar chemical characters are placed in similar ways with comparable interactions with the protein groups. As revealed by the superposition of the docked structures, for example, the hydrophilic moieties that serve as a surrogate for the substrate phosphotyrosine group point toward the PTP loop, including the catalytic cysteine residue (Cys95), whereas the hydrophobic groups stay close to the WPD loop located above the active site. These common features in the calculated binding modes indicate that a potent VHZ inhibitor should include an effective surrogate for the substrate phosphotyrosine group and simultaneously the hydrophobic groups for binding to the WPD loop. To examine the possibility of the allosteric inhibition of VHZ by the identified inhibitors, we carried out docking simulations with the grid maps for the receptor model so as to include the entire part of VHZ. However, the binding configuration in which an inhibitor resides outside the active site was not observed for any of the new inhibitors. These results support the possibility that the inhibitors would impair the catalytic activity of VHZ through specific binding in the active site.

We next turn to the identification of the detailed interactions responsible for the stabilization of the newly found inhibitors in the active site. The calculated binding mode of 1 in the active site of VHZ is shown in Figure 2 . The inhibitor appears to be in a close contact with Cys95-Arg101 and Asp65-Pro68, which belong to the PTP loop comprising the active site and the WPD loop, respectively. We note that one of the terminal carboxylate oxygens of 1 accepts two hydrogen bonds from the side-chain guanidium ion of Arg101 and the backbone amidic nitrogen of Leu97. The other carboxylate oxygen of 1 is found to be stabilized also by the two hydrogen bonds with the backbone amidic groups of Phe99 and Gly100 established in a bifurcated form. Apparently, these four hydrogen bonds involving the carboxylate group are expected to play the role of anchoring the inhibitor in an appropriate pose in the active site. It is also worth noting that the carboxylate carbon of 1 resides in the vicinity of the side-chain thiolate group of Cys95 with the associated interatomic distance of 3.2 Å. Judging from the proximity to the catalytic cysteine residue (Cys95) and the formation of the multiple hydrogen bonds at the bottom of the active site, the benzoate moiety of 1 seems to be an effective surrogate for the phosphorylated tyrosine group in the substrates of VHZ. An additional hydrogen bond is found to be established between one of the aminocarbonyl oxygens of 1 and the side-chain amide group of Gln138, which should also be a significant binding force to stabilize the VHZ-1 complex. The inhibitor 1 can be further stabilized in the active site by the hydrophobic interactions of its nonpolar groups with the side chains of Phe66, Pro68, and Leu97. Thus, the overall structural features derived from docking simulations indicate that the micromolar inhibitory activity of 1 should stem from the multiple hydrogen bonds and hydrophobic interactions established simultaneously in the active site of VHZ. Due to the low molecular weight of ~297, 1 is expected to serve as a good inhibitor scaffold from which much more potent inhibitors can be derivatized. This derivatization should be made in such a way to maximize hydrophobic interactions with nonpolar residues because 1 includes many polar groups and establishes the hydrogen bond interactions in a well-optimized form. The introduction of additional polar groups would have an effect of lowering the inhibitory activity due to the increase in desolvation cost for enzyme-inhibitor complexation.

Figure 2.

Calculated binding mode of 1 in the active site of VH1-like phosphatase Z (VHZ). Carbon atoms of the protein and the ligand are indicated in cyan and green, respectively. Each dotted line indicates a hydrogen bond.

Figure 3 shows the lowest-energy binding mode of 2 in the active site of VHZ. The binding mode of 2 is similar to that of 1 in that the roles of the hydrogen bond donor with respect to the terminal benzene-1,2-diol moiety of 2 are played by the side-chain guanidium ion of Arg101 and the backbone amidic groups of Leu97, Phe99, and Gly100 at the bottom of the active site. Both of the terminal –OH moieties of 2 appear to stabilize the side-chain thiolate ion of Cys95 by donating a hydrogen bond. These structural features of the hydrogen-bond interactions indicate that the benzene-1,2-diol group of 2 can serve as an effective surrogate for the phosphotyrosine group in the substrates of VHZ. A stable hydrogen bond is also established between the side-chain amidic group of Gln138 and the imine moiety of 2, which should also play a role in stabilizing the 5-imino-5,6-dihydro-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one ring at the top of the active site. Hydrophobic interactions in the VHZ-2 complex appear to be established also in a similar fashion to those in the VHZ-1 complex: Its nonpolar groups form the van der Waals contacts with the side chains of Phe66, Pro68, and Leu97. The similarities in structural features of both the hydrogen bond and van der Waals interactions in VHZ-1 and VHZ-2 complexes can thus be invoked to explain the similar inhibitory activities of 1 and 2 ( Table 1 ).

Figure 3.

Calculated binding mode of 2 in the active site of VH1-like phosphatase Z (VHZ). Carbon atoms of the protein and the ligand are indicated in cyan and green, respectively. Each dotted line indicates a hydrogen bond.

In conclusion, we have identified seven novel inhibitors of VHZ 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 have desirable physicochemical properties as a drug candidate and reveal a high potency with IC₅₀ values ranging from 3 to 20 µM. Therefore, each of the newly discovered inhibitors deserves consideration for further development by SAR studies to optimize inhibitory and anticancer activities. Detailed binding mode analyses with docking simulations show that the inhibitors can be stabilized in the active site of VHZ by the simultaneous establishment of multiple hydrogen bonds with the active-site residues and van der Waals contacts with the hydrophobic side chains of the residues in the WPD loop.

Footnotes

Acknowledgements

We thank Prof. Olson at Scripps Research Institute for providing the AutoDock program.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2011-0022858), and a Hanyang University internal grant.

Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at .

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Supplementary Material

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Identification of Potent VHZ Phosphatase Inhibitors with Structure-Based Virtual Screening

Abstract

Keywords

Introduction

Materials and Methods

Results and Discussion

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Supplementary Material