Discovery of Low Micromolar Dual Inhibitors for Wild Type and

Article pubs.acs.org/jcim

Discovery of Low Micromolar Dual Inhibitors for Wild Type and L1196M Mutant of Anaplastic Lymphoma Kinase through StructureBased Virtual Screening Saemina Shin,† Shinmee Mah,‡ Sungwoo Hong,*,‡ and Hwangseo Park*,† †

Department of Bioscience and Biotechnology & Institute of Anticancer Medicine Development, Sejong University, 209 Neungdong-ro, Kwangjin-gu, Seoul 143-747, Korea ‡ Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS) & Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea S Supporting Information *

ABSTRACT: Although anaplastic lymphoma kinase (ALK) is involved in a variety of malignant human cancers, the emergence of constitutively active mutants with drug resistance has rendered it difficult to identify the new medicines for ALKdependent cancers. To find the common inhibitors of the wild type ALK and the most abundant drug-resistant mutant (L1196M), we performed molecular dockingbased virtual screening of a large chemical library in parallel for the two target proteins. As a consequence of augmenting the accuracy of the docking simulation by implementing a sophisticated hydration free energy term in the scoring function, 12 common inhibitors are discovered with the inhibitory activities ranging from submicromolar to low micromolar levels. The results of the binding free energy decomposition indicate that the biochemical potency of ALK inhibitors can be optimized by reducing the dehydration cost for binding to the receptor protein as well as by strengthening the interactions with amino acid residues in the ATPbinding site. The newly identified ALK inhibitors are found to have a little higher inhibitory activity for the L1196M mutant than for the wild type due to the strengthening of the hydrogen bond interactions in the ATP-binding site. Of the 12 common inhibitors, 2-(5-methyl-benzooxazol-2-ylamino)-quinazolin-4-ol (3) is anticipated to serve as a new molecular scaffold to optimize the biochemical potency because it exhibits low micromolar inhibitory activity with respect to both the wild type and L1196M mutant in spite of the low molecular weight (292.3 amu).

1. INTRODUCTION

Accordingly, a great deal of effort has been devoted to the discovery of small-molecule ALK inhibitors as recently reviewed in a comprehensive fashion.11,12 These scientific endeavors led to the identification of structurally diverse ATPcompetitive ALK inhibitors including pyrrolo[2,1-f ][1,2,4]triazine,13,14 4-arylaminopyrimidine,15 pyrimidine-2,4-diamine,16,17 piperidine carboxamide,18 and 3,5-diamino-1,2,4triazole urea19 as the key structural elements. However, many ALK-dependent cancer cells tend to make the ALK mutants that can retain the enzymatic activity in a constitutive fashion by impeding the inhibitor binding in the ATP-binding site.20−23 This makes the cancer cells resistant to anticancer medicines developed as ALK inhibitor. The emergence of drug resistance has prevented the first-generation anti-NSCLC drugs such as crizotinib from being used in clinical treatment, which motivated the development of common inhibitors of the wild type and the drug-resistant mutants of ALK. Among the drugresistant variants, the L1196M mutant is most frequently identified in crizotinib-resistant patients.24 Furthermore, it has

Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase comprising 1620 amino acids and a member of the insulin receptor superfamily.1 It can be activated into the dimeric form due to ligand binding in the extracellular region, which has the effect of autophosphorylating the tyrosine residues in the activation loop (A-loop) of the intracellular kinase domain.2,3 The hyperactivation of ALK often facilitates the proliferation, differentiation, and antiapoptosis of normal cells by inducing the activation of STAT, Ras/MAPK, PI3K/ Akt, and PI3K/PLC-γ pathways.4 The aberrant ALK activity may thus be responsible for cancer pathogenesis. Indeed, the involvement of constitutively active fusion proteins of ALK have been implicated in various human cancers including nonsmall cell lung cancer (NSCLC),5 inflammatory myofibroblastic tumor,6 anaplastic large cell lymphoma,7 squamous cell carcinoma,8 and diffuse large B-cell lymphoma.9 Furthermore, the impairment of ALK activity proved to be effective in the treatment of some cancers.10 This confirmed the usefulness of ALK as a therapeutic target for the discovery of new anticancer drugs. © 2016 American Chemical Society

Received: January 20, 2016 Published: March 25, 2016 802

DOI: 10.1021/acs.jcim.6b00026 J. Chem. Inf. Model. 2016, 56, 802−810

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Journal of Chemical Information and Modeling

acceptor atom such as the backbone aminocarbonyl oxygen within the generally accepted distance limit (3.5 Å) for a hydrogen bond of moderate strength. In a similar way, the side chains of lysine were assumed to be positively charged unless the terminal amine moiety resided in proximity to a hydrogenbond donor. The same criterion was also applied for assigning the protonation states of His residues. Prior to performing the molecular docking for virtual screening, we constructed a chemical library of ALK comprising about 310,000 synthetic compounds and natural products using the latest version (September 2015) of the database distributed by InterBioScreen Ltd. This started from the screening of all 520,000 compounds included in the original chemical database according to Lipinski’s “Rule of Five” to select only the molecules with the physicochemical properties that should be satisfied by potential drug candidates.40 To remove redundancy in the docking library, structurally similar compounds with Tanimoto coefficients larger than 0.75 were clustered into a single representative. All the filtrated molecules were then processed with the CORINA program41 to construct 3D atomic coordinates, which was followed by the atomic charge calculations with Gasteiger−Marsilli method.42 The modified version of the AutoDock program43 was used for virtual screening of common inhibitors of the wild type and L1196M mutant ALK because its outperformance had been demonstrated in various target proteins.44,45 Although ligand solvation effects are critically important in protein−ligand association,46 the scoring function in the current version of AutoDock included a crude form of a hydration free energy term that contained no more than six atom types to cope with protein atoms only. Therefore, a new scoring function was established to enhance the accuracy of virtual screening by substituting the more appropriate hydration free energy function than the original one. This modified scoring function (ΔGbaq) can be expressed as follows.

been difficult to deactivate the L1196M mutant with ALK inhibitors because the substitution of Met for Leu has the effect of lowering the binding affinity due to an increase in steric hindrance in the ATP-binding site. Despite such difficulty, some common inhibitors of the wild type and the drug-resistant mutants of ALK were developed by virtue of various significant synthetic challenges.25−30 The accumulation of information about the structural features of ALK-inhibitor interactions for the wild type and various mutants has also shed a new light on combatting the drug resistance of ALK-dependent cancers.31−36 Most ALK inhibitors were nonetheless discovered through the structural modifications of the known inhibitor scaffolds and the highthroughput screening of chemical libraries without resorting to the sophisticated computational methods useful for establishing rationales for the inhibitory activities. In particular, our understanding of the mechanisms by which the inhibitors of drug-resistant ALK mutants work lags behind that of the wild type ALK inhibitors, although dual inhibition is necessary to cope efficiently with ALK-dependent cancers. The present study was undertaken to find new common inhibitors of the wild type and L1196M mutant ALK by means of structure-based virtual screening in parallel with the two target proteins. Molecular docking for virtual screening has often been unsuccessful due to the imperfections in the protein−ligand binding free energy function to score the putative inhibitors.37 This leads to a poor correlation between the computational predictions and experimental measurements of biochemical potencies. The remarkable feature that discriminates the present virtual screening procedures from the others concerns the implementation of a sophisticated hydration free energy term in computing the binding affinities of putative inhibitors with respect to the wild type and L1196M mutant ALK. This augmentation of the scoring function seems to culminate in the accuracy enhancement in virtual screening due to the effective reflection of the dehydration cost for a putative inhibitor to form the enzyme−inhibitor complex.38 It will be demonstrated that virtual screening with the improved scoring function is useful for enriching the chemical library targeted toward the dual inhibition of the wild type and L1196M mutant ALK.

⎛A Bij ⎞ ij ΔGbaq = WvdW ∑ ∑ ⎜⎜ 12 − 6 ⎟⎟ + Whbond rij ⎠ i = 1 j = 1 ⎝ rij ⎛C

∑ ∑ E(t )⎜⎜ i=1 j=1

2. METHODS Two receptor models were prepared from the X-ray structure of the wild type ALK in complex with CH5424802 (PDB code: 3AOX)31 and that of the L1196M mutant in complex with crizotinib (PDB code: 2YFX)32 to conduct docking simulations for virtual screening of the common inhibitors from a large commercial chemical library. The coordinates of missing residues in the original X-ray structures were generated through homology modeling with the latest version of the MODELER program.39 In this structural building, we adopted an optimization algorithm involving the conjugate gradient method and molecular dynamics simulations to minimize the violations of spatial restraints. To construct the all-atom models for receptor proteins, hydrogens were added to each atom of the wild type and L1196M mutant ALK. The protonation states of titrable Asp, Glu, His, and Lys residues were determined carefully based on the hydrogen-bonding patterns revealed in the original crystal structures of ALK-inhibitor complexes. For example, the side chains of Asp and Glu residues were considered neutral if either of their carboxylate oxygens pointed toward a hydrogen-bond

ij 12 r ⎝ ij

−

qq Dij ⎞ ⎟ + Welec ∑ ∑ i j 10 ⎟ rij ⎠ i = 1 j = 1 ε(rij)rij

+ WtorNtor + Wsol ∑ Si(Occimax − i=1

2 ij

2

∑ Vje−r /2σ ) j≠i

(1)

Here, the weighting factors WvdW, Whbond, Welec, Wtor, and Wsol refer to van der Waals interaction, hydrogen bond, electrostatic interaction, torsional motion, and hydration free energy of a putative inhibitor, respectively. Also, rij represents the interatomic distance, and Aij, Bij, Cij, and Dij are given by the well depth and the equilibrium distance associated with the potential energy function for ALK and ligand atoms. AMBER force field parameters were adopted to calculate the van der Waals interaction energy term as implemented in the original AutoDock program. The hydrogen bond term has the additional weighting factor (E(t)) to describe the angledependent directionality. To compute the electrostatic interaction energy between ALK and a putative inhibitor, we used the sigmoidal function with respect to rij proposed by Mehler et al. as the distance-dependent dielectric constant.47 In the torsional term, Ntor indicates the number of rotatable bonds in the probe molecule. In the hydration free energy term, Si and Vi indicate the atomic solvation energy per unit volume and the 803


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Journal of Chemical Information and Modeling fragmental atomic volume, respectively, while Occimax represents the maximum occupancy of each atom in the putative inhibitor.48 The atomic parameters developed by Chung and Park were used to calculate the hydration free energies of all the probe molecules in the docking library because they had shown a good performance in SAMPL4 blind prediction challenge.49,50 The implementation of this new hydration free energy term is likely to enhance the accuracy of the protein−ligand binding free energy function because the biochemical potency of an inhibitor with many polar moieties tends to be overestimated when the ligand solvation effects are underestimated.38 Using the ligand scoring function in eq 1, docking simulations were carried out in the ATP-binding sites of the wild type and L1196M mutant ALK to score and rank the putative inhibitors in the order of the calculated binding affinities. Because the current research interest was focused on the discovery of common inhibitors, only the compounds included simultaneously in top 500 virtual hits with respect to the two target proteins were selected for biochemical evaluations. Enzyme inhibition assays were performed at Ambit Bioscience Corp. (San Diego, CA, U.S.A.) using the broad and potent kinase inhibitor (staurosporine) as the reference. The putative inhibitors with percent of control (POC) values lower than 20 at 100 μM with respect to both the wild type and L1196M mutant ALK were selected to measure the corresponding IC50 values. These IC50 determinations were carried out based on radiometric kinase assays ([γ-33P]ATP) at Reaction Biology Corp. (Malvern, PA, U.S.A.). At given inhibitor concentrations, biochemical potencies were monitored with the percent remaining kinase activity as compared to the vehicle reaction caused by dimethyl sulfoxide. IC50 values were then calculated through the curve fits with PRISM program (GraphPad Software).

Figure 1. Flowchart for the discovery of common inhibitors of the wild type and L1196M mutant ALK through the two parallel virtual screenings and high-throughput enzyme assays.

for 1−12 was retrieved from the publicly accessible databases such as ChEMBL and PubChem. With respect to excluding the possibility of false positive assay results, compounds 1−12 were confirmed not to contain any substructures included in pan assay interference compounds (PAINS).52 As shown in Table 1, all 12 inhibitors exhibit good inhibitory activities at low micromolar level against both the wild type and L1196M mutant. They seem to deserve further development to optimize the biochemical potency and anticancer activity through the structure−activity relationship (SAR) analysis because they were also screened computationally for possessing the physicochemical properties that should be satisfied by drug candidates. For example, the calculated LogP values of 1−12 range from 1.55 to 4.82 as desirable for a drug candidate. In regard to future development of anticancer medicine, compounds 3 and 10 are anticipated to serve as promising molecular scaffolds from which many more potent inhibitors can be generated by chemical synthesis because of the molecular weights (MWs) lower than 300 amu. We now address the energetic features associated with binding of the newly identified ALK inhibitors in the ATPbinding sites of the wild type and L1196M mutant. Table 2 lists the binding free energies of 1−12 calculated in the gas phase (ΔGbgas) and in aqueous solution (ΔGbaq) as compared to their hydration free energies (ΔGsol). Because the ΔGbaq value of an inhibitor with respect to the target protein is well approximated as the difference between the corresponding ΔGbgas and ΔGsol values, the relative significances of the two energy ingredients can be estimated by decomposition analysis of ΔGbaq. Standard deviations (SDs) of ΔGsol values of 1−12 and those of ΔGbgas values for the wild type and L1196M mutant amount to 2.88, 3.07, and 3.06 kcal/mol, respectively. The similarity of SD values implies that ΔGbgas and ΔGsol terms contribute comparably to ΔGbaq and exemplifies the necessity of the dehydration free energy term to accurately estimate the biochemical potencies of ALK inhibitors. It can be argued in this respect that the chemical modifications to generate more potent ALK inhibitors should be made so as to maximize the ALK−inhibitor interactions and concurrently to minimize the dehydration cost for the new inhibitor to be bound in the ATPbinding site.

3. RESULTS AND DISCUSSION A total of approximately 310,000 molecules were virtually screened with docking simulations around the ATP-binding regions of the wild type and L1196M mutant ALK to select 500 top-scoring virtual hits for each target protein. These two sets contained 118 compounds in common as depicted in Figure 1, all of which were commercially available from the compound supplier (InterBioScreen Ltd., Chernogolovka, Russia). These 118 putative common inhibitors were then tested for the presence of inhibitory activity against the wild type and L1196M mutant ALK at 100 μM by means of the highthroughput binding assays.51 As a consequence of combining the virtual screening and high-throughput binding assays, 12 compounds were identified as common inhibitors of the wild type and L1196M mutant. All these inhibitors revealed good biochemical potency with respect to both target proteins at low micromolar level. The structures and inhibitory activities of the 12 common inhibitors are summarized in Figure 2 and Table 1, respectively. It appears to be a common structural feature for 1−12 that hydrogen-bonding groups and nonpolar aromatic moieties are situated in the middle and at the end of molecular frameworks, respectively. It is therefore likely that both hydrogen bond and hydrophobic interactions would serve as a significant binding force for the common inhibitors to be stabilized in the ATPbinding sites of the wild type and L1196M mutant. None of these molecules has been reported as an ALK inhibitor in the literature or patents. Furthermore, no other biological activity 804


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Figure 2. Chemical structures of 12 common inhibitors of the wild type and L1196M mutant ALK.

Table 1. Calculated LogP (cLogP) and IC50 (μM) Values of 12 Common Inhibitors for Wild Type and L1196M Mutant ALKa

Table 2. Calculated Binding Free Energies of 1−12 in Gas Phase (ΔGbgas) and Aqueous Solution (ΔGbaq) with Respect to Wild Type and L1196M Mutant ALK Together with Hydration Free Energies (ΔGsol)a

IC50 inhibitor 1 2 3 4 5 6 7 8 9 10 11 12 a

cLogP 4.82 3.34 4.37 3.19 3.43 3.89 1.55 4.45 4.78 3.25 3.24 2.40

wild type 1.9 3.9 4.0 4.6 5.6 7.5 6.7 7.6 9.9 8.3 14.1 21.9

ΔGbgas

L1196M mutant 0.8 1.9 2.3 5.7 4.1 4.3 5.4 5.9 7.7 11.1 6.9 12.1

cLogP values were extracted from the ISIS/BASE program. a

The calculated binding free energies of 1−12 compare reasonably well with the experimentally measured biochemical potencies. For example, ΔGbaq values of 1−12 for the L1196M mutant appear to be a little lower than those for the wild type, which is consistent with their relatively higher inhibitory activity with respect to the former than the latter except for 4 and 10 (Table 1). The decrease in ΔGbaq with the change in receptor protein from the wild type to L1196M mutant is basically attributed to a decrease in ΔGbgas in the sense that each common inhibitor has to have a unique ΔGsol value. Most common inhibitors found in this study are therefore expected to bind more tightly in the ATP-binding site of L1196M

ΔGbaq

inhibitor

wild type

L1196M

1 2 3 4 5 6 7 8 9 10 11 12 SD

−27.2 −27.6 −19.8 −23.6 −26.6 −23.4 −24.2 −21.5 −21.8 −18.4 −21.6 −27.1 3.07

−28.1 −28.4 −21.1 −24.1 −26.8 −23.7 −24.7 −22.2 −22.4 −18.8 −22.3 −27.9 3.06

ΔG

sol

−14.5 −15.3 −8.2 −11.4 −14.9 −11.8 −12.6 −10.1 −11.0 −7.8 −11.2 −17.3 2.88

wild type

L1196M

−12.7 −12.3 −11.6 −12.2 −11.7 −11.6 −11.6 −11.4 −10.8 −10.6 −10.4 −9.8 0.85

−13.6 −13.1 −12.9 −12.7 −11.9 −11.9 −12.1 −12.1 −11.4 −11.0 −11.1 −10.6 0.92

All energy data are given in cal/mol.

mutant than in that of the wild type. Because ΔGbgas values exhibit a trend to become more negative in going from 3 and 10 (with MWs lower than 300) to 4, 6−9, and 11 (with MWs ranging from 300 to 400) and furthermore to 1, 2, 5, and 12 (with MWs higher than 400) irrespective of the receptor protein, it can be concluded that the ALK−inhibitor interactions get stronger as the molecular size of inhibitor increases. Nonetheless, ΔGbaq values of 3 and 10 become comparable to those of the larger inhibitors by virtue of the relatively high ΔG sol values, further exemplifying the 805


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Figure 3. Comparative view of the binding modes of 1−7 in the ATP binding sites of (a) wild type and (b) L1196M mutant of ALK. Carbon atoms of 1−7 are indicated in green, cyan, black, gray, pink, orange, and violet, respectively. The positions of residues 1196, Met1199, Gly-loop, and A-loop are also indicated.

Figure 4. Calculated binding modes of 1 in the ATP-binding sites of (a) wild type and (b) L1196M mutant of ALK. Carbon atoms of ALK and 1 are indicated in cyan and green, respectively. Dotted lines indicate hydrogen bonds.

importance of the ligand dehydration term in the scoring function. Consistent with the lowest biochemical potency, 12 is found to have the highest ΔGbaq values with respect to both wild type and L1196M mutant. When ΔGbgas, ΔGsol, and ΔGbaq values of 12 are compared to those of 1 and 2, it becomes apparent that the lowering of inhibitory activity stems from the increased stabilization in water instead of the destabilization in the ATPbinding site. Although ΔGbgas value of 12 decreases significantly due to the presences of additional hydrogen-bonding sulfonamide and −NH2 groups, the strengthening of the interaction in the ATP-binding site is insufficient to compensate for the increased dehydration cost. It is thus confirmed from the decomposition analysis of ΔGbaq that the enhancement of biochemical potency of an ALK inhibitor cannot be achieved by simply strengthening the interactions in the ATP-binding site without proper control of the dehydration cost. Seeking structural insight into the low micromolar activities of the newly identified ALK inhibitors, their interaction patterns in the ATP-binding sites of the wild type and L1196M mutant were addressed in the comparative fashion. Figure 3 shows the lowest-energy binding modes of 1−7 obtained from docking simulations with the modified scoring function. Although 1−7 appear to be stabilized in a little different positions of the ATP-binding site, their binding modes reveal self-consistencies in the context of interactions with the two receptor proteins. For example, at least one polar moiety of each inhibitor points toward the backbone atoms of Met1199 to establish a hydrogen bond in the middle of the gatekeeper region, whereas the terminal nonpolar aromatic rings reside in

close proximity to the activation loop (A-loop) or glycine-rich phosphate-binding loop (Gly-loop). The requirement of the interactions with Met1199, Gly-loop, and A-loop for good biochemical potency was also appreciated in X-ray crystallographic studies of the wild type and L1196M mutant ALK in complex with potent ATP-competitive inhibitors.32,33 As a check for the presence of a peripheral binding pocket that can accommodate inhibitors other than the ATP-binding site, additional docking simulations of 1−12 were performed with respect to the wild type and L1196M mutant using the extensive 3D grid maps to cope with the whole kinase domain of ALK. The clustering analyses of the results for 100 docking runs for all 12 inhibitors reveal that the binding modes accommodating 1−12 in the ATP-binding pocket represent the most stable and concurrently the most probable cluster with the occupation of more than 86% of total population. The preference of 1−12 for the ATP-binding pocket may serve as a support for the possibility that they would impair the kinase activity of ALK in the ATP-competitive fashion. To establish the rationales for good biochemical potencies of the newly found common inhibitors, their calculated binding modes in the ATP-binding site were analyzed in detail. The lowest ΔGbaq conformations of 1 with respect to the wild type and L1196M mutant ALK are compared in Figure 4. It appears to be a common structural feature in ALK-1 and L1196M-1 complexes that the carbonyl oxygen on the six-membered ring of 1 serves as a hydrogen-bond receptor with respect to the backbone amidic nitrogen of Met1199. This is consistent with the previous X-ray crystallographic finding that the capability to form the hydrogen bond with a backbone atom of Met1199 would be requisite for an ALK inhibitor to be bound tightly in 806


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Figure 5. Calculated binding modes of 3 in the ATP-binding sites of (a) wild type and (b) L1196M mutant of ALK. Carbon atoms of ALK and 3 are indicated in cyan and green, respectively. Dotted lines indicate the hydrogen bonds.

Figure 6. Time evolutions of the hydrogen-bond distances between 3 and backbone groups of the activation loop and gatekeeper site of (a) wild type and (b) L1196M mutant ALK.

the ATP-binding pocket.31−36 Hydrophobic interactions are established also in a similar fashion in ALK-1 and L1196M-1 complexes: the nonpolar groups of 1 form the van der Waals contacts with the side chains of Leu1122, Val1130, Ala1148, Ile1171, Val1180, Leu/Met1196, Leu1256, and Phe1271. These interactions seem to be necessary for the good inhibitory activity of 1 because most of such nonpolar residues reside on the Gly-loop, gatekeeper region, or A-loop. Overall, the simultaneous establishment of multiple hydrogen bonds and van der Waals interactions in the ATP-binding site may be invoked to explain the low micromolar and submicromolar biochemical potencies of 1 with respect to the wild type and L1196M mutant ALK, respectively. With respect to the good inhibitory activity, it is also noteworthy in both ALK-1 and L1196M-1 complexes that the terminal 1,3-dioxolane moiety of 1 establishes two hydrogen bonds with the side-chain butylammonium ion of Lys1150 and the backbone amidic nitrogen of Phe1271 that resides in the middle of the DFG (Asp1270-Phe1271-Gly1272) motif in the A-loop. Because these hydrogen bonds are located at the interface between the Gly-loop and A-loop, they seem to play the role of anchor for 1 to be bound tightly in the ATP-binding site. Despite the overall similarity in the binding modes of 1 toward the wild type and L1196M mutant, some remarkable changes in the hydrogen-bonding features are also observed in the L1196M-1 complex in association with the Met substitution for Leu that has the effect of increasing the steric hindrance in the ATP-binding site. For example, both N−H···O hydrogen bonds of Lys1150 and Phe1271 with the 1,3-dioxolane group of 1 appear to get stronger with the change in receptor protein from the wild type to L1196M mutant. The associated N···O

distances contract from 2.98 and 3.24 Å in the ALK-1 complex to 2.63 and 3.06 Å in the L1196M-1 complex, respectively. This strengthening of the two hydrogen bonds can be invoked to elucidate a little higher biochemical potency of 1 for the L1196M mutant than for the wild type because no additional significant difference between ALK-1 and L1196M-1 complexes is observed in the hydrophobic and the other hydrogen bond interactions. It is remarkable to note that the inhibitory activities of 3 with respect to the wild type and L1196M mutant are close to those of 1 (Table 1) despite the substantial MW decrease from 431 to 291 amu. In this regard, 3 is anticipated to serve as a new promising molecular scaffold from which many potent inhibitors can be synthesized. Figure 5 compares the most stable and probable binding modes of 3 in the ATP-binding sites of the wild type and L1196M mutant. We see in both ALK-3 and L1196M-3 complexes that the central amine moiety flanking the two fused aromatic rings and the neighboring nitrogen atom on the oxazole ring of 3 donates and receives a hydrogen bond to the backbone aminocarbonyl oxygen of Glu1197 and from the backbone amidic nitrogen of Met1199, respectively. An additional hydrogen bond is established between the −OH moiety on 1,3-diazine ring of 3 and the backbone aminocarbonyl oxygen of Gly1269. This seems to be also a significant binding force to stabilize ALK-3 and L1196M3 complexes because it resides just ahead of the DFG motif. As in the cases of N−H···O hydrogen bonds in ALK-1 complex, the O−H···O hydrogen bond in the ALK-3 complex appears to get stronger due to the replacement of the receptor with the L1196M mutant, which could be inferred from the shortening of the associated O···O distance from 3.08 Å in ALK-3 to 2.95 807


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decomposition analysis of the calculated binding free energy data that the potency of an ALK inhibitor could be optimized not only by strengthening the interactions with amino acid residues in the ATP-binding site but also by reducing the dehydration cost for binding to the target protein. Consistent with the previous experimental findings, the hydrogen bonds with backbone atoms of Met1199 were found to be a major binding force for the inhibitors to be stabilized in the ATPbinding sites of the wild type and L1196M mutant. The results of extensive docking simulations indicated that the inhibitors could bind a little more tightly to the L1196M mutant than to the wild type by strengthening the hydrogen bonds in the ATPbinding site due to the increased steric hindrance at residue 1196. This differential binding mode with respect to the wild type and L1196M mutant is expected to serve as a primary criterion that should be taken into account in designing the new potent common inhibitors.

Å in the L1196M-3 complex. On the other hand, 3 occupies a smaller volume than 1 in the ATP-binding site and thereby stays more distant from the side chains of Ile1171, Leu/ Met1196, and Phe1271. This inevitably culminates in the weakening of the hydrophobic interactions in the ALK-3 and L1196M-3 complexes, which is in turn responsible for a little lower biochemical potency of 3 than 1. The low micromolar inhibitory activity of 3 for the wild type and L1196M mutant can thus be attributed to the combined effects of the formation of three hydrogen bonds and the reduced dehydration cost. To assess the major binding forces in the ALK-3 and L1196M-3 complexes derived from docking simulations, we carried out 10.2 ns molecular dynamics simulations with the explicit solvent model using the latest version of the AMBER program.53 Figure 6 illustrates the time evolutions of the hydrogen-bond distances of 3 with respect to the backbone groups of the activation loop (aminocarbonyl oxygen of Gly1269) and gatekeeper site (aminocarbonyl oxygen of Glu1197 and amidic −NH moiety of Met1199). All three hydrogen bonds appear to be dynamically stable in both ALK-3 and L1196M-3 complexes with the time-averaged distances ranging from 1.97 to 2.12 Å. Furthermore, the O···H−O (Gly1269), O···H−N (Glu1197), and N−H···N (Met1199) hydrogen bonds are maintained at least for 93%, 97%, and 99% of simulation time when the distance limit for defining a hydrogen bond is assumed to be 2.5 Å. The maintenances of all three hydrogen bonds with dynamic stability are consistent with tight binding of 3 in the ATP-binding sites of the wild type and L1196M mutant of ALK, which can in turn be invoked to explain its micromolar-level inhibitory activity. With respect to improving the biochemical potency of 3 by synthetic modifications, we note that 3 is incapable of establishing a hydrogen bond with the side-chain butyl ammonium ion of Lys1150 in both the ALK-3 and L1196M3 complexes. This may apparently be one of the reasons for the lower inhibitory activity of 3 than 1. Hence, the introduction of a hydrogen-bond accepting moiety at the terminal phenyl ring of 3 would have the effect of raising the binding affinity by inducing an additional hydrogen bond in the ATP-binding site. It is also worth remarking that 3 stays in the ATP-binding site without significant hydrophobic interactions with the nonpolar side chains of Ile1171, Leu/Met1196, and Phe1271 that appeared to establish van der Waals contacts with 1. Further potency enhancement is therefore anticipated when a hydrophobic moiety would be substituted at the terminal phenyl ring of 3. The introduction of a nonpolar moiety has the advantage over the hydrogen-bonding groups in the context that the former has little effect on the dehydration cost, while the substitution of the latter may lead to a decrease in binding affinity caused by the increased stabilization in water. Using 3 as the new molecular scaffold, we plan to perform SAR investigations to identify the new common inhibitors of the wild type and L1196M mutant ALK with low nanomolar biochemical potency.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00026. SMILES codes of 1−12. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +82-2-3408-3766. Fax: +82-2-3408-4334. E-mail: [email protected] (S.H.). *Telephone: +82-42-350-2811. Fax: +82-42-350-2812. E-mail: [email protected] (H.P.). Author Contributions

The manuscript was written through contributions of all authors. S.S. designed experiments, performed calculations and experiments, analyzed data, and wrote the paper. S.M. performed experiments and analyzed data. S.H. designed calculations and experiments, analyzed data, and wrote the paper. H.P. designed calculations and experiments, analyzed data, and wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education, Science and Technology (NRF-2011-0022858) and by Institute for Basic Science (IBS-R010-G1).

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REFERENCES

(1) Iwahara, T.; Fujimoto, J.; Wen, D.; Cupples, R.; Bucay, N.; Arakawa, T.; Mori, S.; Ratzkin, B.; Yamamoto, T. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 1997, 14, 439−449. (2) Stoica, G. E.; Kuo, A.; Aigner, A.; Sunitha, I.; Souttou, B.; Malerczyk, C.; Caughey, D. J.; Wen, D.; Karavanov, A.; Riegel, A. T.; Wellstein, A. Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin. J. Biol. Chem. 2001, 276, 16772−16779. (3) Stoica, G. E.; Kuo, A.; Powers, C.; Bowden, E. T.; Sale, E. B.; Riegel, A. T.; Wellstein, A. Midkine binds to anaplastic lymphoma kinase (Alk) and acts as a growth factor for different cell types. J. Biol. Chem. 2002, 277, 35990−35998.

4. CONCLUSIONS By means of structure-based virtual screening with molecular docking, we have identified 12 common inhibitors of the wild type and L1196M mutant ALK with biochemical potencies ranging from low micromolar to submicromolar levels. To enhance the possibility of finding the actual inhibitors, we modified the scoring function by implementing a proper molecular hydration free energy term. It was found from 808


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Discovery of Low Micromolar Dual Inhibitors for Wild Type and

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