It has been widely known that human epidermal growth factor receptor 2 (HER2) inhibitors exhibit distinct antitumor responses against HER2-positive breast cancer. To date, Lapatinib (Tykerb ®) has been approved by the U.S. Food and Drug Administration (FDA) as a reversible HER2 inhibitor for treating breast cancer. However, HER2 L755S, T798I and T798M mutations confer drug resistance to lapatinib, restricting its efficacy toward HER2-positive breast cancer. Thus, novel therapy toward mutant HER2 is highly desired. Although several irreversible HER2 inhibitors have been developed to overcome these drug resistance problems, most of them were reported to cause severe side effects. In this study, three pharmacophore models based on HER2 L755S, T798I and T798M mutant structures were constructed and then validated through receiver operating characteristic (ROC) curve analysis and Güner-Henry (GH) scoring methods. Subsequently, these well-validated models were utilized as 3D queries to identify novel irreversible HER2 inhibitors from National Cancer Institute (NCI) database. Finally, two potential irreversible HER2 inhibitor candidates, NSC278329 and NSC718305, were identified and validated through molecular docking, molecular dynamics (MD) simulations and ADMET prediction. Furthermore, the analyses of binding modes showed that both NSC278329 and NSC718305 exhibit good binding interactions with HER2 L755S, T798I and T798M mutants. All together, the above results suggest that both NSC278329 and NSC718305 can serve as novel and effective irreversible HER2 inhibitors for treating breast cancers with HER2 L755S, T798I and T798M mutants. In addition, they may act as lead compounds for designing new irreversible HER2 inhibitors by carrying out structural modifications and optimizations in future studies.
The World Health Organization (WHO) reports that breast cancer is one of the most common malignancies among women worldwide [
However, the efficacy of several anti-HER2 therapeutics, such as small molecule HER2 tyrosine kinase inhibitors (TKIs), is limited by the occurrence of several HER2 point mutations, such as L755S, T798I and T798M [
So far, due to the lack of HER2 L755S, T798I and T798M mutant structures, the strategies to develop novel irreversible HER2 inhibitors were limited to experimental methods, such as lead modification, enzyme inhibitory analysis, and cell proliferation assay [
The crystal structure of wild-type HER2-TAK285 (PDB ID: 3RCD; resolution: 3.21Å) was obtained
from the RCSB Protein Data Bank and prepared using “Prepare Protein” algorithm of Discovery Studio 2017 (DS2017). Using the “Define Site” tool of DS2017, the active site of HER2 structure was defined as a collection of several critical residues, such as Leu755, Thr798, and Cys805 etc., enclosed within a sphere of 11 Å radius around the co-crystallized ligandTAK285. Then, the co-crystalized ligand TAK285 and crystal water molecules were removed and only the wild-type HER2 structure was retained as the target template. Based on the structure of wild-type HER2, the structures of HER2 L755S, T798I and T798M mutants were constructed using the “Build Mutant” option implemented in DS2017. Subsequently, the qualities of these HER2 mutant structures were evaluated by the on-line UCLA SAVES server. All the HER2 mutant structures showed only 0.4% of residues in the disallowed region (
Due to the lack of receptor-ligand binding information, each of the known 40 irreversible HER2 inhibitors was individually docked into the active sites of HER2 L755S, T798I and T798M mutant structures and a maximum of 10 binding poses were generated for each receptor-ligand complex using CDOCKER. For each receptor-ligand complex, the generated docking pose with the best CDOCKER interaction energy was selected for pharmacophore modeling. Thus, a total of 120 docking poses were selected for the generation of structure-based pharmacophore models using the “Receptor-Ligand Pharmacophore Generation” protocol of DS2017 [18 , 19]. For each receptor-ligand binding complex, a maximum of 10 pharmacophore models with 4-6 pharmacophore features were automatically generated with default parameters. Finally, a total of 36, 36, and 38 pharmacophore models based on HER2 L755S, T798I, and T798M mutant structures were constructed, respectively.
To select the best structure-based pharmacophore models for HER2 L755S, T798I, and T798M mutant, a test 3D database comprising both 40 known irreversible HER2 inhibitors and 254 decoys (from DecoyFinder-2.0) was constructed using “Build 3D Database” (maximum 255 conformations for each compound) of DS2017. Then, the constructed pharmacophore models were used for screening the test 3D database using “Screen Library” program of DS2017 with default parameters. Subsequently, Güner-Henry (GH) scoring and receiver operating characteristic (ROC) analysis methods were adopted to evaluate the screening results of each model. Finally, three models (named as models I, II and III), which were constructed based on the HER2 L755S-compound 39, HER2 T798I-compound 10 and HER2 T798M-compound 6 complexes, respectively, displayed the best GH scores and “excellent” ROC curves and were selected for further virtual screening.
The flowchart of the overall virtual screening procedure in this study is summarized in
compounds were selected from models I, II, and III, respectively, as potential irreversible HER2 inhibitors for the further molecular docking studies (
The selected 143, 72 and 191 potential irreversible HER2 inhibitors were separately docked into the active sites of HER2 L755S, T798I and T798M mutant structures, respectively, using the CDOCKER program of DS2017 with CHARMm force field. Subsequently, the CDOCKER interaction energies (kcal・mol−1), which are also known as CDOCKER scores, were obtained from these docking studies. These docking results were further compared to those of the 40 known irreversible HER2 inhibitors, including HER2 L755S-compound 22, HER2 T798I-compound 14 and HER2 T798M-compound 19. These complexes displayed the lowest CDOCKER interaction energies among all of the 40 known irreversible HER2 inhibitors. Finally, the ones which exhibited lower CDOCKER interaction energies than those known irreversible HER2 inhibitors were selected as hit compounds.
In order to investigate the binding conformations in the active sites of the HER2 mutants, the selected hit compounds were subjected to further visual inspection based on the main criteria that the distance between Michael acceptors of each selected compound and the sulfhydryl group of HER2 Cys805 must be within the ideal range (5.5 Å) for Michael addition [
MD simulations were performed to confirm the binding stabilities of the selected potential irreversible HER2 inhibitors using the Gromacs 2016.3 software package [24 , 25]. With Amber Tools 2017, the AMBER99SB-ILDN force field [
ADMET properties of the selected potential irreversible HER2 inhibitors were obtained by uploading the simplified molecular input line entry specification (SMILES) data of each compound to the online webserver admetSAR [
In the past decades, lapatinib has been used as an effective reversible HER2 inhibitor for the treatment ofHER2-positive breast cancers. However, HER2 L755S, T798I and T798M point mutations have been reported to drive resistance toward lapatinib, thus restricting its clinical uses. Previous studies have indicated that irreversible HER2 inhibitors were able to form covalent bonds with these HER2 mutant structures through Michael addition, thus can overcome the resistance caused by these HER2 mutations. However, most of the known irreversible HER2 inhibitors were reported to cause lethal side effects due to bad ADMET properties. Therefore, there was a desire for developing novel irreversible HER2 inhibitors which are safe and potent. It this study, homology modeling, structure-based pharmacophore modeling, virtual screening, molecular docking, MD simulations and ADMET analysis were combined to discover novel irreversible HER2 inhibitors targeting HER2 L755S, T798I and T798M mutants. To the best of our knowledge, this is the first attempt to utilize structure-based approach to discovery potent irreversible HER2 inhibitors with novel structural scaffolds and desired chemical features for treating breast cancer with HER2 L755S, T798I and T798M mutants.
Structure-based pharmacophore models are generated from the structures of the protein-ligand complexes through investigating the important interactions and excluded volumes in their binding sites [
To obtain receptor-ligand docking poses, 40 known irreversible HER2 inhibitors were individually docked into the constructed HER2 L755S, T798I and T798M mutant structures using CDOCKER protocol. Then, the generated 120 docking complexes were applied to structure-based pharmacophore modeling, and a total of 36, 36, and 38 models based on HER2 L755S, T798I, and T798M mutant structures were constructed, respectively. Then, the performance of these models was further validated by GH scoring method and ROC analysis [32 - 35].
For HER2 L755S mutant, the optimal structure-based pharmacophore models (model I) was constructed based on the HER2 L755S-compound 39 complex (
For HER2 T798I mutant, the optimal structure-based pharmacophore model (model II) was constructed from HER2 T798I-compound 10 complex (
For HER2 T798M mutant, the optimal structure-based pharmacophore model was built from the HER2 T798M-compound 6 complex (
Parameter | Model I | Model II | Model III |
---|---|---|---|
Total molecules in database (D) Total number of actives in database (A) Total number of hit molecules from database (Ht) Total number of active molecules in hit list (Ha) % yield of actives (%Y) [(Ha/Ht) × 100] % ratio of actives (%A) [(Ha/A) × 100] False negatives [A-Ha] False positives [Ht-Ha] Enrichment factor (EF) [(Ha × D)/(Ht × A)] Goodness of hit score (GH)a | 294 40 52 39 75.00 97.50 1 13 5.14 0.77 | 294 40 47 39 82.98 97.50 1 8 5.68 0.84 | 294 40 50 39 78.00 97.50 1 11 5.34 0.79 |
D is number of compounds in a database, A is the number of active compounds in the database, Ht is the number of hits retrieved, Ha is the number of actives in hit list, %Y is the fraction of hit relative to the size of database (hit rate or selectivity), %A is the ratio of actives retrieved in hit list, EF is the enrichment of active bin by model relative to random screening, GH is the Güner-Henry score. a [ ( H a 4 H t × A ) ( 3 A + H t ) × ( 1 − ( H t − H a D − A ) ) ] ; GH score > 0.7 indicates a statistically good model.
In order to investigate the influence caused by point mutation, compounds 6, 10, 39 were docked into the active sites of WT HER2 structure. Then the docking conformations were compared with those of HER2 mutants. As shown in
Virtual screening is a versatile technique to identify novel and potent lead compound for particular drug target [
Molecular docking is a well-established method to predict the molecular-level interactions of small molecules in the receptor active sites. In order to investigate the binding interactions between receptors and ligands, these potential irreversible HER2 inhibitors were individually docked into the active sites of HER2 L755S, T798I and T798M mutant structures and CDOCKER interaction energies (kcal・mol−1) obtained in the docking experiments were utilized to evaluate their binding interactions as presented in
HER2 L755S | HER2 T798I | HER2 T798M | |||
---|---|---|---|---|---|
Compounds | CDOCKER scores | Compounds | CDOCKER scores | Compounds | CDOCKER scores |
22 | −61.859 | 14 | −59.644 | 19 | −59.719 |
NSC278329 | −62.748 | NSC702108 | −60.981 | NSC659505 | −60.220 |
NSC381866 | −65.115 | NSC702137 | −60.748 | NSC692910 | −59.838 |
NSC642003 | −63.099 | NSC718305 | −68.817 | NSC718305 | −66.841 |
NSC659397 | −62.345 |
When docking to the HER2 T798I mutant structure, NSC702108 (−60.981 kcal/mol), NSC702137 (−60.748 kcal/mol) and NSC718305 (−68.817 kcal/mol) all had lower CDOCKER interaction energies than the optimal known irreversible HER2 inhibitor compound 14 (−59.644 kcal/mol) (
For the HER2 T798M mutant, NSC659505 (−60.220 kcal/mol), NSC692910 (−59.838 kcal/mol) and NSC718305 (−66.841 kcal/mol) all had lower CDOCKER interaction energies than compound 19 (−59.719 kcal/mol) (
Molecular dynamics (MD) simulation is an accurate method for investigating the physical movements of proteins-ligand complexes, giving a view of the dynamical evolution of the binding processes. In this study, HER2 L755S-NSC278329, HER2 L755S-NSC642003, HER2 T798I-NSC718305 and HER2 T798M-NSC718305 docking complexes were subjected to 50 ns MD simulations in an explicit hydration environment to investigate their binding stabilities. The dynamic stabilities and MD simulation trajectories of these complexes were analyzed by their backbone root mean square deviations (RMSD) [
ADMET properties were calculated to investigate the pharmacokinetics of a prospective drug compound in the human body [
Parameter | Lapatinib | NSC278329 | NSC642003 | NSC718305 |
---|---|---|---|---|
Human Intestinal Absorption Blood-Brain Barrier Caco-2 Permeability P-glycoprotein Substrate Renal organic cation transporter CYP450 2C9 Substrate CYP450 2D6 Substrate CYP450 3A4 Substrate CYP450 1A2 Inhibitor CYP450 2C9 Inhibitor CYP450 2D6 Inhibitor CYP450 2C19 Inhibitor CYP450 3A4 Inhibitor CYP450 Inhibitory Promiscuity hERG inhibition I hERG inhibition II AMES Toxicity Carcinogensa | HIA+ BBB+ Caco2- Substrate Non-inhibitor Non-substrate Non-substrate Substrate Non-inhibitor Non-inhibitor Non-inhibitor Non-inhibitor Inhibitor High Inhibitor Inhibitor Non-toxic Non-carcinogens | HIA+ BBB- Caco2- Substrate Non-inhibitor Non-substrate Non-substrate Substrate Non-inhibitor Non-inhibitor Non-inhibitor Inhibitor Inhibitor Low Non-inhibitor Non-inhibitor Non-toxic Non-carcinogens | HIA+ BBB- Caco2- Non-substrate Non-inhibitor Non-substrate Non-substrate Substrate Non-inhibitor Non-inhibitor Non-inhibitor Inhibitor Non-inhibitor High Non-inhibitor Non-inhibitor Non-toxic Carcinogens | HIA+ BBB+ Caco2- Non-substrate Non-inhibitor Non-substrate Non-substrate Substrate Non-inhibitor Non-inhibitor Non-inhibitor Non-inhibitor Inhibitor Low Non-inhibitor Non-inhibitor Non-toxic Non-carcinogens |
documents in 1997 and 1999, respectively, off-target transporter interactions and drug-drug interactions have been considered as the important criteria for drug development [
Cross docking analysis was performed to gain a deeper insight into the specificity of these selected irreversible HER2 inhibitors. In this study, NSC718305 was docked into HER2 L755S mutant while NSC278329 was docked into HER2 T798I and HER2 T798M mutants. Based on the results of cross docking, NSC718305 exhibited good CDOCKER scores of −71.664 kcal/mol by forming hydrogen bonding interactions with Lys753, Ser783, Cys805, Asp863 residues of HER2 L755S mutant (
Interestingly, NSC718305 was the only compound to be screened by model II and model III. These two pharmacophore models totally differ from each other not only by the number of their features but also by their geographies. As shown in
HER2 L755S, T798I and T798M point mutations were found to cause resistance toward FDA-approved lapatinib. Although several irreversible HER2 inhibitors have been synthesized to overcome such resistance problem, most of them were associated with lethal side effects, such as diarrhea, liver damage etc. Therefore, developing of novel irreversible HER2 inhibitors with less side effects and better ADMET properties against these HER2 mutants are highly desired. In this study, NSC278329 and NSC718305 were identified as novel irreversible HER2 inhibitors through a series of computational approaches. Unlike to the other experimental approaches, structure-based approach applied in this study has the advantages of searching compounds with novel structural scaffolds and desired chemical features. Furthermore, ADMET prediction adopted in this study has the ability to filter compounds with reasonable drug properties to decrease the risks of lethal side effects, such as off-target transporter interactions, drug-drug interactions and toxicity. Through cross docking, NSC278329 and NSC718305 were
proved to exhibit good binding interactions toward HER2 L755S, T798I and T798M mutants, suggesting they can extensively treat breast cancers with HER2 L755S, T798I and T798M mutants [
The authors greatly thank the Ministry of Science and Technology (MOST 103-2221-E-027-0901-MY3 and MOST 104-2221-E-027-073-MY3) and National Taipei University of Technology and Taipei Medical University (NTUT-TMU-101-10 and NTUT-TMU-102-10) for their financial supports.
The authors declare no conflicts of interest regarding the publication of this paper.