Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.     

Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to "coincidence detection," allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.     

New molecular scaffolds for the design of Mycobacterium tuberculosis type II dehydroquinase inhibitors identified using ligand and receptor based virtual screening

Using ligand and receptor based virtual screening approaches we have identified potential virtual screening hits targeting type II dehydroquinase from Mycobacterium tuberculosis, an effective and validated anti-mycobacterial target. Initially, we applied a virtual screening workflow based on a combination of 2D structural fingerprints, 3D pharmacophore and molecular docking to identify compounds that rigidly match specific aspects of ligand bioactive conformation. Subsequently, the resulting compounds were ranked and prioritized using receptor interaction fingerprint based scoring and quantitative structure activity relationship model developed using already known actives. The virtual screening hits prioritized belong to several classes of molecular scaffolds with several available substitution positions that could allow chemical modification to enhance binding affinity. Finally, identified hits may be useful to a medicinal chemist or combinatorial chemist to pick up the new molecular starting points for medicinal chemistry optimization for the design of novel type II dehydroquinase inhibitors.     

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies,enzyme, and cell assays

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 10⁶, M^?1 (±.21). However, UBA-domain of MARK4-F3 doesn’t show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC₅₀ values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.     

High performance screening, structural and molecular dynamics analysis to identify H1 inhibitors from TCM Database@Taiwan

New-type oseltamivir-resistant H1N1 influenza viruses have been a major threat to human health since the 2009 flu pandemic. To resolve the drug resistance issue, we aimed to identify a new type of inhibitors against H1 from traditional Chinese medicine (TCM) by employing the world's largest TCM database () for virtual screening and molecular dynamics (MD). From the virtual screening results, sodium (+)-isolaricireinol-2 alpha-sulfate, sodium 3,4-dihydroxy-5-methoxybenzoic acid methyl ester-4-sulfate, sodium (E)-7-hydroxy-1,7-bis(4-hydroxyphenyl)hept-5-ene-3S-sulfonate, and 3-methoxytyramine-betaxanthin were identified as potential drug-like compounds. MD simulation of the binding poses with the key residues Asp103 and Glu83, as well as other binding site residues, identified higher numbers of hydrogen bonds than N-Acetyl-D-Glucosamine (NAG), the natural ligand of the esterase domain in H1. Ionic bonds, salt bridges, and electrostatic energy also contribute to binding stability. Key binding residues include Lys71, Glu83, Asp103, and Arg238. Structural moieties promoting H-bond or salt bridge formations at these locations greatly contribute to a stable ligand-protein complex. An available sodium atom for ionic interactions with Asp103 can further stabilize the ligands. Based on virtual screening, MD simulation, and interaction energy evaluation, TCM candidates demonstrate good potential as novel H1 inhibitors. In addition, the identified stabilizing features can provide insights for designing highly stable H1 inhibitors.     

Human microtubule affinity-regulating kinase 4 is stable at extremes of pH

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.     

Energetic contributions of amino acid residues and its cross-talk to delineate ligand-binding mechanism

Receptor-based QSAR approaches can enumerate the energetic contributions of amino acid residues toward ligand binding only when experimental binding affinity is associated. The structural data of protein-ligand complexes are witnessing a tremendous growth in the Protein Data Bank deposited with a few entries on binding affinity. We present here a new approach to compute the E nergetic CONT ributions of A mino acid residues and its possible C ross-T alk (ECONTACT) to study ligand binding using per-residue energy decomposition, molecular dynamics simulations and rescoring method without the need for experimental binding affinity. This approach recognizes potential cross-talks among amino acid residues imparting a nonadditive effect to the binding affinity with evidence of correlative motions in the dynamics simulations. The protein-ligand interaction energies deduced from multiple structures are decomposed into per-residue energy terms, which are employed as variables to principal component analysis and generated cross-terms. Out of 16 cross-talks derived from eight datasets of protein-ligand systems, the ECONTACT approach is able to associate 10 potential cross-talks with site-directed mutagenesis, free energy, and dynamics simulations data strongly. We modeled these key determinants of ligand binding using joint probability density function (jPDF) to identify cross-talks in protein structures. The top two cross-talks identified by ECONTACT approach corroborated with the experimental findings. Furthermore, virtual screening exercise using ECONTACT models better discriminated known inhibitors from decoy molecules. This approach proposes the jPDF metric to estimate the probability of observing cross-talks in any protein-ligand complex. The source code and related resources to perform ECONTACT modeling is available freely at https://www.gujaratuniversity.ac.in/econtact /.     

Novel Inhibitors Induce Large Conformational Changes of GAB1 Pleckstrin Homology Domain and Kill Breast Cancer Cells

The Grb2-associated binding protein 1 (GAB1) integrates signals from different signaling pathways and is over-expressed in many cancers, therefore representing a new therapeutic target. In the present study, we aim to target the pleckstrin homology (PH) domain of GAB1 for cancer treatment. Using homology models we derived, high-throughput virtual screening of five million compounds resulted in five hits which exhibited strong binding affinities to GAB1 PH domain. Our prediction of ligand binding affinities is also in agreement with the experimental K _D values. Furthermore, molecular dynamics studies showed that GAB1 PH domain underwent large conformational changes upon ligand binding. Moreover, these hits inhibited the phosphorylation of GAB1 and demonstrated potent, tumor-specific cytotoxicity against MDA-MB-231 and T47D breast cancer cell lines. This effort represents the discovery of first-in-class GAB1 PH domain inhibitors with potential for targeted breast cancer therapy and provides novel insights into structure-based approaches to targeting this protein.     

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4)

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.     

Identification of novel leads applying in silico studies for Mycobacterium multidrug resistant (MMR) protein

Multidrug efflux mechanism is the main cause of intrinsic drug resistance in bacteria. Mycobacterium multidrug resistant (MMR) protein belongs to small multidrug resistant family proteins (SMR), causing multidrug resistance to proton (H⁺)-linked lipophilic cationic drug efflux across the cell membrane. In the present work, MMR is treated as a novel target to identify new molecular entities as inhibitors for drug resistance in Mycobacterium tuberculosis. In silico techniques are applied to evaluate the 3D structure of MMR protein. The putative amino acid residues present in the active site of MMR protein are predicted. Protein–ligand interactions are studied by docking cationic ligands transported by MMR protein. Virtual screening is carried out with an in-house library of small molecules against the grid created at the predicted active site residues in the MMR protein. Absorption distribution metabolism and elimination (ADME) properties of the molecules with best docking scores are predicted. The studies with cationic ligands and those of virtual screening are analysed for identification of new lead molecules as inhibitors for drug resistance caused by the MMR protein.     

Designing and optimization of novel human LMTK3 inhibitors against breast cancer – a computational approach

Estrogen receptor-α (ERα) is expressed more in patients with breast cancer and its level correlated with endocrine resistance. LMTK3 is reported as breast cancer target with regulation of estrogen receptor-α (ERα) through phosphorylation. In this computational study, structure-based inhibitor screening was performed on human LMTK3 using ZINC database. ATP-binding cavity with critical residues involved in the LMTK3 phosphorylation was used as target site for the screening. From the large ligand library, the best compounds were screen with three-phase virtual screening methods in Dockblaster, AutoDock Vina and AutoDock, respectively. The evaluation of ligands was carried out by binding energy and weak interactions, such as hydrogen bond interactions and hydrophobic contacts, in the target site that favors LMTK3 inhibition. Top compounds were found to be more effective in druglikeness activity by ADME prediction. The stability and binding affinity of ligand complexes were optimized by trajectory analysis such as RMSD, Rg, SASA and interhydrogen bonds from molecular dynamics simulations. The behavior of protein motion after ligand binding was illustrated by eigenvectors from principal component analysis (PCA). In addition, binding free energy of the LMTK3–ligand complexes were calculated by MM/PBSA methods and results supported the strong binding in dynamic system. Thus, the computational studies illustrated the structural insights on LMTK3 inhibition mechanism by ligands ZINC04670539, ZINC05607079 and ZINC04344028, also proposed as potent lead candidates. Our findings step towards developing novel LMTK3 inhibitors and identified lead candidates can be future breast cancer drugs with further experimental studies.     

Microtubule Affinity-Regulating Kinase 4: Structure, Function, and Regulation

MAP/Microtubule affinity-regulating kinase 4 (MARK4) belongs to the family of serine/threonine kinases that phosphorylate the microtubule-associated proteins (MAP) causing their detachment from the microtubules thereby increasing microtubule dynamics and facilitating cell division, cell cycle control, cell polarity determination, cell shape alterations, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. MARK4L is a 752-residue-long polypeptide that is divided into three distinct domains: (1) protein kinase domain (59–314), (2) ubiquitin-associated domain (322–369), and (3) kinase-associated domain (703–752) plus 54 residues (649–703) involved in the proper folding and function of the enzyme. In addition, residues 65–73 are considered to be the ATP-binding domain and Lys88 is considered as ATP-binding site. Asp181 has been proposed to be the active site of MARK4 that is activated by phosphorylation of Thr214 side chain. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation. On the other hand, the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas suggesting its involvement in cell cycle. Several biological functions are also associated with MARK4 including microtubule bundle formation, nervous system development, and positive regulation of programmed cell death. Therefore, MARK4 is considered as the most suitable target for structure-based rational drug design. Our sequence, structure- and function-based analysis should be helpful for better understanding of mechanisms of regulation of microtubule dynamics and MARK4 associated diseases.     

Developing novel approaches to improve binding energy estimation and virtual screening: a PARP case study

Poly-(ADP-ribose)-polymerase (PARP) is a promising anti-cancer target as it plays a crucial role in the cellular reparation and survival mechanisms. However, the development of a robust and cost effective experimental technique to screen PARP inhibitors is still a scientific challenge owing to the difficulties in quantitative detection of the enzyme activity. In this work we demonstrate that the computational chemistry tools including molecular docking and scoring can perform on par with the experimental studies in assessing binding constants and in the recovery of active compounds in virtual screening. Using the recently introduced Lead Finder software we were able to dock a set of 142 well characterized PARP inhibitors and obtain a good correlation between the calculated and experimentally measured binding energies with the rmsd of 1.67 kcal mol⁻¹. Additionally, fine-tuning of the energy scaling coefficients within the Lead Finder scoring function has further decreased rmsd to the value of 0.88 kcal mol⁻¹. Moreover, we were able to reproduce the selectivity of ligand binding between the two isoforms of the enzyme-PARP1 and PARP2-suggesting that the Lead Finder software can be used to design isoform-selective inhibitors of PARP. An impressive enrichment was obtained in the virtual screening experiment, in which the mentioned set of PARP inhibitors was mixed with a commercial library of 300,000 compounds. We also demonstrate that the virtual screening performance can be significantly improved by an additional structural filtration of the docked ligand poses through detection of the crucial hydrogen bonding interactions with the enzyme.     

Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to l ‐arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named “AraC‐TAL1”) was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand‐binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence‐activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL‐responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X‐ray diffraction was used to solve the crystal structure of the apo AraC‐TAL1 ligand‐binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild‐type AraC ligand‐binding domain (root‐mean‐square deviation 0.93 Å), suggesting that AraC‐TAL1 behaves similar to wild‐type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence–function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC.     

Dynamic and Multi-Pharmacophore Modeling for Designing Polo-Box Domain Inhibitors

The polo-like kinase 1 (Plk1) is a critical regulator of cell division that is overexpressed in many types of tumors. Thus, a strategy in the treatment of cancer has been to target the kinase activity (ATPase domain) or substrate-binding domain (Polo-box Domain, PBD) of Plk1. However, only few synthetic small molecules have been identified that target the Plk1-PBD. Here, we have applied an integrative approach that combines pharmacophore modeling, molecular docking, virtual screening, and in vitro testing to discover novel Plk1-PBD inhibitors. Nine Plk1-PBD crystal structures were used to generate structure-based hypotheses. A common pharmacophore model (Hypo1) composed of five chemical features was selected from the 9 structure-based hypotheses and used for virtual screening of a drug-like database consisting of 159,757 compounds to identify novel Plk1-PBD inhibitors. The virtual screening technique revealed 9,327 compounds with a maximum fit value of 3 or greater, which were selected and subjected to molecular docking analyses. This approach yielded 93 compounds that made good interactions with critical residues within the Plk1-PBD active site. The testing of these 93 compounds in vitro for their ability to inhibit the Plk1-PBD, showed that many of these compounds had Plk1-PBD inhibitory activity and that compound Chemistry_28272 was the most potent Plk1-PBD inhibitor. Thus Chemistry_28272 and the other top compounds are novel Plk1-PBD inhibitors and could be used for the development of cancer therapeutics.     

Pharmmaker: Pharmacophore modeling and hit identification based on druggability simulations

Recent years have seen progress in druggability simulations, that is, molecular dynamics simulations of target proteins in solutions containing drug‐like probe molecules to characterize their drug‐binding abilities, if any. An important consecutive step is to analyze the trajectories to construct pharmacophore models (PMs) to use for virtual screening of libraries of small molecules. While considerable success has been observed in this type of computer‐aided drug discovery, a systematic tool encompassing multiple steps from druggability simulations to pharmacophore modeling, to identifying hits by virtual screening of libraries of compounds, has been lacking. We address this need here by developing a new tool, Pharmmaker, building on the DruGUI module of our ProDy application programming interface. Pharmmaker is composed of a suite of steps: (Step 1) identification of high affinity residues for each probe molecule type; (Step 2) selecting high affinity residues and hot spots in the vicinity of sites identified by DruGUI; (Step 3) ranking of the interactions between high affinity residues and specific probes; (Step 4) obtaining probe binding poses and corresponding protein conformations by collecting top‐ranked snapshots; and (Step 5) using those snapshots for constructing PMs. The PMs are then used as filters for identifying hits in structure‐based virtual screening. Pharmmaker, accessible online at http://prody.csb.pitt.edu/pharmmaker , can be used in conjunction with other tools available in ProDy.     

Binding of the proline-rich segment of myelin basic protein to SH3 domains: spectroscopic, microarray, and modeling studies of ligand conformation and effects of posttranslational modifications

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.     

Combined pharmacophore modeling, 3D-QSAR and docking studies to identify novel HDAC inhibitors using drug repurposing

Abstract

Histone deacetylases (HDACs), a critical family of epigenetic enzymes, has emerged as a promising target for antitumor drugs. Here, we describe our protocol of virtual screening in identification of novel potential HDAC inhibitors through pharmacophore modeling, 3D-QSAR, molecular docking and molecular dynamics (MD) simulation. Considering the limitation of current virtual screening works, drug repurposing strategy was applied to discover druggable HDAC inhibitor. The ligand-based pharmacophore and 3D-QSAR models were established, and their reliability was validated by different methods. Then, the DrugBank database was screened, followed by molecular docking. MD simulation (100?ns) was performed to further study the stability of ligand binding modes. Finally, results indicated the hit DB03889 with high in silico inhibitory potency was suitable for further experimental analysis.

Communicated by Ramaswamy H. Sarma     

Ligand-based virtual screening,molecular docking,QSAR and pharmacophore analysis of quercetin-associated potential novel analogs against epidermal growth factor receptor

The present study was to explore expectation and examination of therapeutic potential quercetin analogs as efficient anticancer agents against human epidermal growth factor receptor (EGFR), which is a consistent hallmark for moderating the non-small-cell lung carcinoma (NSCLC). Here, ligand-based virtual screening, pharmacophore approach and molecular docking were established as rational strategies for recognition of small analogs against the ligand binding domain of EGFR (PDB code: 1XKK). Adverse effects, toxicogenomics and pharmacokinetics reported that 10 candidates showed reliable consequences with less side effects and more efficient for target receptor. Protein–ligand interaction profiles revealed that the probable H-bonds, atomic-π contacts, salt bridges and van der Waals interactions sustain the complexity and stability of receptor structure; thus, they could complicate to generate single alteration acquired for drug resistance. In silico anticancer properties explain the lead scaffolds which are assumed to be flexible and experimentally proved chemicals. The overall consequences indicated that recognized leads could be utilized as reference skeletons for new inhibitors envisaging toward EGFR to ameliorate NSCLC and other malignant disorders.