p38a MAP kinase inhibitors to overcome EGFR tertiary C797S point mutation associated with osimertinib in non-small cell lung cancer (NSCLC): emergence of fourth-generation EGFR inhibitor

Iqrar Ahmada , Matin Shaikha, Sanjay Suranaa, Arabinda Ghoshb and Harun Patela

ABSTRACT

The third-generation EGFR (epidermal growth factor receptor) inhibitors selectively and irreversibly target EGFR-T790M and other activating EGFR mutations. Osimertinib is the only FDA-approved thirdgeneration inhibitor, which has a good potency against the EGFR-T790M mutant with minimal toxicities and excellent selectivity for wild-type EGFR. EGFR tertiary Cys797 to Ser797 (C797S) point mutation emanate rapidly after the treatment of osimertinib, which is an undruggable mutation to all three existing generation drugs. Recently, trisubstituted imidazoles were reported based on an off-target hit of a p38a MAPK (mitogen-activated protein kinase) inhibitor as the fourth-generation EGFR-TKIs to overcome the C797S resistance by inhibiting the clinically relevant triple mutant kinase L858R/T790M/ C797 EGFR. Here, we are reporting the clinical trial p38a MAPK kinase inhibitors SD-06, Amgen 16, RWJ67657 and SCIO-323 as L858R/T790M/C797S EGFR TK inhibitors to overcome the problem of drug resistance in non-small cell lung cancer (NSCLC).

KEYWORDS
p38a MAPK kinase
inhibitor; EGFR T790M/C797S; molecular docking; MMGBSA; dynamic simulation

1. Introduction

Lung cancer is the second most common cancer in men and women worldwide and the leading cause of cancer-related death (https://www.cancer.net/cancer-types/lung-cancer-nonsmall-cell/statistics, consulted on 22 June 2020). Nearly 85% of all lung cancers are identified as non-small cell lung cancer (NSCLC) with a 5-year survival rate of less than 15% (Pallis et al., 2009; Shamoo et al., 2006; Siegel et al., 2015; Torre et al., 2015). Abnormal activation of the signaling pathway for the epidermal growth factor receptor (EGFR) in NSCLC is well validated and an important therapeutic target for the drug discovery of anticancer drugs (Hatanpaa et al., 2010; Hynes et al., 2005). EGFR inhibitors such as Gefitinib, Erlotinib, Afatinib have been approved by USFDA and achieved significant clinical benefit in NSCLC patients with EGFR activating mutations (i.e. L858R and delE746-A750) and/or secondary threonine 790 to methionine 790 (T790M) mutation, respectively (Shen et al., 2019; Takeda & Nakagawa, 2019). A great deal of effort to overcome T790M mutation drug resistance has been made, and third-generation EGFR-TKIs, such as WZ4002, CO1686 (Rociletinib), AZD9291 (Osimertinib), HM61713 (Olmutinib), EGF816 (Nazartinib), ASP8173 (Naquotinib) and PF0674775 have been discovered (Finlay et al., 2014; Jia et al., 2016; Lee et al., 2014; Planken et al., 2017; Walter et al., 2013; Wang et al., 2016; Zhang, Wang, et al., 2018; Zhou et al., 2009). These EGFR-TKIs with novel structures conquers the T790M mutation resistance through covalent binding with Cys-797. The mode of action of these EGFR-TKIs (third generation) revealed that the acrylamide moiety (electrophilic warhead) undergoes a Michael addition reaction with the side chain of the Cys-797 residue in physiological conditions to increase the target residence time and restore inhibitory activity against the EGFR-T790M mutants. Meanwhile, they retain excellent selectivity over wild-type EGFR to mitigate the side effects associated with second-generation irreversible inhibitor (Patel et al., 2017; Song et al., 2016; Hynes et al., 2005).
Particularly, pyrimidine base osimertinib demonstrates approximately 75% overall response rate in T790M EGFR mutation-positive and sensitizing EGFR mutations, while sparing wild-type EGFR in NSCLC. It was approved by USFDA and European Medical Agency in March 2017 and April 2018, respectively, for metastatic EGFR T790M-positive NSCLC, which has progressed on or after EGFR-TKI therapy (Odogwu et al., 2018; Scott, 2018).
While promising survival results and response rates have been reported in osimertinib-treated patients, unfortunately, acquired resistance (tertiary Cys797 to Ser797 (C797S) point mutation) occurs after about 10 months, which interferes in the covalent bond formation (Niederst et al., 2015; Thress et al., 2015). Evidence of this mutation was isolated before 3 years in NSCLC patients by Thress et al. They reported that this tertiary mutation occurs in the frame of exon 20 and accounts for 10%–26%, where cysteine at codon 797, inside the ATP-binding site, is replaced by serine that results in the loss of the covalent bond formation of osimertinib (Thress et al., 2015). Predictably, the C797S mutation often confers cross-resistance to other irreversible third-generation TKIs, such as Rociletinib, Plmutinib and Nazartinib, by averting their binding to the EGFR active site (Bersanelli et al., 2016; Jiang et al., 2018; Wang et al., 2017; Yang et al., 2018). These mutations are thought to be responsible for steric interference leading to a decreased compound affinity for the EGFR kinase domain (Jiang et al., 2018; Yang et al., 2018). However, this acquired mutation remains a significant challenge in the development of precision medicine as an emerging ‘unmet clinical need’ for NSCLC patients (Bersanelli et al., 2016; Jiang et al., 2018; Wang et al., 2017; Yang et al., 2018). Therefore, the development of the fourth-generation EGFR-TKIs with a new scaffold and high potential are still needed.
Based on the literature survey and clinical trial data (Goldstein & Gabriel, 2005; Heider et al., 2017; Kong et al., 2013; Lin et al., 2014; Margutti & Laufer, 2007; Roberts & Der, 2007; Souza et al., 2012; Xing, 2016; Yang et al., 2014; Zhang et al., 2007), we have observed that the pharmacophoric features of p38a MAPK (mitogen-activated protein kinase) inhibitors (Figures 1 and 2) and their binding site resemble with L858R/T790M/C797S mutant EGFR in NSCLC. Hence, here we have hypothesized and in silico validated the clinical trial p38a MAPK inhibitors as L858R/T790M/C797S EGFR TK inhibitors to overcome the problem of drug resistance in NSCLC (Padhi & Hazra, 2019).

2. Hypothesis

2.1. Binding site analysis

We have observed that the p38a MAP kinase enzyme resembles with mutant T790M/C797S EGFR’s ATP competitive binding site in terms of hinge region, hydrophobic region I (HR-I) and hydrophobic region II (HR-II). The common features in the binding site include: (1) the N- and C-terminal domains of p38a MAPK and mutant L858R/T790M/C797S EGFR are connected via a hinge region as shown in Figure 3 (Bagley et al., 2010). (2) The common features between inhibitors and enzymes include: (a) key interactions between p38a MAPK and its inhibitor (SB203580) include hydrogen bond between the pyridine nitrogen of SB203580 and Met-109 residue of the hinge region (Figure 3); (b) hydrophobic interaction between the 4-fluorophenyl of SB203580 with gatekeeper residue Thr-106 of the HR-I (Figure 3); (c) methyl sulfinyl phenyl of the SB203580 is involved in the hydrophobic interaction with a phosphate-binding site of the HR-II (Figure 3; Bagley et al., 2010).
Similarly, trisubstituted imidazole Compound 1 interacts with the L858R/T790M/C797S EGFR, where the aminopyridine moiety of the trisubstituted imidazoles forms the hydrogen bond with the hinge residue Met-793 of L858R/T790M/C797S EGFR and 4-fluorophenyl of the trisubstituted imidazoles (similar to the SB203580) is involved in the hydrophobic interaction with the T790M mutated residue (Thr790 to Met790) in the HR-I (Figure 3). While the Michael acceptor of Compound 1 is interacting with the phosphate-binding site in the HR-II (Figure 3; Juchum et al., 2017).

2.2. Pharmacophore analysis

We have collected the p38a MAPK and L858R/T790M/C797S EGFR TK inhibitors from the literature (Goldstein & Gabriel, 2005; Heider et al., 2017; Kong et al., 2013; Lin et al., 2014; Margutti & Laufer, 2007; Roberts & Der, 2007; Souza et al., 2012; Xing, 2016; Yang et al., 2014; Zhang et al., 2007). Close observation of structural features of the trisubstituted imidazoles (p38a MAPK inhibitors) indicates that they have almost the same pharmacophoric features as like that of the L858R/T790M/C797S EGFR TK inhibitors (Figure 4). The common features include: (1) Hydrophobic group I (4-fluoro phenyl), which is required to form the hydrophobic interaction with the mutated gatekeeper residue T790M of the L858R/T790M/C797S EGFR TK (Figures 3 and 4). (2) Hydrophobic group II (methyl sulfinyl phenyl and phenyl thio ethanol), which is required to interact with the mutated serine residue (C797S) and phosphate-binding site of the L858R/T790M/C797S EGFR TK (Figures 3 and 4). (3) Pyridine ring, which is involved in the hydrogen bond interaction with the Met-793 of the hinge region of the L858R/T790M/C797S EGFR TK (Figures 3 and 4). (4) Trisubstituted imidazole is common basic ring present in both the inhibitors, which holds all the functionality on it (Figures 3 and 4).
Similarly, we have studied another series of the p38a MAP kinase inhibitors containing the pyrido-pyrimidine ring and surprisingly they have also the similar pharmacophoric features as that of the L858R/T790M/C797S EGFR TK inhibitors as shown in Figure 5 (Goldstein & Gabriel, 2005; Heider et al., 2017; Kong et al., 2013; Lin et al., 2014; Margutti & Laufer, 2007; Roberts & Der, 2007; Souza et al., 2012; Xing, 2016; Yang et al., 2014; Zhang et al., 2007). Similarity in the structure of the enzymes (p38a MAPK and L858R/T790M/C797S EGFR) and pharmacophoric features of both the inhibitors indicate that structural optimization of the p38a MAPK inhibitors may result in the potent T790M/C797S EGFR TK inhibitors (Juchum et al., 2017).

3. Validation of hypothesis (literaturebased evidences)

3.1. Optimization of the L858R/T790M/C797S EGFR TK inhibitors from p38a MAPK inhibitors

Stefan Laufer and collaborators developed the trisubstituted imidazoles with a rigidized 7-azaindole hinge binding motif as a new structural class of L858R/T790M/C797S EGFR TK inhibitors by a target hopping approach from p38a MAPK inhibitor templates (Juchum et al., 2017). They initially optimized the Compound 6 from the well-known p38a MAPK inhibitors (SB203580 and ML3403) by C-2 modifications of SB203580 imidazole core and aromatization with purine bioisosteres as new C-5 rigidized amino-pyridinyl scaffolds on imidazole core of ML3403 (Figure 6; Selig et al., 2012). Based on selectivity screening of the highly potent reversible p38a MAPK inhibitor 6, they identified EGFR inhibition as an offtarget effect of this compound. High potency, as well as moderate physicochemical properties and cellular activity against p38a MAPK, lead them to pick this compound as a first lead structure for further improvements in terms of the inhibition of EGFR mutants. Compound 6 is having IC50 ¼ 49.2 nM against the wild-type EGFR, IC50 ¼ 24.1 nM against the L858R-EGFR and moderate activity against the double mutant L858R/T790M EGFR TK (IC50 ¼ 916 nM) (Juchum et al., 2017). They then shifted the kinase target of a known p38a MAPK inhibitor (Compound 6) into potent EGFR inhibitors using effective synthetic routes to obtain more flexible synthetic procedures that allow the modification at different parts of the molecule and tolerate less robust functional groups. Hence, they synthesized a set of different trisubstituted imidazoles with a 7-azaindole hinge binding motif and studied the structure–activity relationship of this class compounds against different mutant EGFRs (Gunther et al., 2017; Gunther et al.,€ 2016; Juchum et al., 2017). This target hopping approach leads to the development of the fourthgeneration class of EGFR inhibitors (Compound 2), which is having high binding affinities to the clinically challenging NSCLC triple mutants (IC50 value of 7.64 nM) as shown in (Figure 6).

4. In silico validation of hypothesis

4.1. Mining of p38a MAPK inhibitors (clinical trial data) to get L858R/T790M/C797S EGFR TK inhibitors

Based on the above observation and inspired by the work of Stefan Laufer and collaborators (Juchum et al., 2017) we have collected the p38a MAPK inhibitors, which are in clinical trials and in silico evaluated for L858R/T790M/C797S EGFR TK inhibitory potential (Goldstein & Gabriel, 2005; Heider et al., 2017; Kong et al., 2013; Lin et al., 2014; Margutti & Laufer, 2007; Roberts & Der, 2007; Souza et al., 2012; Xing, 2016; Yang et al., 2014; Zhang et al., 2007).

4.1.1. Molecular docking

Based upon the foregoing observation, we have performed docking study of clinical trials p38a MAPK inhibitor toward the T790M/C797S mutant EGFR. The ligands were prepared using the LigPrep module (Schrodinger, LLC, New York, NY,€ USA, 2009) by adding hydrogen atoms, removing salt, generating stereoisomers, ionizing at pH (7 ± 2) and determining valid 3D conformation (Sastry et al., 2013). Additionally, the geometry of the ligands was minimized using the standard molecular mechanic’s energy function OPLS-2005 force field. The crystal structure of L858R/T790M/C797S mutant EGFR (PDB ID: 5XGN) was obtained from Protein Data Bank (Kong et al., 2017). The protein structure was prepared using the Protein Preparation Wizard (PPrep) module in Maestro software. Finally, the protein structure was minimized using the OPLS-2005 force field (Schrodinger, LLC, New York, NY, USA,€ 2009) Glide’s Receptor Grid Generation module was used to generate the receptor grid at the active site of co-crystalline ligand with the centered dimension cubic grid box of 10 Å 10 Å 10 Å (Sastry et al., 2013). Finally, the low-energy conformation of the ligands was selected and docked into the grid generated from protein structures using standard precision (SP) docking mode. The evaluation was carried out with a glide SP docking score and a single absolute best pose is produced as the output for a specific ligand (Elokely & Doerksen, 2013; Friesner et al., 2006).

4.1.2. Docking validation

The docking procedure was validated using two methods viz. (i) Overlay method: the crystallized ligand Go6976 from the T790M/C797S mutant EGFR sketched and re-docked into the active site of T790M/C797S mutant EGFR (PDB ID: 5XGN). The parameters utilized in the current docking study should be successfully validated if the docked conformation was perfectly overlapped over the crystallized bioactive conformation of the ligand available in the T790M/C797S mutant EGFR complex. (ii) Chemical resemblance: the molecular docking method is validated when the docked ligand should have same interactions with the residues of T790M/C797S mutant EGFR as that present in the downloaded crystallized T790M/C797S mutant EGFR. (Al-Khodairy et al., 2013; Jain & Mujwar, 2020).

4.1.3. Binding free energy calculation

Molecular mechanics with generalized born surface area (MM-GBSA) is the most popular method to estimate the ligand binding energies, which includes the OPLS3 power field and VSGB solvent model (Li et al., 2011). The prime MM-GBSA simulation was carried out by using the Glide pose viewer file to calculate the total binding free energy. These poses were taken as inputs for the energy minimization of the protein–ligand complexes (Ecomplex), the free protein (Eprotein) and the free ligands (Eligand). The binding free energy DGbind was determined according to the following equation: The MM-GBSA calculations are used to estimate relative binding affinity of ligands to the receptor (reported in kcal/ mol). As the MM-GBSA binding energies are approximate free energies of binding, a more negative value indicates stronger binding (Casalvieri et al., 2020; Li et al., 2011).

4.1.4. Molecular dynamics (MD) simulations

MD simulations for the best dock protein–ligand complex was carried out using the Desmond program, an explicit solvent MD package (version 3.1, Desmond Molecular Dynamics System, Schrodinger) along with fixed optimized potentials for€ liquid simulation (OPLS-2005) force field (Bowers et al., 2006; Chow et al., 2008; Shivakumar et al., 2010). The system was built up for simulation using a predefined water model (simple point charge [SPC]) as solvent in an orthorhombic box with periodic boundary conditions specifying the shape and size of box as 10 Å 10Å 10 Å distance. The desirable electrically neutral system for simulation was built with 0.15 M NaCl (physiological concentration of monovalent ions) in 10Å buffer regions between the protein atoms and box sides using the system-built option. Steepest Descent and limited-memory Broyden-Fletcher Goldfarb-Shanno algorithms were applied in a hybrid manner to achieve the relaxation of the system (Bowers et al., 2006; Shivakumar et al., 2010). A constant 300 K temperature and 1 atm pressure was maintained during the simulation using the Nose-Hoover thermostat algorithm and Martyna-Tobias-Klein barostat algorithm, respectively (Evans & Holian, 1985; Martyna, 1994). Long-range and short-range Coulombic interaction was controlled using smooth particle mesh Ewald method with 9.0 Å endpoint values (John et al., 2015). The simulation was achieved under NPT ensemble for 100 ns and trajectory information was obtained with the rest of 10ps applying the Berendsen thermostat and barostat methods (Ivanova et al., 2018).

5. Results and discussion

5.1. Molecular docking and validation of methodology

Tertiary mutation, L858R/T790M/C797S EGFR, is undruggable EGFR mutation in NSCLC. It arises rapidly after treatment with osimertinib and other third-generation irreversible EGFR inhibitors targeting T790M mutation (Takeda & Nakagawa, 2019). It is a dominant resistance mechanism to irreversible third-generation EGFR-TKIs that disturbed the formation of the key covalent bond which drives target potency and selectivity (Takeda & Nakagawa, 2019). Stefan Laufer and coworker designed a series of trisubstituted imidazole’s as novel mutant L858R/T790M/C797S EGFR inhibitors based on an off-target hit of a p38a MAPK inhibitor (Figure 6; Gunther et al., 2017; Gunther et al.,€ 2016; Juchum et al., 2017). Xiaoyun Lu et al. identified a pyrimido-pyrimidinone derivative JND3229, a new highly potent L858R/T790M/C797S EGFR inhibitor with singledigit nM potency, which is another molecule resembling with p38a MAP kinase inhibitors (Figure 5; Lu et al., 2018). Based on these observations, we inferred that other p38a MAP kinase inhibitors, which are in clinical trials could work against the clinically challenged triple mutant NSCLC. Therefore, we used PDB ID: 5XGN to conduct molecular docking of clinical trial p38a MAPK inhibitors against the triple mutant EGFR. The docking procedure was validated using Overlay method and Chemical resemblance method based on re-docking of cocrystallized ligand (Go6976). Docked conformation of the Go6976 was perfectly overlaid over its bioactive conformation obtained from the crystallized macromolecular complex as shown in Supplementary Figure S1. Both the ligands also showed similar binding interactions with the triple mutant EGFR TK. Thus, the molecular docking methodology was successfully validated for the current in silico studies.
According to docking result analysis, interestingly, all the p38a MAPK inhibitors have shown noteworthy docking score against L858R/T790M/C797S EGFR except SKF-86002, Acumapimod, Talmapimod, Ralimetinib, TAK-715, Doramapimod and Neflamapimod (Table 1 and Figure 7). Most of the p38a MAPK inhibitors show the hydrogen bond interaction with the hinge residue Met-793 and Asp-855 residue of the DFG motif of the L858R/T790M/C797S EGFR. Pyrazole-based SD-06 (presented by 5.2. Binding free energy calculation by MMPfizer) had a higher binding affinity with 8.022kcal/mol docking GBSA method score. According to Kong and collaborators, the C797S mutation MM-GBSA analysis was performed on all of the protein–ligand increases the local hydrophilicity around residue 797 without complexes to evaluate the affinity of ligands to the target protein affecting the structure and function of the kinase domain (Kong receptors (mutant L858R/T790M/C797S EGFR). The MM-GBSAet al., 2013). Likewise, terminal 2-hydroxy piperidino-ethanone of SD-06 has formed hydrogen bond interaction with polar hydroxyl based binding free energy (DGbind) calculations were performed using ligand docked complexes. The major energy components, group of leu718 and hydrophilic interaction with a water molecule present in the vicinity of mutated C797 residue (Figure 7). such as lipophilic interaction energy (DGbind Lipo), van der Waals Additionally, nitrogen of pyrazolopyrimidine forms a hydrogen interaction energy (DGbind vdW), Coulomb or electrostatics interbond interaction with the Met793 ‘hinge’ residue of the EGFR kin- action energy (DGbind Coulomb), generalized born electrostatic ase (Figure 7). The 4-chlorophenyl group of SD-06 was directed solvation energy (DGbind Solv GB) and covalent interaction binding toward the hydrophobic back pocket composed of Glu-762, Met- energy (DGbind Covalent) altogether contribute to the calculation of MM-GBSA-based relative binding affinity. The binding energies 790, Thr-854 and Phe-856 residues. and the contributing factors calculated for the protein dock complexes are mentioned in Table 2. Among the complexes studied, three complexes showed high binding free energies namely, Amgen 16 (DGbind of 82.66kcal/mol), Pexmetinib (DGbind of 87.45kcal/mol) and Talmapimod (DGbind of 81.85kcal/mol).

5.3. MD simulation

To confirm binding modes of ligands and to check the stability of the protein–ligand complexes, we performed MD simulations utilizing the Desmond program. The top dock scorer SD-06-EGFR L858R/T790M/C797S complex was subjected to MD simulation for 100 ns. The root mean standard deviation (RMSD) plot of SD-06-EGFR L858R/T790M/C797S complexes is displayed in Figure 8. The plot exhibited minor fluctuation up to 3.5 Å, which demonstrates the stability of the ligand within the binding pocket. The root mean square fluctuation (RMSF) of an amino acid residue was used to calculate the average value of all atomic fluctuations for a given amino acid (Padhi et al., 2012, 2013; Patel, Ahmad, et al., 2020; Patel, Shaikh, et al., 2020; Shahraki et al., 2018). The protein RMSF is shown in Figure 9. Results indicated that Met-1007 and Asp-1008 residues were fluctuating at 4.86 Å during the simulation. However, these protein residues have not been involved in ligand interactions. High fluctuations were observed in N- and C-terminal regions compared to any other part of the protein. If the fluctuation of the active site and the main chain atoms was mild, it indicates that the conformational change was slight (Zhang, Ma, et al., 2018). Overall, the fluctuation of amino acid residues during the interaction was observed to be below 2.0 Å, which is perfectly acceptable (Vora et al., 2019). Furthermore, protein interaction with the ligand has been monitored throughout the simulation. According to Kong and collaborators, EGFR T790M/C797S acquired tertiary mutation (L858R/T790M/ C797S) increases the local hydrophilicity around residue 797 without disturbing the structure and function of the kinase domain. Therefore, the construction of small molecule inhibitors that enhance the binding affinity to other regions of the EGFR kinase domain are key elements in the development of fourth-generation EGFR inhibitors (Kong et al., 2013). SD 06 showed distinct water-mediated hydrogen bond interaction with the Asp-800 and Leu-718. Pyrimidine nitrogen of SD 06 was involved in direct hydrogen bond interaction with the Met-793 residue located in the hinge region of the L858R/ T790M/C797S EGFR TK (Figure 10). Supplementary Figure S2 shows the total number of specific contacts protein makes with ligand in each and every trajectory frame. The contribution of amino acids in each trajectory frame of 100 ns MD simulation as shown in the bottom panel of Supplementary Figure S2, which represents the number of contacts and their density, i.e. a darker shade of orange indicates more than one contact in that frame. Key interaction was seen during each frame of simulation with Met-793 residue of the receptor, it was one of the stable interactions observed during the complete simulation process. Other interactions were also found with Leu-718, Phe-723, Met-790, Asp-800 and Leu-844 but they were not persistent during the entire simulation.
The Five molecular properties (ligand RMSD, the radius of gyration [rGyr], molecular surface area [MolSA], solventaccessible surface area [SASA] and polar surface area [PSA]) of ligand were also examined to illustrate the stability of the SD-06 in the T790M/C797S mutant EGFR during the simulation of 100 ns as shown in Supplementary Figure S3. Ligand RMSD indicates root mean square deviation of a ligand with respect to the reference conformation (usually the initial frame is used as the reference and it is regarded as time t ¼ 0). The RMSD values of SD-06 were below 2 Å. Radius of gyration is used to evaluate the ‘extendedness’ of a ligand and is equivalent to its principal moment of inertia; the radius of gyration throughout the 100 ns simulation remained constant and ranged from 4.60 to 4.90 Å for SD-06. MolSA, SASA and PSA plots also suggested the stability of SD-06 during the simulation process. To better understand the stability of ligand during simulation, trajectories were collected at regular intervals 20 ns of time till 100 ns so as to analyze the active site fluctuations (cluster analysis). These protein structures, when superimposed showed RMSD in the range of 2.0627–0.7651 Å (Figure 11).

6. Conclusion

The L858R/T790M/C797S mutation in the tyrosine kinase domain of EGFR was reported to be a leading resistance mechanism to the irreversible EGFR inhibitors of the thirdgeneration class (osimertinib). L858R/T790M/C797S mutation increases the local hydrophilicity around residue 797, without affecting the structure and function of the kinase domain. In the present study, we have correlated the binding site of the p38a MAPK and triple mutant L858R/T790M/C797S EGFR. We have observed that p38a MAP kinase resembles with mutant L858R/T790M/C797S EGFR ATP competitive binding site in terms of hinge region, HR-I and HR-II. At the same time, inhibitors of both the class have same pharmacophoric features. Therefore, we hypothesized that p38a MAPK inhibitor could act on clinically challenged (EGFR L858R/T790M/C797S) tertiary mutation to overcome resistance problems in NSCLC. Our proposed hypothesis was also supported by the work of Stefan Laufer and collaborators, who developed the trisubstituted imidazoles with a rigidized 7-azaindole hinge binding motif as a new structural class of L858R-T790M-C797S EGFR TK inhibitors by a target hopping approach from p38a MAPK inhibitor templates. Our hypothesis was further validated by the molecular docking study, which revealed that p38a MAPK inhibitors (SD-06, Amgen 16, RWJ67657 and SCIO-323) have similar affinity to the date reported mutants L858R/ T790M/C797S EGFR inhibitors. Further, MD studies were performed in order to check the overall stability of the protein–ligand complex. MD simulation for 100 ns further suggested that docked compound SD-06 was stable into the L858RT790M-C797S EGFR TK enzyme. Here, we conclude that using target hopping strategy, p38a MAPK inhibitors SD-06, Amgen 16, RWJ67657and SCIO-323 could be further explored as a starting point for the discovery and development of the undruggable L858R/T790M/C797S EGFR TK inhibitors in the treatment of NSCLC.

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