Recent Progress of Small-Molecule Epidermal Growth Factor

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Recent progress of small-molecule epidermal growth factor receptor (EGFR) inhibitors against C797S resistance in non-small-cell lung cancer Lingfeng Chen, Weitao Fu, Lulu Zheng, Zhiguo Liu, and Guang Liang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01310 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 23, 2017

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Journal of Medicinal Chemistry

Recent progress of small-molecule epidermal growth factor receptor (EGFR) inhibitors against C797S resistance in non-small-cell lung cancer

Miniperspective Lingfeng Chen a, b, Weitao Fu a, Lulu Zheng a, Zhiguo Liu a, Guang Liang a, b, * a

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou

Medical University, Wenzhou, Zhejiang 325035, China b

School of Chemical Engineering, Nanjing University of Science and

Technology, Nanjing, Jiangsu, 210094, China

*Corresponding authors: Prof. Guang Liang Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, P. R. China. Tel./fax: +86 577 86699396. E-mail addresses: [email protected] (G. Liang).

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Abstract: The epidermal growth factor receptor (EGFR) has been a particular interest for drug development for treatment of non-small-cell lung cancer (NSCLC). The current third-generation EGFR small-molecule inhibitors, especially osimertinib, are at the forefront clinically for treatment of patients with NSCLC. However, a high percentage of these treated patients developed a tertiary cystein797 to serine790 (C797S) mutation in the EGFR kinase domain. This C797S mutation is thought to induce resistance to all current irreversible EGFR TKIs. In this miniperspective, we present key mechanisms of resistance in response to third-generation EGFR TKIs, and emerging reports on novel EGFR TKIs to combat the resistance. Specifically, we analyze the allosteric and ATP-competitive inhibitors in terms of drug discovery, binding mechanism, and their potency and selectivity against EGFR harboring C797S mutations. Lastly, we provide some perspectives on new challenges and future directions in this field.

Keywords: Epidermal growth factor receptor; non-small-cell lung cancer; C797S mutation; Tyrosine Kinase Inhibitor; drug resistence.


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1. Introduction Lung cancer accounts for more than one quarter of cancer-related deaths worldwide, and the 5-year relative survival is currently less than 20%.1 Non-small-cell lung cancer (NSCLC) is the most common subtype of lung cancer, which accounts for about 80-85%.2 A member of the ErbB family, the epidermal growth factor receptor (EGFR) is fundamentally important in cell proliferation, survival, migration, adhesion, and differentiation, and is one of the most valuable drug targets for the treatment of NSCLC.3 At least one activating mutation in the tumor genome of patients, such as single amino acid replacement in exon 21 (L858R) or deletion in exon 19 (delE746-A750, del19) is required for a discernible response to treatment with EGFR tyrosine kinase inhibitors (TKIs).4 In 2009 and 2013, FDA approved first-generation TKIs gefitinib and erlotinib for EGFR activating mutation-positive NSCLC patients, which resulted in massive and rapid tumor volume shrinkage.5 However, patients typically develop secondary T790M gatekeeper drug resistance after about 12 months of treatment.6 Also, the clinical application of second-generation irreversible inhibitor afatinib (BIBW2992)7 is limited due to its poor therapeutic window. Please insert Figure 1 here Currently, third-generation EGFR TKIs are being developed for effective management of T790M-associated resistance while sparing wild-type EGFR (Figure 1). WZ40028 is the first reported third-generation compound, with 30-100 times more potency against EGFR T790M than to wild-type EGFR. In a Phase II trial of 1



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(rociletinib, also known as CO-1686)9, structurally similar to WZ4002, treatment of T790M-positive patients sustain a response rate of 59% (27 out of 46 patients) and high disease control rate (93%), but T790M-negative patients show lower respective rates of 29% and 59%. However, Clovis has halted the further development of rociletinib after the FDA rejected the request for accelerated approval and required more randomized data.10 Clinical studies also indicated that rociletinib may cause hyperglycemia by inhibition of off-targets including insulin-like growth factor receptor and insulin receptor kinases.11, 12 2 (Osimertinib, also known as AZD9291)13, with a distinct mono-anilino-pyrimidine scaffold, became the first globally-approved TKI for EGFR T790M-positive NSCLC patients in 2015 following the progress of previous generations of TKI. Moreover, 3 (olmutinib, also known as HM61713)14, with a thieno[3,2-d]pyrimidin core, is approved by the Korean Ministry of Food and Drug Safety approved in 2016 for treatment of NSCLC patients with T790M mutation. Other

third-generation

EGFR

TKIs

including

4

(Nazartinib,

EGF816:

NCT02108964)15, 5 (Naquotinib, ASP8273: NCT02500927)16, 6 (Avitinib, AC0010: NCT02274337)17, and 7 (PF06747775, NCT02349633)18 are currently in early clinical trials.

2. Acquired resistance to third-generation EGFR inhibitors Despite the therapeutic successes of third-generation EGFR TKIs, acquired resistance remains a significant clinical issue in patient’s quality of life. Investigations of 1-3 from clinical trials and in vitro experimental models indicate multiple


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mechanisms of resistance, in part attributed to the known heterogeneity of the NSCLC cell population. Table S1 summarizes the most recognized resistance mechanisms of third-generation EGFR TKIs: 1) EGFR-dependent ligand/receptor amplification and tertiary amino acid missense mutations in the ATP site of the kinase domain, e.g., C797S,19,

20

L718Q,21

G796D,22

L792F/H,23,24

L798I,20

L692V20,

EGFR

amplification25; 2) EGFR-independent (bypass) pathway, attributed to alterations of other signaling molecules, such as MET, FGFR,28 IGF1R29 signaling; and 3) phenotype transformation, e.g., epithelial-mesenchymal transition (EMT) and transformation to small cell lung cancer (SCLC).9,

30

Among all these possible

mechanisms, tertiary C797S mutation in the EGFR kinase domain is the most difficult one to deal with and accounts for more than 20% incidence rate from clinical data.14, 31

2.1 Current understanding of EGFR C797S-mediated resistance Third-generation EGFR TKIs have a Michael receptor warhead component, which can react with active thiols of Cys797 within the EGFR ATP binding domain, thereby forming a covalent bond. However, mutant EGFR with C797S cannot form a covalent bond with third-generation EGFR TKIs, since serine fail to form a covalent bond in a Michael addition under physiological conditions, thus providing a key mechanism of resistance. In 2014, Byrd et al reported a cysteine-to-serine mutation in Bruton’s tyrosine kinase (BTK) which confers resistance to the tyrosine kinase inhibitor, ibrutinib, in chronic lymphocytic leukemia (CLL).32, 33 The finding indicates that the resistance from BTK C481S is mechanistically consistent with that from the



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EGFR C797S mutation. Thress et al.

19

performed droplet digital PCR (ddPCR) on

cell-free plasma DNA (cfDNA) collected from 15 osimertinib-treated patients in the phase I/II AURA trial (NCT01802632). The findings indicate that the acquired C797S mutation occurs in a marked proportion of osimertinib-resistant patients (6 out of 15 cases, 40%). In another case report, a female patient with stage IV lung adenocarcinoma, already harboring EGFR 19 exon deletion and T790M double mutations, develops a third mutation (C797S) following treatment with 9 months with 2.34 Similalry, Ahn et al.35 reported a case of C797S mutation as resistance mechanism of 3. In a phase I/II trial, a 57-year-old female with EGFR 19 exon deletion develops a T790M mutation after gefitinib treatment, and acquires a C797S mutation after 17 months of treatment with 3. Recent clinical study using 2 as first-line treatment of EGFR mutant positive advanced NSCLC (NCT01802632) demonstrated a robust ORR and prolonged PFS in patients. But 2 of 9 resistant patients were detected having EGFR C797S mutation.36 2.2 Occurrence of multiple mechanisms of resistance within a cancer In addition to the acquired C797S mutation, the EGFR-independent pathway also leads to resistance to third-generation TKIs. A study based on tissue biopsies of post-treatment with 2, indicated that MET amplification is one of the acquired mechanisms of resistance.26 The strategy of combined EGFR-TKIs with inhibition of signaling by the EGFR-independent pathway appears to be effective in delaying or overcoming the acquired resistance, driving a growing interest in combined therapy to improve patient outcomes. Although most strategies relied on combining a blocker of


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EGFR in combination with an inhibitor of the EGFR-independent pathway, Moores and coworkers recently developed an antibody with ability to inhibit primary/acquired EGFR mutations as well as the MET pathway.15,

37, 38

Table S2 lists the current

clinical trials launched to evaluate combined therapies of third-generation EGFR-TKIs with inhibitors of EGFR-independent pathway. For 2, more than 13 early-phase clinical trials are currently ongoing, including targeting TORC1/2 (NCT02503722),

Bcl2

(NCT02520778),

VEGFR2

(NCT02143466),

MET

(NCT02143466),

Abl1/Src

(NCT02789345), (NCT02954523),

MEK JAK-1

(NCT02917993), FGFR (NCT02664935), and AKT (NCT02664935). The therapeutic results are highly anticipated in the near future.

3. Emerging EGFR-TKIs to combat C797S resistance EGFR C797S point mutation is the most common mechanism of acquired resistance to third-generation EGFR-TKIs. Niederst et al.

39

reported that the allelic

context of the acquired C797S mutation after treatment with third-generation EGFR-TKIs may impact subsequent clinical treatment decisions. For example, with the case of C797S and T790M mutations in trans, cancer cells are resistant to third-generation TKIs, but sensitive to a combination treatment of first- and third-generation TKIs. And if the mutations are in cis, the current EGFR-TKIs are ineffective in suppressing tumor progression.39 Wang et al.

40

was the first to report

the clinical efficacy of combination of first- and third- generation EGFR-TKIs targeting EGFR T790M and C797S in trans. However, after 3 months of PFS, the



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patient experienced exacerbation with the emergence of EGFR C797S located in cis with T790M. Thus, new agents to overcome the L858R/T790M/C797S or del19/T790M/C797S mutatations are critically important. Emerging structural studies of EGFR mutants are providing invaluable insights of binding modes and pharmacological significance. Recently, Kong and coworkers41 generated complex crystal structures of EGFR mutants T790M/C797S (PDB: 5XGN) and observed that the C797S mutation increases the local hydrophilicity around residue 797, without affecting the structure and function of the kinase domain. Therefore, in addition to the importance of covalent binding with Cys797, construction of small molecule inhibitors which enhance the binding affinity to other regions of the EGFR kinase domain as well as identification of new allosteric sites are key elements in the development of fourth-generation EGFR inhibitors. 3.1 EAI045 To search for an allosteric EGFR inhibitor, Jia et al. compound

library,

identifying

8

42

screened a 2.5 million

(EAI001,

2A)43,

Figure

N-(4,5-dihydrothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)-2-phenylacetamide.

It

has

selectivity against the L858R/T790M mutant but spares the wild-type form. High concentration

ATP

screening

further

confirm

that

8

is

a

potentially

non-ATP-competitive (allosteric) inhibitor. The co-crystal structure of 8 with the EGFR T790M-mutant kinase (PDB ID: 5D41) shows the inhibitor bound within the allosteric pocket near the classic ATP binding pocket, which is partially formed by the outward displacement of the


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αC-helix structure (Figure 2D).42 8 binds as a “three-bladed propeller” or “Y shaped” configuration.14 As shown in Figure 2B, the aminothiazole fragment stretches to the T790M gatekeeper residue, accounting for the selectivity for the T790M mutant. The phenyl group inserts into a hydrophobic gap at the back of the ATP binding pocket, thereby interacting with the Leu-777 and Phe-856 residues, while the third region, the oxoisoindolinyl moiety, extends along the αC-helix towards the solvent exposed area. In addition, the amido group of 8 forms a hydrogen bond with Asp-855 in the Asp-Phe-Gly (DFG) motif.42 Further medicinal chemistry optimization of its phenyl group yielded a more potent inhibitor 9 (EAI045, Figure 2C, EGFR L858R/T790M IC50 = 3 nM), and maintains 1000-fold selectivity against wild-type EGFR. However, the binding site of 9 was created by the movement of the αC-helix when EGFR kinase was in the inactive conformation. And two subunits of the ErbB asymmetric dimer makes the allosteric binding site inaccessible, thus the efficacy of single 9 agent is limited. With the combination use of dimer-disrupting antibody cetuximab, each EGFR kinase domain can be successfully accessed by 9. Compound 9 synergizes with cetuximab to promote tumor regression in L858R/T790M EGFR and L858R/T790M/C797S EGFR mutants in mouse models.44 Because of its distinct binding site, the authors also speculate that combination use of ATP-site-directed inhibitors and 9 is expected to overcome the acquired resistance. Interestingly, the second-generation EGFR inhibitor neratinib not only occupies the ATP site, but also extends into the allosteric pocket, occupying two of the three blades of 9.45 Thus, we speculate that structure-based



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design of fourth-generation EGFR-TKIs enabling interactions with both ATP and allosteric sites will be of great value. Please insert Figure 2 here 3.2 Brigatinib Duong-Ly et al.

46

recently screened 183 small-molecule compounds against 76

drug-resistant mutant kinases and reported the potential repurposing opportunities for FDA-approved kinase inhibitors. Based on this information, Uchibori et al.47 conducted a focused drug screening to examine their efficacy against triple-mutant (del19/T790M/C797S) EGFR. Among 30 selected compounds, 10 (brigatinib) potently inhibits the proliferation of Ba/F3 cells harboring the triple-mutation. Different from the inhibition mechanism of 9, 10 acts on EGFR kinase domain by competitive inhibition of ATP binding47 Please insert Figure 3 here 10 was developed as a next-generation inhibitor targeting anaplastic lymphoma kinase (ALK), thus several other ALK-TKIs with similar chemical structures to 10 were also investigated, namely 11 (TAE684), 12 (ASP3026), 13 (AP26113-analog), 14 (ceritinib) and 15 (AZD3463) (Figure 3).48, 49 Initial structure-activity relationship (SAR) analysis reveals functional groups important for maintaining the activity to the triple-mutant EGFR. The replacement of a 4-methylpiperazin group (10) to a dimethylamino moiety (13) barely affected the inhibitory potency against del19/T790M/C797S

triple-mutant

EGFR.

However,

introduction

of

the

isopropylsulfonyl (11) and indole (15) group decreases activity against the


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triple-mutant EGFR, suggesting that phosphine oxide group in 10 is more favored. The removal of chloride (12) leads to a great loss of activity, and moreover, the isopropoxy group in 14 might work as a negative factor. Overall, this area needs more systematic SAR analysis and investigation. Studies of in silico docking indicate that the 2-aminopyrimidine core of 10 forms two classic hydrogen bonds with the backbone amide of residue Met793 and fits into the pocket without steric hindrance to C797S or T790M. The phosphine oxide moiety completely occupies the triphosphate-binding region in the ATP-binding pocket, providing electrostatic or van der Waals interactions. Phosphine oxide-linked phenyl and piperidin groups contribute less to the affinity than other components and allows for further structural modification. In a nude mice model implanted with EGFR del19/C797S/T790M-expressing PC9 cells, treatment with 10 at 75 mg/kg/day showed significant inhibition of the tumor growth, compared with 2 (50 mg/kg/day) treated group. In addition, administration of 10 (75mg/kg) combined with cetuximab (1 mg per mouse) three times a week synergistically suppressed the growth of EGFR del19/C797S/T790M-expressing cells in vivo.47 These in vivo evidences demonstrated the potential of 10. A Phase 2 trial is ongoing to assess the preliminary anti-tumor activity of 10, both in non-small cell lung cancer (NSCLC) with ALK gene rearrangement or mutated EGFR (NCT01449461). However, 10 is an inhibitor targeting multiple kinases, including ALK, ROS1, FLT3, and EGFR. We believe that 10 might be an appropriate lead compound for the design of next-generation EGFR inhibitors. It would be crucial to develop more specific and potent inhibitors against



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EGFR triple mutants by medicinal chemistry methods. 3.3 4-aminopyrazolopyrimidines Please insert Figure 4 here 4-aminopyrazolopyrimidine is one of the important kinase hinge range binding pharmacophores.50 The most extensively studied 4-aminopyrazolopyrimidine molecule, ibrutinib, has excellent antitumor activities in EGFR mutant NSCLC and is now in phase I/II clinical trial for NSCLC (NCT02321540).51 Based on ibrutinib’s scaffold, Wang et al.

52

discovered compound 17 with high potency against EGFR

drug-resistant mutant L858R/T790M (IC50 < 0.3 nM). However, 17 only has moderate anti-proliferative activity in EGFR-BaF3 isogenic cell lines harboring C797S mutation (IC50 > 2 µM). Engel et al.

53

reported three 4-aminopyrazolopyrimidines and evaluated their

efficacy for EGFR drug-resistant mutants, including recently discovered C797S tertiary mutation. Compounds 18-20 display excellent inhibition in H1975 cells (GI50 values of 0.21 µM, 0.49 µM, and 0.14 µM respectively). Notably, compound 20, decorated with 2-napthalene, has promising activity against the L858R/T790M/C797S mutant kinase with IC50 of 88 nM. However, 20 is structurally similar to 17, which has only moderate activity. Clearly, additional investigation of compound 20 in C797S mutant harboring cell lines as well as in vivo is needed for further confirmation.

3.4 Tri-substituted imidazoles


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Please insert Figure 5 here Through screening for kinase selectivity of a p38α MAP kinase inhibitor 21, Selig et al.

54

found EGFR as one of its off-targets. Since 21 is a promising lead

compound with low toxicity, Laufer and his colleagues

55, 56

employed target hopping

strategies to discover novel EGFR inhibitors against EGFR L858R/T790M and clinically challenging L858R/T790M/C797S mutants. By transferring the aromatic phenol group of 21 to aliphatic alcohol chains, active compounds 22 (2-hydroxypropyl) and 23 (4-hydroxypropyl) are produced that inhibit the triple mutant with IC50 values of 7.6 nM (22) and 8.4 nM (23), respectively. In comparison, 2 exhibits about 35-fold weaker potency than 22 and 23 with an IC50 value of 278 nM 56

. Molecular docking studies suggest that the aliphatic alcohol chains form beneficial

hydrogen bonds to Asn-842, thereby increasing inhibitory activity.55 At the same time, the Laufer group

55

screened about 2000 compounds that are

structurally-related to 21 against EGFR kinase and discovered compound 24 as a promising lead structure for further modification. SAR examination has led to development of active compounds consisting of multiple hydrophobic and polar interactions with several regions within the EGFR ATP binding domain. Compounds 25-2757 with or without the Michael receptor reversibly inhibit the EGFR L858R/T790M/C797S triple mutant with low nanomolar IC50 values. Our modeling studies of 25 with EGFR T790M/C797S reveal that the aminopyridine moiety binds to the hinge region with bidentate hydrogen bonds, and the fluorophenyl group contributes to the hydrophobic interactions with surrounding residues (Figure 6AB).



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In addition, a polar contact occurs between the hydroxyl group and the Arg-841. And two hydrogen bonds are formed between the carbanyl group and the mutated Ser-797. In terms of the discovery of C797S-targeting inhibitors, compound without a Michael receptor (e.g., 26) may be more selective and safe since they lose the ability to covalently binding to off-target kinases. The trisubstituted imidazoles may serve as potential lead for the further development of highly potent fourth-generation EGFR inhibitors. Further optimization needs to reduce the inhibitory activity on wild-type EGFR. Please insert Figure 6 here 3.5 2-aryl-4-aminoquinazolines Please insert Figure 7 here Recently, Park et al. 58 relied on a two-track virtual screening strategy to search for EGFR del/T790M/C797S mutant selective inhibitors with low binding free energies but high binding free energies to WT EGFR. After virtual and experimental screening of a 670,000 compound chemical database, hit compound 28 possesses promising sub-micromolar inhibitory activity against the EGFR del/T790M/C797S mutant and 163-fold selectivity over WT EGFR. SAR examination indicates that the most potent compounds 29, 30, and 31 display more than 1000-fold selectivity for the triple mutant over the wild type, as well as nanomolar inhibitory activity.58 Binding mode analysis (Figure 6CD) reveals the terminal phenolic moiety of 31 forming two hydrogen bonds with Met-793 and Gln-791, respectively. Moreover, the pyridin-3-yl-methylamine moiety forms two more hydrogen bonds with the hydroxy


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group of the mutated Ser-797. The removal of -OH group or change of the -NH to oxygen leads to loss of inhibitory activity, further confirming the strong interactions between compounds, Ser-797 and Met-793 residues.58

4. Real-time circulating tumor DNA as marker for NSCLC Personalized medicine in cancer therapy relies on the customization of healthcare guided by molecular analyses.59 In the clinical setting, tissue-based tumor sample cannot reflect the overall heterogeneity of the tumor, and cannot be obtained repeatedly because of its invasive procedure. In contrast, the detection of circulating tumor DNA (ctDNA), commonly known as ‘liquid biopsies’, can help monitor response to therapy, identify acquired drug resistance in a timely way, and enable clinicians to repeatedly and non-invasively obtain blood samples to track the progression of cancers.60 Over the past 5 years, technologies to analyse ctDNA, especially droplet digital PCR (ddPCR) and next-generation sequencing (NGS), have advanced significantly. Specifically, ddPCR can monitor the dynamic individual mutations, but requires to know the mutant allele in advance. However, NGS has strong advantages over ddPCR in discovering novel mutated variants and has becoming a key tool for precision medicine. Though the clinical utility of NGS remains to be verified in more comprehensive studies, this approach may become preferable over ddPCR during the treatment of NSCLC due to the high complexity and unpredictable of C797S mutations, including multiple DNA variants, cis versus trans, different co-existing C797S mutations, and other tertiary EGFR mutations.61



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The US FDA recently has approved the use of liquid biopsies for detection of EGFR mutations in NSCLC patients. Such methods provide a comprehensive view of specific resistance mutations during a treatment period, such as acquired EGFR gatekeeper and C797S mutations associated with EGFR-TKIs treatment.59, 62, 63

5. Conclusions Treatment of NSCLC patients with the T790M mutation with third-generation EGFR-TKIs is highly promising, but drug resistance still occurs. Novel technologies which detect ctDNA are highly useful by monitoring dynamic mutations in disease progression. Among the escaped mechanisms, C797S point mutation is the most frequent mechanism of acquired resistance to third-generation TKIs attributed to loss of the irreversible binding site of third-generation TKIs. Moreover, when C797S and T790M alleles occur in cis, there is no current therapeutic drug available to suppress disease progression. Thus, there is an urgent need to develop therapeutic agents effective against EGFR tertiary mutants. The relatively new development of next generation small molecule EGFR-TKIs with different scaffolds and binding mechanisms appears hopeful. The ongoing combination trials with third-generation TKIs may change the treatment paradigm for EGFR mutant NSCLC in the near future (Figure 8). Please insert Figure 8 here Highly potent non-ATP-competitive MEK1/2 allosteric inhibitors (cobimetinib and refametinib) have been developed and tested in numerous clinical trials over the


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past decade.64, 65 The recently developed EGFR allosteric inhibitor 9, which binds to the analogous site, showed excellent therapeutic efficacy to lung cancers driven by the triple L858R/T790M/C797S mutant when synergized with cetuximab. Through mutant targeted drug screening and structure-based de novo optimization, other novel ATP-competitive molecules with a distinct scaffold from third-generation EGFR-TKIs are identified and demonstrated excellent inhibitory activity against acquired C797S resistant mutants. Despite these advances, several major limitations and challenges remain; 1) Allosteric inhibitor, 9, is capable of inhibiting the EGFR L858R/T790M/C797S mutation, but may lose its efficacy against del/T790M/C797S due to the reduced space of the allosteric pocket. The competitive inhibitor, 10, tends to be less sensitive to L858R/T790M/C797S mutant than del19/T790M/C797S. Additionally, both 9 and 10 exhibit mechanistic synergy when combined with cetuximab, but the potency of single therapy is not satisfying in vivo. Thus, there remains a big challenge to develop fourth-generation TKIs with broader drug-resistant mutant selectivity and greater inhibitory capacity. 2) Several newly discovered molecules, i.e., tri-substituted imidazoles and 4-aminopyrazolopyrimidines, exhibit potent activity against the C797S mutant, but has low or no selectivity against WT EGFR. With the recognized importance of EGFR T790M and C797S mutants in acquired resistance, development of TKIs with high selectivity for the mutants is critical in the next generation drug discovery. 3) And finally, more comprehensive in vivo studies of newly developed agents are needed to validate their in vitro efficacy.



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Author information Corresponding authors *G.L.: E-mail: [email protected]; phone, +86-577-86699396.

Notes The authors declare that there are no conflicts of interest.

Biographies Lingfeng Chen received his B.S. and M.S. degree in Pharmacy from Wenzhou Medical University in China. Now he is studying for a Ph.D. degree at Nanjing University of Science and Technology in China. The focus of his research is on structure-based drug design, lead optimization and biological activity screening of tyrosine kinase inhibitors. During his master degree study, he has designed and synthesized a number of EGFR and FGFR kinase inhibitors as potential anti-cancer candidates. Weitao Fu obtained his B.S. degree and M.S. degree in Pharmacy at Wenzhou Medical University in China. Now he is studying for Ph.D. degree in Pharmacy at Zhejiang University in China. His research interests lie in the area of computer-aided drug design, including molecular docking, structure-based virtual screening, molecular dynamics simulations, with primary focus on the structure-based design of anti-prostate cancer targeting androgen receptor.


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Lulu Zheng received her B.S. and M.S. degree in Pharmacy from Wenzhou Medical University in China. The focus of her research is on anticancer drug screening especially against Epidermal Growth Factor Receptor and Fibroblast Growth Factor Receptor. Using kinase and cellular based assays and xenograft animal models, she has discovered several promising candidates for further exploration. Zhiguo Liu earned his B.S. degree in pharmaceutical engineering from Changchun University in 2004, and then received his Ph.D. degree in Medicinal Chemistry from Chonnam National University (Gwangju, Korea) in 2011. Now, he is a full professor in School of Pharmaceutical Sciences of Wenzhou Medical University, China. Dr. Liu’s research interests are the design and synthesis of new chemotherapeutic drugs as well as the discovery of new bioactive natural products. He has published more than 40 peer-reviewed articles as the first or corresponding author in the fields of medicinal chemistry and organic chemistry. Guang Liang earned his B.S. degree in applied chemistry from Beijing University of Chemical Technology in 2002, and received his Ph.D. in biological chemistry from Nanjing University of Science and Technology (Nanjing, China) in 2008. Now, he is a full professor and the dean of School of Pharmaceutical Sciences in Wenzhou Medical University, China. His research interest is focused on design and discovery of novel anti-inflammatory and anti-cancer small molecules. He has published more than 100 peer-reviewed articles as the corresponding author in the fields of medicinal chemistry and biochemical pharmacology.



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Acknowledgments This work was supported by the National Natural Science Funding of China (81622043, 81773579), and the Natural Science Funding of Zhejiang Province (LR16H310001, LY17B020008).

Abbreviations Used NSCLC, non-small-cell lung cancer; EGFR, epidermal growth factor receptor; TKIs, tyrosine kinase inhibitors; WT, wild type; ctDNA, circulating tumor DNA; cfDNA, cell-free plasma DNA; ddPCR, droplet digital PCR; NGS, next-generation sequencing; EMT, epithelial-mesenchymal transition; SCLC, transformation to small cell lung cancer; BTK, Bruton’s tyrosine kinase; CLL, chronic lymphocytic leukemia; PFS, progression free survival; ALK, anaplastic lymphoma kinase; SAR, structure-activity relationship; FAK, focal adhesion kinase; SFK, Src family kinase; PTEN, phosphatase and tensin homolog deleted on chromosome ten; FGFR, fibroblast growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; VEGFR, Vascular Endothelial Growth Factor Receptor; JAK-1, Janus kinase 1.

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L858R/T790M/C797S mutants: an example of target hopping. J. Med. Chem. 2017, 60, 4636-4656. 57. Gunther, M.; Lategahn, J.; Juchum, M.; Doring, E.; Keul, M.; Engel, J.; Tumbrink, H. L.; Rauh, D.; Laufer, S. Trisubstituted pyridinylimidazoles as potent inhibitors of the clinically resistant L858R/T790M/C797S EGFR mutant: targeting of both hydrophobic regions and the phosphate binding site. J. Med. Chem. 2017, 60, 5613-5637. 58. Park, H.; Jung, H. Y.; Mah, S.; Hong, S. Discovery of EGF receptor inhibitors



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that are selective for the d746-750/T790M/C797S mutant through structure-based de novo design. Angew. Chem. Int. Ed. Engl. 2017, 56, 7634-7638. 59. Perez-Callejo, D.; Romero, A.; Provencio, M.; Torrente, M. Liquid biopsy based biomarkers in non-small cell lung cancer for diagnosis and treatment monitoring. Transl Lung Cancer Res. 2016, 5, 455-465. 60. Siravegna, G.; Marsoni, S.; Siena, S.; Bardelli, A. Integrating liquid biopsies into the management of cancer. Nat. Rev. Clin. Oncol. 2017, 14, 531-548. 61. Cai, X.; Janku, F.; Zhan, Q.; Fan, J. B. Accessing genetic information with liquid biopsies. Trends Genet. 2015, 31, 564-575. 62. Oellerich, M.; Schutz, E.; Beck, J.; Kanzow, P.; Plowman, P. N.; Weiss, G. J.; Walson, P. D. Using circulating cell-free DNA to monitor personalized cancer therapy. Crit. Rev. Clin. Lab. Sci. 2017, 54, 205-218. 63. Remon, J.; Caramella, C.; Jovelet, C.; Lacroix, L.; Lawson, A.; Smalley, S.; Howarth, K.; Gale, D.; Green, E.; Plagnol, V.; Rosenfeld, N.; Planchard, D.; Bluthgen, M. V.; Gazzah, A.; Pannet, C.; Nicotra, C.; Auclin, E.; Soria, J. C.; Besse, B. Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA. Ann. Oncol. 2017, 28, 784-790. 64. Zhao, Y.; Adjei, A. A. The clinical development of MEK inhibitors. Nat. Rev. Clin. Oncol. 2014, 11, 385-400. 65. Rice, K. D.; Aay, N.; Anand, N. K.; Blazey, C. M.; Bowles, O. J.; Bussenius, J.; Costanzo, S.; Curtis, J. K.; Defina, S. C.; Dubenko, L.; Engst, S.; Joshi, A. A.; Kennedy, A. R.; Kim, A. I.; Koltun, E. S.; Lougheed, J. C.; Manalo, J. C.; Martini, J.


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F.; Nuss, J. M.; Peto, C. J.; Tsang, T. H.; Yu, P.; Johnston, S. Novel carboxamide-based allosteric MEK inhibitors: discovery and optimization efforts toward XL518 (GDC-0973). ACS Med. Chem. Lett. 2012, 3, 416-421.

Figure Legends Figure 1. Third-generation EGFR-TKIs and their clinical trials status. The image of EGFR T790M/C797S kinase domain was generated by Pymol (PDB: 5XGN). Figure 2. Structure and binding mode of the allosteric inhibitors. (A) Chemical structure of 8. (B) Overall view of 8 bound to EGFR T790M/V948R kinase (PDB, 5D41). 8 is shown in green, kinase protein in pink, αC-helix in slate. Hydrogen bond between 8 and DFG is shown as red dotted line. (C) Chemical structure of 9. (D) The overview of the classic EGFR ATP binding pocket (green) and the created allosteric site (yellow). Figure 3. Chemical structures of inhibitors 10-15 and their cell growth inhibition activities against Ba/F3 cells expressing del19/T790M/C797S EGFR-mutants using the CellTiter-Glo assay. Figure 4. Chemical structures of inhibitors 16-20 and their activities against EGFR kinase and its mutants. The enzymes activities of 18-20 against EGFR kinase and its mutants were performed using HTRF KinEASE-TK assay. Figure 5. Chemical structures of inhibitors 21-27 and their activities against EGFR kinase and its mutants. The enzymes activities of 21-27 were tested in a radiolabeled 33P(ATP) kinase assay or HTRF KinEASE-TK assay.



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Figure 6. Inhibitors binding with EGFR C797S/T790M mutant. (A) Chemical structure of 25 and its depicted binding mode. (B) The binding mode of 25 with the EGFR C797S/T790M mutant. (C) Chemical structure of 31 and its depicted binding mode. (D) The binding mode of 31 with the EGFR C797S/T790M (PDB: 5XGN) mutant. Figure 7. Chemical structures of inhibitors 28-31 and their activities against EGFR kinase and its mutants using radiolabeled 33P(ATP) kinase assay. Figure 8. Intratumoral heterogeneity and future treatment paradigm of patients with EGFR-mutant NSCLC. Each colored ball represents a distinct clone with newly acquired resistance mechanism.

Table of Contents graphic


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Figure 1. Third-generation EGFR-TKIs and their clinical trials status. The image of EGFR T790M/C797S kinase domain was generated by Pymol (PDB: 5XGN). 872x876mm (96 x 96 DPI)



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Figure 2. Structure and binding mode of the allosteric inhibitors. (A) Chemical structure of EAI001. (B) Overall view of EAI001 bound to EGFR T790M/V948R kinase (PDB, 5D41). EAI001 is shown in green, kinase protein in pink, αC-helix in slate. Hydrogen bond between EAI001 and DFG is shown as red dotted line. (C) Chemical structure of EAI045. (D) The overview of the classic EGFR ATP binding pocket (green) and the created allosteric site (yellow). 968x835mm (96 x 96 DPI)


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Figure 3. Chemical structures of inhibitors 10-15 and their cell growth inhibition activities against Ba/F3 cells expressing del19/T790M/C797S EGFR-mutants using the CellTiter-Glo assay. 119x58mm (300 x 300 DPI)



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Figure 4. Chemical structures of inhibitors 16-20 and their activities against EGFR kinase and its mutants. The enzymes activities of 18-20 against EGFR kinase and its mutants were performed using HTRF KinEASETK assay. 155x128mm (300 x 300 DPI)


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Figure 5. Chemical structures of inhibitors 21-27 and their activities against EGFR kinase and its mutants. The enzymes activities of 21-27 were tested in a radiolabeled 33P(ATP) kinase assay or HTRF KinEASE-TK assay. 151x89mm (300 x 300 DPI)



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Figure 6. Inhibitors binding with EGFR C797S/T790M mutant. (A) Chemical structure of 25 and its depicted binding mode. (B) The binding mode of 25 with the EGFR C797S/T790M mutant. (C) Chemical structure of 31 and its depicted binding mode. (D) The binding mode of 31 with the EGFR C797S/T790M (PDB: 5XGN) mutant. 942x828mm (96 x 96 DPI)


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Figure 7. Chemical structures of inhibitors 28-31 and their activities against EGFR kinase and its mutants using radiolabeled 33P(ATP) kinase assay. 64x16mm (300 x 300 DPI)



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Figure 8. Intratumoral heterogeneity and future treatment paradigm of patients with EGFR-mutant NSCLC. Each colored ball represents a distinct clone with new acquired resistance mechanism. 1340x876mm (96 x 96 DPI)


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Recent Progress of Small-Molecule Epidermal Growth Factor

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