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. 2024 Aug 30;15(10):3371–3394. doi: 10.1039/d4md00384e

Next-generation EGFR tyrosine kinase inhibitors to overcome C797S mutation in non-small cell lung cancer (2019–2024)

Debasis Das a,✉, Lingzhi Xie a, Jian Hong a
PMCID: PMC11376191  PMID: 39246743

Abstract

Lung cancer is a leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for the major portion (80–85%) of all lung cancer cases. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are commonly used as the targeted therapy for EGFR-mutated NSCLC. The FDA has approved first-, second- and third-generation EGFR-TKIs as therapeutics options. Osimertinib, the third-generation irreversible EGFR-TKI, has been approved for the treatment of NSCLC patients with the EGFRT790M mutation. However, due to the EGFRC797S mutation in the kinase domain of EGFR, resistance to osimertinib is observed and that limits the long-term effectiveness of the drug. The C797S mutation is one of the major causes of drug resistance against the third-generation EGFR TKIs. The C797S mutations including EGFR double mutations (19Del/C797S or L858R/C797S) and or EGFR triple mutations (19Del/T790M/C797S or L858R/T790M/C797S) cause major resistance to the third-generation EGFR-TKIs. Therefore, the discovery and development of fourth-generation EGFR-TKIs to target triple mutant EGFR with C797S mutation is a challenging topic in medicinal chemistry research. In this review, we discuss the discovery of novel fourth-generation EGFR TKIs, medicinal chemistry approaches and the strategies to overcome the C797S mutations. In vitro activities of EGFR-TKIs (2019–2024) against mutant EGFR TK, anti-proliferative activities, structural modifications, binding modes of the inhibitors and in vivo efficacies in animal models are discussed here.

Prospects of novel fourth-generation EGFR-TKIs overcoming C797S-mediated resistance in non-small cell lung cancer.graphic file with name d4md00384e-ga.jpg

1. Introduction

Lung cancer is a very aggressive disease and it is the leading cause of cancer-related deaths worldwide.1 As per the report from the International Agency for Research on Cancer (IARC), approximately 2.5 million new lung cancer cases were recorded worldwide in 2022.2

Lung cancer is prevalent in highly populated countries including India3 and China. The burden of lung cancer has been increased over the past three decades in China.4 Cigarette smoking is considered as the number one risk factor for lung cancer in smokers. For non-smokers, risk factors include exposure to secondhand smoke, radon, radioactive radiation, carcinogens and heredity. The major portion (80–85%) of the total lung cancer patients is detected with non-small cell lung cancer (NSCLC) and the remaining 10–15% of the lung cancer patients are detected with small cell lung cancer (SCLC).5 The relative survival rate for NSCLC is low due to its late stage detection. Brain metastases are commonly detected in approximately 25–40% of NSCLC patients after two years of disease progression.6 Earlier NSCLC cases were treated by surgery, chemotherapy and radiation therapy or their combinations. Due to the advancement of cancer biology and detection techniques in recent years, the diagnosis of NSCLC cases has become easier7 and the treatment options for NSCLC cases have been evolved.8 Targeted therapy, including tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs) and monoclonal antibodies (mAbs), are widely used as treatment options.9

At the molecular level, the progression of NSCLC is closely associated with mutation and overexpression of the epidermal growth factor receptor (EGFR) gene. EGFR is a transmembrane receptor tyrosine kinase, encoded from the EGFR gene. EGFR regulates a number of cellular processes, such as cell proliferation, metabolism, apoptosis and survival. EGFR consists of an extracellular ligand binding domain, a transmembrane (TM) domain and an intracellular kinase domain.10 EGFR forms homodimers and heterodimers with other human ErbB family receptors after binding with an epidermal growth factor (EGF), and subsequently activates TK domains. Activated EGFR promotes autophosphorylation and initiates complex downstream signalling pathways, including the mitogen-activated protein kinase signalling pathway (RAS–RAF–MEK–ERK1/2) and phosphatidyinositol-2-kinase (PI3K)–AKT–mTOR pathway.11 Overexpression, amplification and activating mutations of EGFR are commonly observed in NSCLC cases. Mutation of EGFR occurs within exons 18–21 of the TK-domain of EGFR (Fig. 1). Approximately 90% of the activating EGFR mutations in NSCLC cases involve the “common mutations”, including exon19 deletions (19Del/del19/Del19) and a point mutation, substitution of leucine to arginine at 858 (L858R) in exon 21. So, EGFR tyrosine kinase has become a target for NSCLC treatment, and several tyrosine kinase inhibitors (EGFR-TKIs) have been developed to control unwanted EGFR activities.

Fig. 1. Schematic representation of the EGFR domains, exons and mutations in NSCLC.

Fig. 1

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The US Food and Drug Administration (FDA) approved a few EGFR TKIs in recent years. The first-generation EGFR-TKIs, gefitinib (1) and erlotinib (2) (Fig. 2), are reversible, quinazoline-based reversible inhibitors that are sensitive to EGFR common mutations (19Del, L858R).12,13 These inhibitors significantly delay the progression of disease compared to platinum-based and docetaxel chemotherapy. Despite the initial good response to the treatment with first-generation TKIs, nearly 50% of the NSCLC patients gained an additional gate keeper mutation (T790M) after 10–16 months of treatment. The mutation enhances the affinity of EGFR tyrosine kinase (EGFR-TK) with ATP and weakens the binding of the first-generation EGFR TKIs, causing drug resistance. The FDA approved the quinazoline-based irreversible covalent inhibitors, afatinib (3) and dacomitinib (4), as the second-generation EGFR-TKIs to reduce the T790M mutation problem.14,15

Fig. 2. A schematic representation of the evolution of the 1st, 2nd, 3rd and 4th-generation EGFR TKIs against EGFR mutations, and the FDA approved drugs for NSCLC treatments.

Fig. 2

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Available Michael acceptors on the second-generation EGFR TKIs increase the stability of inhibitor and EGFR-TK complex by forming covalent bonds with the thiol-moiety of the Cys797 residue. The second-generation inhibitors display nano-molar potency, but show poor selectivity to EGFRs. The second-generation TKIs inhibit mutant EGFR, as well as the wild type EGFR (EGFRwt). Due to lack of selectivity, these EGFR TKIs cause dose-related side effects such as skin irritation/rash and diarrhoea. As the second-generation EGFR-TKIs do not eliminate the T790M mutation issue completely, third-generation EGFR-TKs were thus discovered. A pyrimidine scaffold with a Michael acceptor is commonly used as the structural motif for the third-generation EGFR TKIs to form the Cys797 site. The FDA approved osimertinib (5), the first third-generation EGFR-TKI16 to patients who are resistant to the first- and second-generation EGFR TKIs.16,17 Recently, the FDA approved another two EGFR-TKIs with a pyrimidine scaffold, mobocertinib (6) and lazertinib (7), for EGFR exon 20 insertion and targeting T790M mutation.18,19 Osimertinib already demonstrated remarkable efficacy and low toxicity. However, an acquired drug resistance was observed after 10 months after administration of osimertinib to the patients. The mechanisms of resistance to third-generation EGFR-TKIs are complex. For the most common cases, about 40% of the drug resistance is due to C797S mutation.20 These C797S mutations include double mutations (19Del/C797S or L858R/C797S) and/or triple mutations (19Del/T790M/C797S or L858R/T790M/C797S). A few uncommon mutations (L718Q, G796D, etc.) and bypass activation are observed for third-generation inhibitors.21 After EGFRC797S mutation, the 3rd-generation drugs cannot form covalent bonds with the EGFR, reducing the drug and ATP competitive activity. To overcome the C797S mutation problem, the next-generation or fourth-generation EGFR TKIs are being developed.

The fourth-generation EGFR TKIs are discovered to overcome the acquired mutation of C797S.22–25 The research area is flourishing very fast, and many new chemical structures of EGFR TKIs have been discovered recently. In this article, we summarise the discovery works of the fourth-generation EGFR TKIs that have been published in different journals from 2019 to 2024, to overcome drug resistance due to the C797S mutation. The latest information and publications up to May 15, 2024 are reported here.

2. Fourth-generation EGFR-TKIs

Despite the excellent performance of osimertinib, a 3rd-generation EGFR TKI-acquired resistance remains a major issue of the quality of life of patients. No approved clinical strategy available for the patients who developed resistance to third-generation TKI treatments and acquired triple mutations. Hence, the next-generation EGFR TKIs are expected to overcome the triple mutations, including EGFRDel19/T790M/C797S and EGFRL8585R/T790M/C797S mutation.26,27

2.1. ATP-competitive fourth-generation EGFR TKIs

2.1.1. 4-Aminopyrimidine derivatives

(a). CH7233163 (8)

Kashima et al. identified CH7233163 (8) as a potent EGFR TKI against the EGFRDel19/T790M/C797S mutant.28 In a biochemical assay, CH7233163 exhibited strong potency against EGFRDel19/T790M/C797S with IC50 = 0.28 nM, and it was much better than the allosteric inhibitor EAI045 (IC50 > 1000 nM). The crystal structure analysis of CH7233163 appeared as a non-covalent ATP competitive inhibitor (Fig. 3).

Fig. 3. Chemical structure of CH7233163 and interactions of EGFR-L858R/T790M/C797S (a cartoon diagram in light green) with CH7233163, blue-colour-coded stick model (PDB: 6LUB).28.

Fig. 3

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In a Del19/T790M/C797S NIH3T3 xenograft mouse model, a significant tumor regression was observed by CH7233163 at 100 mg kg−1 dose. No sign of body weight change and toxicity was observed during the study. They could prepare a crystal grade complex of EGFRL858R/T790M/C797S with CH7233163, but they observed purification difficulty for the complex of CH7233163 with EGFRDel19/T790M/C797S. The interaction of CH7233163 with EGFRL858R/T790M/C797S is shown in Fig. 3(b). CH7233163 reached the ATP-binding pocket and interacted with the gatekeeper residue T790, and it was confirmed as an ATP-competitive inhibitor (PDB.ID: 6LUB).

(b). BLU-945 (9)

Blueprint Medicines developed BLU-945 (9, Fig. 4), a fourth-generation EGFR-TKI that targeted the EGFRL858R/T790M/C797S mutation and other resistance mutations.29 Using the KINOMEscan screening platform, >25 000 small-molecule kinase inhibitors were screened over 400 kinases30 and compound 10 was identified as the “Blueprint Library Hit” with moderate potency, EGFRL858R/T790M (IC50 = 290 nM) and EGFRL858R/T790M/C797S (IC50 = 266 nM) mutations and good stability (HLM Clint (μL min−1 mg) = 4.0), where HLM Clint was the measurement of clearance obtained from isolated human liver microsome. An extensive SAR studies and structure optimization led to a sulfone-containing compound 11 with excellent stability and potency (Table 1). However, compound 11 suffered from low oral bioavailability (F = 2%). The MDCK-MDRI assay revealed that compound 11 had poor passive permeability and high efflux ratio, indicating a risk or P-gp mediated active efflux. Then, they addressed the TPSA of the core by replacing one of the nitrogen atoms with carbon to an isoquinoline derivative 12 that improved the oral bioavailability in rat (F = 85%, Cl = 20 mL min−1 kg−1). Further modification and addition of a 2-methyl on the azetidine ring gave compound 13 with an improvement of potency and WT selectivity (Tables 1 and 2).

Fig. 4. Discovery of BLU-945 from hit at blueprint medicines and structure optimization sequence.

Fig. 4

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Observed SAR data for improved bioavailability and stability30.
11 12 13
Enz EGFR LR/TM IC50 (nM) 0.1 0.2 0.3
Enz EGFR LR/TM/CS IC50 (nM) 0.1 0.2 0.2
Enz EGFRwt IC50 (nM) 1050 385 505
MDCK-MDR1 PA-B/efflux 2/32 17/4 9/3
Rat IV PK Cl (mL min−1 kg) (Clu), t1/2, F (%) 67 (838), 1.6 h, 2% 20(833), 3.0 h, 85% 25 (847), 1.3 h, 50%
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In vitro antiproliferative activity of 9, 11, 12, 13 in different cell lines; IC50 in nM.
Compound H1975 (EGFRLR/TM) A431 (EGFRwt)
11 4.8 1608
12 2.7 1362
13 1.0 1780
BLU-945 (9) 1.1 544
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However, compound 13 showed UGT-mediated glucuronidation in humans, resulting in high in vivo clearance. To overcome the problem, methylation of the hydroxyl group on 13 was done and BLU-945 (9) was discovered. BLU-945 showed very good enzyme inhibition against EGFRL858R/T790M (IC50 = 0.4 nM), EGFRL858R/T790M/C797S (IC50 = 0.5 nM), and selectivity over wild-type EGFR >900 times (IC50 = 683 nM). In the cell assay, BLU945 showed about 500 times better selectivity against mutant EGFR (IC50 = 1.1 nM) over wild-type A431 cell (IC50 = 544 nM). BLU-945 potently inhibited EGFR phosphorylation in Ba/F3 (L585R/T790M/C797S) and Ba/F3 (ex19Del/T790M/C797S) cell lines at IC50 values of 3.2 nM and 4.0 nM, respectively. An improvement of the ADME PK profile was observed in BLU-945, and the data are given in Table 3. BLU-945 showed low to moderate clearance and better t1/2 (3.5 h).

In vitro and in vivo profiles of compounds 9 and 13.
13 BLU-945 (9)
Human LM Clint/Hep Clint 7.2/41 89/23
Rat LM Clint/Hep Clint 43/33
Cyno LM Clint/Hep Clint 55/80 109/51
Cyno IV PK Cl (ml min−1 kg) (Clu) 31.8 (611) 4.9 (138)
Cyno PK t1/2 (h) 2.9 3.5
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In the in vivo efficacy study over 14 days in mice bearing NCI-H1975 xenografts, BLU-945 resulted in tumor stasis at 30 mg kg−1 BID dose and tumor regression at 100 mg kg−1 dose. The activity of BLU-945 was studied in NOD SCID mice bearing engineered Ba/F3 (EGFRL858R/T790M/C797S) and BaF3 (EGFRex19del/T790M/C797S) osimertinib-resistant tumor models. In both models, BLU-945 produced strong tumor regression. Blu-945 was evaluated with patient-derived cell-line xenografts (PDX) models with EGFR-driven NSCLC in mice. Substantial tumor growth inhibition was observed after the treatment at 75 mg kg−1 and 100 mg kg−1 BID doses for 56 days. With these promising in vivo efficacy results and suitable safety margins, BLU-945 was chosen for clinical studies. BLU-945 is currently being evaluated in a phase 1/2 clinical trial (NCT 04862780) for treatment of resistant EGFR-driven NSCLC.29

(c). Aminobenzimidazole macrocycle to BI-4020 (16)

A fourth-generation EGFR inhibitor, BI-4020, was developed at Boehringer Ingelheim (BI) to overcome EGFR-activating mutations-T790M/C797S. Engelhardt et al. screened an in-house molecule library of BI, and identified the aminobenzimidazole 14 (Fig. 5) as a promising inhibitor against mutant EGFR.31 The compound 14 showed selectivity in the kinase 238 assay and good biochemical results against the triple mutants EGFRL858R/T790M/C797S (IC50 = 2100 nM), EGFRdel19/T790M/C797S (IC50 = 250 nM). The biomarker modulation was measured in EGFRdel19/T790M/C797S-dependent BaF3 cell lines with an IC50 = 790 nM. They modified the core and rigidified the whole molecule by macrocyclization, and identified compound 15. The biochemical activity of the macrocycle structure 15 was improved significantly, and it showed 17-fold improvements over the open-chain structure. Further optimization of the macrocyclic structure 15 led to the discovery of BI-4020 (16) as a highly potent inhibitor against EGFR triple mutants. BI-4020 showed strong anti-proliferative activity against BaF3-EGFR19Del/T790M/C797S (IC50 = 0.2 nM), BaF3-EGFRwt (IC50 = 190 nM), and A431-EGFRwt (IC50 = 200 nM) cells (Table 4).31 BI-4020 displayed approximately 4000-fold more active osimertinib against triple mutant EGFR.

Fig. 5. Schematic representation of the discovery of BI-4020, fourth-generation active site binding EGFR inhibitor; interaction of BI-4020 with EGFRL858R/T790M/C797S (PDB: 7KXZ).

Fig. 5

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Comparison between BI-4020 and osimertinib, IC50 in nM (ref. 31).
Drugs BaF3-EGFRwt BaF3-EGFRdel19/TM/CS PC-9 EGFRdel19/TM/CS A431-EGFRwt
BI-4020 (16) 190 0.2 1.3 200
Osimertinib 81 780 >1000 84
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The in vivo efficacy of BI-4020 was studied in the EGFRDel19/T790M/C797S mutant human NSCLC PC-9 EGFRdel19/T790M/C797S model. A significant tumor regression in the PC9 EGFR19Del/T790M/C797S xenograft mouse models was observed by BI-4020 at a dose of 10 mg kg−1. After 19 days treatment, BI-4020 induced tumor growth inhibition (TGI) of 121% (P = 0.0005), whereas osimertinib showed almost no effect on the tumor growth inhibition (TGI) of 6% in the same model (p > 0.05) at 25 mg kg−1 daily doses.

Recently, Suzhuki et al.32 reported that brigatinib-resistant mutants could be overcome by BI-4020, as a single treatment or in combination with an anti-EGFR antibody. They studied the potential therapeutic strategies to overcome the resistance mutations by using next-generation macrocyclic EGFR inhibitors. The binding mode of BI-4020 and mutant EGFRL858R/T790M/C797S was obtained by molecular dynamic (MD) simulation. BI-4020 fitted into the ATP-binding pocket of mutant EGFR, and formed hydrogen bonds with the K745, T854, M793 residues (Fig. 5, PDB: 7KZX).

(d). Diarylpyrimidine-2,4-deivatives/N2,N4-diphenylpyrimidine-2,4-diamine derivatives

In the past few years, N2,N4-diphenylpyrimidine-2,4-diamine derivatives have been extensively studied in relation with anti-cancer, anti-inflammatory and antimalarial diseases. The scaffold has been used for making several ALK, JAK2, and CDK9 inhibitors.33,34 The FDA approved the drugs, fedratinib, ceritinib and brigatinib, bearing the scaffold. Deuterated derivates of the scaffold have been used for making ALK inhibitors.35,36

Brigatinib (17, Fig. 6) is a highly potent second-generation anaplastic lymphoma kinase (ALK) inhibitor that can penetrate the blood brain barrier.37 The US FDA approved the drug for the first line treatment for adults with ALK+ metastatic NSCLC in 2020.38 Uchibori et al. showed that brigatinib effectively inhibited the kinase activity of EGFRDel19/T790M/C797S, and also exhibited anti-proliferative activity against EGFR-mutated cell lines, including BaF3 (EGFRDel19/T790M/C797S) at IC50 = 67.2 nM.39 However, the in vivo efficacy of brigatinib against mutant EGFR was not satisfactory. Combination treatment with EGFR-targeted antibodies, such as cetuximab, was required to achieve marked effects in the mouse animal models.

Fig. 6. Brigatinib and other substituted pyrimidine-structures as 4th-generation EGFR-TKIs.

Fig. 6

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To improve the bioavailability, Li et al.40 explored the SAR around the phenyl unit of brigatinib. One of the most selective compounds, 18, suppressed EGFR mutants effectively. The EGFR mutation inhibition activity of compound 18 was about 54 times higher than that of brigatinib. In addition, 18 showed better anti-proliferation activity against Ba/F3, PC9, PC9-EGFRDel19/T790M/C797S, and H1299 cells than osimertinib and was similar to brigatinib (Table 5). In the pharmacokinetic experiments, compound 18 exhibited an oral bioavailability value of 81.7%, and an area under curve (AUC) value of 5219 h ng mL−1 (Table 6).

Antiproliferative activities of compound 18 in different cell lines40.
IC50 (μM) BaF3-EGFRLR/TM/CS BaF3-EGFRDel19/TM/CS PC9 PC9-EGFRDel19/TM/CS H1299 (EGFRwt)
18 0.258 ± 0.167 0.141 ± 0.081 1.192 ± 0.727 0.603 ± 0.294 2.115 ± 0.302
Brigatinib 0.286 ± 0.176 0.155 ± 0.046 0.829 ± 0.148 1.110 ± 0.028 1.049 ± 0.16
Osimertinib 2.893 ± 0.601 2.885 ± 0.689 0.011 ± 0.005 3.210 ± 0.853 >10.0
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Pharmacokinetic parameters of compound 18 in SD rats (n = 3 per group)40.
Doses IV, 0.5 mg kg−1 PO, 3 mg kg−1
T 1/2 (h) 2.67 ± 0.45 2.16 ± 0.2
T max (h) 2.67 ± 1.15
C max (ng mL−1) 288 ± 16 868 ± 181
AUC0-t (h ng mL−1) 929 ± 91 4552 ± 114
AUC0-α (h ng mL−1) 1060 ± 144 5219 ± 1536
CL (mL min−1 kg) 7.97 ± 1.17
MRT0-α (h) 3.65 ± 0.56 4.37 ± 0.62
Vss (mL kg−1) 1720 ± 40
F% 81.7 ± 20.7
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Liu et al.41 reported LS-106 (19) as an effective 4th-generation C797S mutation specific EGFR inhibitor. LS-106 inhibited the kinase activities of mutant EGFR19del/T790M/C797S (IC50 = 2.4 nM) and EGFRL858R/T790 M/C797S (IC50 = 3.1 nM). It could effectively block the phosphorylation of mutant EGFR in dose-dependent manner. The anti-proliferative effects of LS-106 against BaF3-EGFR19del/T790M/C797S (IC50 = 90 nM) and BaF3-EGFRL858R/T790M/C797S (IC50 = 120 nM) were better than that of osimertinib. In the PC-9-OR lung cancer xenograft mouse model, oral administration of LS-106 effectively suppressed the tumor growth at the dose of 30 mg kg−1 or 60 mg kg−1, and achieved tumor growth inhibition (TGI) of up to 83.5% and 136.6%, respectively.

To overcome osimertinib drug resistance due to C797S mutations on EGFR, Heppner et al.42 demonstrated a feasible strategy of binding the inhibitor to the lysine 745 (K745) residue of EGFR. Ferlenghi et al.43 designed and synthesized 2-anilinopyrimidines bearing a sulfonyl fluoride for targeting K745. Among them, the sulfonyl fluoride UPR1444 (20) was identified as the most potent sulfonyl-fluoride derivative that inhibited EGFRL858R/T790M/C797S through formation of a sulfonamide bond with Lys745. UPR1444 (20) displayed inhibitory activity similar to osimertinib against EGFRwt. UPR1444 showed better potency than osimertinib in BaF3 (EGFRL858R/T790M/C797S) cells. The series of sulfonyl-fluoride derivatives had the polar interaction to Lys745 of mutant EGFR. Finlay et al.44 designed and synthesized a series of compounds, and identified 21 as a potent inhibitor against the triple mutant EGFR19del/T790M/C797S, with IC50 < 1 nM. Compound 21 showed better inhibitory effect than brigatinib and oral exposure. Antiproliferative activity of 21 was observed against the PC9-VanR triple mutant (Exon19del/T790M/C797S) cell line with IC50 = 739 nM. The compound had good renal clearance and good permeability. In the xenograft mouse models of NCI-H1975 (L858R/T790M) double mutation and NCI-H1975 cell (L858R/T790M/C797S) triple mutation, compound 21 inhibited the growth of the triple mutation tumor by 52% and double mutation tumor by 87%.

Guo et al. studied different anilinopyrimidine derivatives by changing the aryl groups on the pyrimidine unit of brigatinib and identified one of the potent derivatives, compound 22, against EGFRDel18/T790M/C797S (IC50 = 0.31 nM) and EGFRL858R/T990M/C797S (IC50 = 0.09 nM) mutants.45 Compound 22 demonstrated good anti-preoperative activity in BaF3-EGFRDel18/T790M/C797S (IC50 = 14 nM).

TQB3804 (23) is a 4th-generation, orally bioavailable, mutant-selective EGFR inhibitor used to overcome osimertinib resistance.46 TQB3804 is effective against EGFRDel19/T790M/C797S (IC50 = 0.46 nM) and EGFRL858R/T990M/C797S (IC50 = 0.13 nM) mutations. It showed similar enzymatic activity for EGFRwt (IC50 = 1.07) to osimertinib and anti-proliferative activity against the BaF3 (EGFRd746-750/T790M/C797S), NCI-H1975(EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750) and A431 (EGFRwt) cell lines with IC50 of 26.8, 163, 45 and 147 nM, respectively. TQB3804 significantly inhibited tumor growth in triple mutant BaF3 (EGFRd746-750/T790M/C797S), NCI-H1975(EGFRd746-750/T790M/C797S) and PC9 (EGFRd746-750/T790M/C797S) CDX models and LUPF104 PDX model. TQB3804 has been entered in the phase I clinical trials.

(e). N 2,N4-Diphenylpyrimidine-2,4-diamine deuterated derivatives

“Deuterium switch” involves replacing hydrogen with a deuterium atom at certain sites of the drug molecule, and improves the metabolic stability bioavailability. The deuterium switch technique has been used for improving pharmacokinetic (PK) profiles and sometimes lowering toxicities of the marketed drugs.47,48 Application of the “deuterium switch” in medicinal chemistry is widespread to identify cost effective, efficacious drugs.49 Recently, deterabenazine50 and donafenib51 have been approved by FDA and NMPA, respectively. The deuterated drug design technique was adapted for developing new ALK inhibitors at our laboratoy.35 Liu et al. reported a series of new N2,N4-diphenylpyrimidine-2,4-diamine deuterated derivatives.36 Most of the compounds exhibited antiproliferative activity against BaF3-EGFRL858R/T790M/C797S and BaF3-EGFR19Del/T790M/C797S cell lines with nanomolar IC50 values. Among them, compound 24 (Fig. 6) represented good metabolic stability and strong activity against mutant cells (IC50 = 11–16 nM). Compound 24 inhibited tumor growth up to TGI = 75.1% at 40 mg kg−1 in xenograft mouse models, and it was better than non-deuterated compound 25 with TGI = 73.2% at 80 mg kg−1 dose. The inhibitor could downregulate the protein phosphorylation of EGFR and m-TOR signalling pathways.

(f). Pyrimidine derivatives similar to the osimertinib structure

The FDA approved osimertinib, a third-generation TKI for NSCLC.17 After C797S mutation, the patients become resistant to osimertinib treatment. Several groups tried to modify differ parts of the osimertinib structure to overcome the C797S mutation problem. Ding et al.52 replaced the acrylamide motif of osimertinib with heteroaryl acyl groups to overcome the Cys797 mutation problem. The amide moiety was kept as such to form hydrogen bonding. In the series, the reversible EGFR TKI, compound 26 (Fig. 7) showed inhibitory activity against EGFRwt (IC50 = 29 nM), mutant EGFRL858R/T790M (IC50 = 10 nM) and EGFRL858R/T790M/C797S (IC50 = 242 nM). Another compound 27 showed remarkable inhibitory activity against EGFRL858R/T790M/C797S (IC50 = 137 nM) that was nearly three times better than osimertinib (IC50 = 410 nM). The compounds exhibited dose-dependent activities of cell apoptosis, G1/G0 phase arrestation, and inhibiting migration in A549 and/or H1975 cells. They put these compounds 26 and 27 as promising compounds for further study.

Fig. 7. Pyrimidine derivatives similar to the osimertinib structure.

Fig. 7

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(g). Osimertinib and brigatinib core hybridization & macrocyclization

Chen et al.53 reported the conformational constrained 4-(1-sulfonyl-3-indol)yl-2-phenylmainopyrimidine derivatives as potent fourth-generation EGFR inhibitors targeting T790M/C797S mutations. They designed and synthesized a novel EGFRC797S inhibitor 28 (Fig. 8) by hybridizing osimertinib and brigatinib structural units. This compound showed a promising inhibitory effect against EGFRC797S mutant kinase, however, with poor cellular activity against BaF3(EGFRL858R/T790M/C797S, IC50 = 2610 nM). They adapted a macrocyclization strategy to improve the cellular activity. A less basic morpholine was incorporated to replace the 1-methyl-4-(piperidin-4-yl)piperazine motif to improve the cell permeability, and alkyl ethers with different lengths were selected for the linker to explore the conformation effects in the series. By the optimization of the indole ring connection to the phenyl unit, compound 29 was obtained. Compound 29 showed low kinase selectivity and toxicity to parental BaF3 cells (IC50 = 0.293 μM). Analysis of the co-crystal structure of macrocycle 29 with EGFRT790M/C797S indicated that 29 formed hydrogen bonds with the Met793 residue and sulfonyl group on the K795 residue (PDB: 7VRA). Addition of a methyl group on the phenyl ring on compound 29 led to the identification of compound 30 with increased kinase selectivity and potency. Compound 30 inhibited triple mutant EGFR19Del/T790M/C797S (IC50 = 15.8 nM) and EGFRL858R/T790M/C797S (IC50 = 23.6 nM), and suppressed the proliferation of BaF3-EGFR19Del/T790M/C797S (IC50 = 0.052 μM) and BaF3-EGFRL858R/T790M/C797S (IC50 = 0.036 μM) cell lines. The study provided a lead compound to combat EGFRC797S-mediated resistance in NSCLC patients.

Fig. 8. Osimertinib and brigatinib hydride structure and macrocycle 30; co-crystal structure of macrocycle 29 with EGFRT790M/C797S (PDB: 7VRA).

Fig. 8

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(h). 4-Aminoquinazoline derivatives

4-Aminoquinazoline derivatives are widely used as kinase inhibitors.54 Many first- and second-generation EGFR TKIs have been developed using the 4-aminoquinazoline scaffold.22 Park et al.55 virtually screened a chemical library of about 370 000 compounds, and identified promising compound 31 (Fig. 9) with micromolar activity against EGFR19Del/T790M/C797S. Upon further optimization of the series, they identified compound 32 and compound 33 at nanomolar activity against EGFR19Del/T790M/C797S with IC50 values of 3.38 nM and 4.84 nM, respectively.

Fig. 9. 4-Aminoquinazoline derivative from virtual screening.

Fig. 9

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Zhang et al.56 designed and synthesized a series of 2-aryl-4-amino substituted quinazoline derivatives to overcome the drug resistance due to the L858R/T790M/C797S triple mutation. They identified compound 34 (Fig. 10) and compound 35 as potent compounds against EGFRL858R/T790M/C797S. Compound 35 showed good activity against H1975-EGFRL858R/T790M (IC50 = 3.3 μM) and H1975-EGFRL858R/T790M/C797S (IC50 = 1.2 μM). Compound 35 exhibited good microsomal stabilities in rat, mouse and human (Table 7). However, compound 35 showed low AUC and poor bioavailability (18.6%, Table 8).

Fig. 10. 4-Aminoquinazoline derivative via structure modification.

Fig. 10

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Stabilities of compound 35 in liver microsomes (LM) of various species56.
Liver microsomes T 1/2 (min) CLint (ml min−1 kg)
Human 66.6 20.8
Mouse 35.4 39.2
Rat 34.1 40.6
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Pharmacokinetic parameters of compound 35 in male SD rats56.
Doses IV, 1.0 mg kg−1 PO, 5 mg kg−1
T 1/2 (h) 0.6 0.9
T max (h) 0.1 0.7
C max (ng mL−1) 535 199
AUC0-t (h ng mL−1) 378 351
AUC0-α (h ng mL−1) 379 353
CL (mL min−1 kg) 2648 14 700
MRT0-α (h) 0.7 1.4
Vz 2377 19 685
F% 18.6%
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Dou et al.57 developed a series of 4-amilinoquinoline derivatives as fourth-generation EGFR TKIs targeting C797S mutation. Starting from a reversible Pan EGFR/HER2 inhibitor vandetanib (36), they designed a series that simultaneously occupies the ATP binding site and the allosteric site. The most potent compound 37 (Fig. 11) showed a high anti-proliferative effect against BaF3-EGFRL858R/T790M/C797S (IC50 = 0.75 μM) and BaF3-EGFR19Del/T790M/C797S (IC50 = 0.09 μM) cells, and was better than vandetanib. In the BaF3-EGFR19Del/T790M/C797S xenograft model, compound 37 significantly inhibited tumor growth up to TGI = 67.95% at 30 mg kg−1 dose.

Fig. 11. EGFR TKIs via vandetanib core modification.

Fig. 11

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Li et al. discovered a series of potent noncovalent reversible EGFR TKIs with an anilinoquinazoline core.58 They designed the series by combining vandetanib and the Y-shaped allosteric inhibitor EAI045 core structures together. The most active compound of the series, compound 38 displayed significant inhibitory activity against EGFRL858R/T790M/C797S (IC50 = 2.3 nM) and cytotoxicity against BaF3-EGFRL858R/T790M/C797S (IC50 = 0.64 μM). BDTX-1535 is a 4th-generation irreversible, brain penetrant, EGFR MasterKey inhibitor that targets EGFR resistance mutation in NSCLC and in GBM. BDTX-1535 is currently in phase 1 clinical trial in patients (NCT05256290).59,60

(i). Pyrimidopyrimidinone and 5-methylpyrimidopyridone derivatives

A pyrimidopyrimidinone scaffold containing derivative JND3229 (39) was identified as a potent fourth-generation EGFR TKI with IC50 = 5.8 nM against EGFRC797S mutation.61 It suppressed the proliferation of BaF3 cells harbouring the EGFRL858R/T790M/C797S and EGFR19Del/T790M/C797S mutations with IC50 = 0.51 and 0.32 μM, which were comparable to the IC50 values of brigatinib under the same experiment condition. JND3229 suppressed the growth of NSCLC cell lines NCI-H1975 (EGFRT790M) cells with an IC50 = 0.31 μM. In an X-ray crystallographic study (PDB ID: 5ZTO), the interactions between JND3229 and the EGFRL858R/T790M/C797S protein (Fig. 12) indicated that JND3229 was accommodated in the ATP binding site of mutated EGFR with a reversible “U-shaped” configuration.61 The 3-chlorophenyl unit was directed towards the hydrophobic pocket, and the pyrido[2,3-d]pyrimidine-7-one core formed a bidentate hydrogen bond interaction with the “hinge” residue Met793 of the protein. In vivo efficacy of JND3229 was studied in a BaF3-EGFR19D/T790M/C797S xenograft mouse model. The animals were treated with JND3229 at a dose of 10 mg kg−1 twice daily by intraperitoneal injection or by vehicle control for 10 days. EAI045 (60 mg kg−1, once daily by oral gavage) combination with cetuximab (1 mg kg−1, once every other day by intraperitoneal injection) was used as a positive control. A significant tumor growth inhibition was observed by JND3229 with TGI = 42.2%, whereas EAI045/cetuximab combination gave TGI = 22.3% only. JND3229 was well tolerated, and no bodyweight loss or other toxic signs were detected during study.

Fig. 12. JND3229 core modification and identification of compound 41.

Fig. 12

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Extensive SAR studies on JND3229 were conducted by Hu et al. to give a series of reversible 2-oxo-3,4-dihydorpyrimido[4,5-d]pyrimidine derivates.62 One of the most potent compounds 40 suppressed EGFRL858R/T790M/C797S kinase with (IC50 = 3.1 nM). Furthermore, compound 40 displayed effective inhibitory activity against the BaF3 (EGFRL858R/T790M/C797S) and BaF3 (EGFRdel19/T790M/ C797S) cell lines with IC50 = 290 nM and 316 nM, respectively. A scaffold modification of JND3229 was done, and a number of 5-methylpyrimidone derivatives were synthesized as new selective 4th-generation EGFR-TKIs.63 One of the best compounds, 41 exhibited an IC50 = 27.5 nM against EGFRL858R/T790M/C797S.

Wang et al.64 identified a hit compound 42 (Fig. 13) through a high-throughput screening (HTS) of their in-house compound library against EGFRL858R/T790M/C797S. After SAR studies and structural optimization, they discovered a highly potent and oral EGFRL858R/T790M/C797S inhibitor compound 43 with a novel chemical scaffold. The compound 43 showed low nanomolar activity against the EGFRL858R/T790M/C797S mutant with (IC50 = 0.01 μM). Compound 43 significantly inhibited the phosphorylation of the EGFR, AKT, and STAT3 signalling pathways. This compound induced cell cycle arrest and cell apoptosis at the G0/G1-phase progression. In the PC-9 EGFRL858R/T790M/C797S xenograft mouse model, compound 43 induced significant tumor regression, and osimertinib as the positive control was less effective than compound 43 at the same dose.

Fig. 13. Pyrrolo[2,3-d]pyrimidine derivative as EGFR TKI.

Fig. 13

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(j). Substituted 9H-purine derivatives

A series of 2,9-disubstituted-8-phenylthio/phenylsulfinyl-9H-purines were reported by Hei et al.65 They synthesized thirty-one compounds and among them, compound 44 (Fig. 14) displayed potency against EGFRL858R (IC50 = 1.9 nM) and HCC827 cell (IC50 = 29 nM). Then, they optimized the structure and identified compound 45 with moderate inhibitory activity against EGFRL858R/T790M/C797S (IC50 = 114 nM). To strengthen the inhibitory activity, Lei et al.66 designed a series of compounds (general structure 46) with extra hydrogen bonds to the C797S residue of the triple mutant EGFR. They put sulfonyl, acyl or 2-hydroxyacetyl groups at the 9-position of the 9H-purine scaffold to form hydrogen bonds with Ser797. They synthesized a series of novel 9-heterocyclyl substituted 9H-purines, and identified compounds 47, 48 and 49 as promising inhibitors against EGFRL858R/T790M/C797S with IC50 values of 18, 22 and 41 nM, respectively, better than osimertinib (EGFRL858R/T790M/C797S IC50 = 110 nM). Compound 47 showed potent anti-proliferation against HCC826 and H1975 with IC50 = 0.88 nM and 200 nM, respectively.

Fig. 14. Substituted 9H-purine derivatives against triple mutant EGFR.

Fig. 14

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2.2. EGFR allosteric binding site inhibitors

Typically, tyrosine kinase receptors have three sites: (a) active site or ATP competitive site, (b) inactivation site, and (c) allosteric site. The existing TKIs often target the ATP binding site of the kinase. The approved 1st, 2nd, 3rd-generation EGFR-TKIs target ATP binding sites. The binding of drugs or inhibitors is not possible at the inactivation site. So, the inactivation site is not considered as a valid target for developing tyrosine kinase inhibitors. The allosteric binding pocket of EGFR is located far away from the ATP binding site. Targeting the allosteric site is a relatively new approach towards developing novel EGFR TKIs against mutations. Allosteric site binding inhibitors are unaffected by the common ATP-site mutations and overcome the EGFR-TK resistance problem. So, EGFR allosteric inhibitors offer promise to serve as mutant-selective fourth-generation EGFR inhibitors or next-generation EGFR inhibitors.67,68

Utilizing the high-throughput screening (HTS) technique, Jia et al. screened a 2.5 million compounds library and identified N-(4,5-dihydrothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)-2-phenylacetamide EAI001 (50, Fig. 15) as a novel non-ATP competitive allosteric EGFR inhibitor.69 EAI001 showed high selectivity and potency against EGFRL858R/T790M and EGFRwt with IC50 = 24 mM and >50 mM, respectively.

Fig. 15. Allosteric binding site inhibitors EAI001 & EAI005.

Fig. 15

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Optimization of the EAI001 structure, specially the phenyl group, led to a potent inhibitor EAI045 (51, Fig. 15) as the fourth-generation EGFR inhibitor overcoming T790M and C797S resistance.70 EAI045 (51) displayed 1000-fold selectivity against EGFRL858R/T790M (IC50 = 3 nM) over wild-type EGFR (IC50 = 4.3 mM).71 EAI045 showed modest activity against L858R and T790M mutants with IC50 values of 0.75 μM and 1.7 μM, respectively. In vivo efficacy of EAI045 was demonstrated in EGFRL858R/T790M and EGFRL858R/T790M/C797S mutants in mouse models.72 EAI045 was active against one subunit of an EGFR heterodimer/asymmetric dimer.69 EAI045 reduced tumor growth in a mouse model of L858R/T790M—mutant-driven lung cancer. During activation, EGFR forms asymmetrical dimers where the allosteric site is less accessible. This blocks the binding of EAI045. So, EAI045 exerted minimal cellular and in vivo efficacy as a single agent. The addition of cetuximab prevents dimer formation, and EAI045 demonstrated good activity in EGFRL858R/T790M models. In combination with cetuximab, EAI045 exhibited a synergistic effect for tumor shrinkage in the mouse model carrying L858R/T790M/C797S mutation.

To et al.73 designed and synthesized an EGFR allosteric site binding EGFR inhibitor, JBJ-04-112-05 (52, Fig. 16) by linking the 5-indole substituted isoindolinone moiety on EAI001. The compound JBJ-04-112-05 showed IC50 = 15 nM against the L858R/T790M mutant EGFR. Optimization of the isoindolinone series identified JBJ-04-125-02 (53) with a phenyl piperazine substituent extending into a channel alongside the α-helix. JBJ-04-125-02 (53) displayed a stronger in vitro potency of IC50 = 0.26 nM against the T790M/L858R mutant EGFR. Despite its poor oral bioavailability, the in vivo efficacy of the single drug and combination therapy of osimertinib and JBJ-04-125-02 were carried out in the H1975 mouse model upon daily oral dosing at 100 mg kg−1. The combination JBJ-04-125-02 with osimertinib delivered enhanced effects relative to individual agents in a set of in vitro and in vivo studies.74 To improve permeability and bioavailability, To et al.75 prepared a potent N-methyl piperidine JBJ-09-063 (54) by reducing the number of H-bond donors in the molecule. However, the changes could not bring a significant impact on oral exposure. JBJ-09-063 exhibited enhanced potency EGFRL858R/T790M IC50 = 0.063 nM and EGFRL858R/T790M/C797S IC50 = 0.083 nM. JBJ-09-063 achieved tumor regression in osimertinib-resistant triple mutant EGFRL858R/T790M/C797S models upon daily oral dosing at 50 mg kg−1. At the same dose, minimal effects were observed by JBJ-09-063 in an A431 (EGFRwt) model.

Fig. 16. Discovery of JBJ-09-063.

Fig. 16

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Following a structure-based drug design approach, Wittlinger et al. designed a “two-in-one” mutant-selective EGFR inhibitor that extended both orthosteric and allosteric sites.76 They designed and prepared a series of compounds by fusing portions of the allosteric inhibitor EAI045 with the pyridinyl imidazole scaffold of LN2057 (55, Fig. 17). By linking these two scaffolds via the phenyl unit, they could remove a chiral centre and identified a novel compound 56. Compound 56 was demonstrated as a potent and selective inhibitor against EGFRL858R/T790M (IC50 = 1.5± 0.3 nM) and EGFRL858R/T790M/C797S (IC50 = 4.9 ± 1.0 nM) mutations, and was about 10 times better than EGFRwt (IC50 = 47 ± 8 nM). Compound 56 displayed better potency than LN2057 (IC50 = 130 ± 4 nM) and EAI045 (IC50 = 13 ± 0.8 nM) against the triple mutant EGFR.

Fig. 17. Combination of allosteric binding and orthosteric binding sites.

Fig. 17

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De Clercq et al. reported an allosteric inhibitor DDC4002 (57) (Fig. 18) based on the diazopinone scaffold that showed selectivity against triple mutant EGFR.77 Using a structure-based medicinal chemistry approach, they optimized the core and identified an inhibitor 58 with high selectivity and observed potency against EGFRL858R/T790M (IC50 = 11 nM) and EGFRL858R/T790M /C797S (IC50 = 13 nM) (Fig. 18).

Fig. 18. Diazopinone and quinazolinone scaffolds.

Fig. 18

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Gero et al.78 identified a new series of quinazolinone derivatives as allosteric EGFR inhibitors by the scaffold hopping approach. One of the best compounds in the series, compound 59 showed very good potency against mutant EGFRL858R/T790M (IC50 = 0.1 nM) and cellular activity in BaF3-EGFRL858R/T790M and BaF3-EGFRL858R/T790M/C797S with IC50 = 0.32 μM and 0.02 μM, respectively. The 5-F quinazolinone 59 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg kg−1.

3. PROTACs

PROteolysis Targeting Chimeras (PROTACs) technology is a well-established method for degradation of targeted proteins, and it is used as one of the popular technologies in new drug development.79,80 PROTAC is a heterobifunctional molecule containing two warheads that are connected through a linker. Due to the distinct targeting selective mechanism of actions, PROTACs possess an advantage over common kinase inhibitors (Fig. 19).81 PROTACs are designed to bind simultaneously to a target protein to be degraded and an E3 ubiquitin ligase.82 PROTAC may be used for overcoming dose-related toxicity and drug resistance. In the past two decades, many PROTACs have been developed to target kinases, proteins and nuclear receptors.83 Recently, the FDA approved AV-110, the first orally bioavailable PROTAC against the androgen receptor, for the treatment of metastatic castrate-resistant prostate cancer in phase 1/2 clinical studies.84

Fig. 19. Schematic representation of the PROTAC structures and the mechanism of action for target selective degradation.

Fig. 19

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Many PROTACs targeting EGFR have been developed in the past few years.85 Mainly, these EGFR PROTACs are designed based on first-, second- and third-generation EGFR inhibitors, which effectively degrade EGFRDel19, EGFRL858R, and EGFRL858R/T790M (Fig. 20). Cheng et al.86 reported on the gefitinib-based novel E3 ligase von Hippel–Lindau (VHL)-recruiting EGFR degrader MS39 (60, Fig. 20) and the first-in-class E3 ligase cereblon (CRBN)-recruiting EGFR degrader MS154 (61). These PROTACs induced the degradation of mutant EGFR, but not wild type-EGFR. The quantified DC50 values for MS39 in HCC-827 and H3255 cell were 5.0 nM and 3.3 nM, respectively. The DC50 values of MS154 in HCC-827 and H3255 cells were 11 nM and 25 nM, respectively. Many EGFR-PROTACs have been discovered based on second-generation and third-generation EGFR kinase inhibitors.87–89 Among them, compounds 62–65 gained attention due to lower DC50 values and good activity in cell lines. The DC50 of SIAIS126 (63) was <30 nM for CRBN-recruiting EGFRL858R/T790M degradation.90 Zhao et al. reported CP17 as the most potent EGFR mutant degrader with EGFRDel19 DC50 = 0.49 nM in HCC827 cells and EGFRL858R/T790M DC50 = 1.56 nM in H1975 cells.91 Zhang et al. designed, synthesized and evaluated a series of CRBN-recruiting EGFR degraders and identified compound 65 as the best in the series.92 The compound 65 induced EGFRL858R/T790M degradation with DC50 = 13.2 nM and inhibited NCI-H1975 cell proliferation with IC50 = 46.82 nM. The in vivo efficacy of compound 65 was demonstrated in a NCI-H1975 xenograft model with TGI = 63.7%.

Fig. 20. Some EGFR PROTACs based on first-, second-, and third-generation EGFR inhibitors.

Fig. 20

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Several PROTACs have been discovered to degrade mutant EGFR. However, to date, there have been relatively few PROTACs as EGFRC797S mutant degraders (Fig. 21). Jang et al. developed an allosteric EGFR degrader DDC-01-163 (66) that selectively inhibited the proliferation of L858R/T790M (IC50 = 96 nM) and L858R/T790M/C797S (IC50 = 41 nM) mutant BaF3 cells.93 When the PROTAC DDC-01-163 was combined with osimertinib, the anti-proliferative activity was enhanced in mutant BaF3 cells. A brigatinib-based PROTAC 67 was designed and synthesized against triple-mutated EGFRDel19/T790M/C797S by Zhang et al.94 One of the best representative compounds, 67, induced EGFR degradation (DC50 = 8 nM) dose-dependently, and showed good antiproliferative activity against BaF3-EGFRDel19/T790M/C797S cells (IC50 = 20 nM). Du et al.95 reported on the orally bioavailable EGFR RPOTAC, HJM-561 (68) that degraded EGFR C797S triple mutants (Del19/T790M/C797S and L858R/T790M/C797S). HJM-561 inhibited the proliferation of BaF3-EGFRDel19/T790M/C797S and BaF3-EGFRL858R/T790M/C797S cells. An in vivo efficacy study of HJM-561 resulted in a significant reduction of the tumor volume by 58% and 84% at 20 mg kg−1 and 40 mg kg−1 doses, respectively, in BaF3-EGFR(Del19/T790M/C797S) cell-derived xenograft models. The antitumor efficacy was comparable with that of TQB3804. Zhu et al. reported on the novel PROTAC compound 69 targeting C797S mutation.96 Compound 69 exhibited DC50 = 10.2 nM against EGFRL858R/T790M/C797S and IC50 = 10.3 nM against H1975-TM. The in vivo antitumor activity of compound 69 was evaluated in H1975-TM xenograft mouse models. After 31 days of study, the tumor growth was significantly reduced and the tumor growth inhibition (TGI) rates for 25 mg kg−1 and 100 mg kg−1 were achieved at 48.1% and 66.4%, respectively.

Fig. 21. Some EGFR-PROTACs based on fourth-generation EGFR inhibitors targeting C797S mutation.

Fig. 21

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4. Crystal structure analysis of the C797S mutant with inhibitor complexes

Recently, several groups have reported on the crystals structures of EGFR mutant kinases and EGFR TKIs. Here, we have discussed some of the crystal structures related to T790M and C797S mutations.

Yan et al. reported on the molecular dynamic simulations of osimertinib with T790M mutation.97 Osimertinib interacted with the gate keeper residue Met790 in T790M (PDB: 6jx4; Fig. 22a), but not with Thr790 in the wild-type EGFR. The covalent bond formation with the cysteine residue and hydrogen bond interactions at Met793, C797 and Tyr801 were clearly observed. Another crystal structure of osimertinib with triple mutant EGFR with C797S mutation indicated that residue Tyr801 was too far away to form hydrogen bonding (PDB: 6LUD; Fig. 22b).28

Fig. 22. (a) Crystal structure of EGFRT790M in complex with osimertinib (PDB ID: 6JX4).97 (b) Crystal structure of EGFRL858R/T790M/C797S in complex with osimertinib (PDB ID: 6LUD).28.

Fig. 22

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Suzuki et al. determined the crystal structure of mutant EGFR (T790M/C797S) with brigatinib (Fig. 23).32 Brigatinib formed two hydrogen bonds with the main chain residue at M793. The M790 and S797 residues were located away from brigatinib (PDB: 8H7X).

Fig. 23. Binding mode of brigatinib with EGFRT790M/C797S (PDB ID: 8H7X).32.

Fig. 23

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Hu et al.62 modified the JND3229 structure and identified a highly potent compound 40 against EGFRC797S mutation. The X-ray crystal structure of JND329 with mutant EGFR is shown in Fig. 24. They analysed the co-crystal structure of 40 with EGFRT790M/C797S. The compound 40 bound to mutant EGFR with a reversible “U-shaped” configuration with the hinge residue Met793. The 2-chloropheyl group was directed towards the hydrophobic pocket of residues Lys745, Glu762, Leu788, Met766 and Met790. The carbonyl amide in 40 formed a hydrogen bond with Ser720 mediated by a water molecule, while the NH of amide in JND3229 formed a hydrogen bond with Leu718 mediated by a water molecule too.

Fig. 24. X-ray crystalline structure of JND3229 complexed with EGFRT790M/C797S (PDB ID: 5ZTO).62.

Fig. 24

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As per X-ray crystallographic data analysis, the allosteric inhibitor EAI001 formed a selective complex with the mutant EGFR allosteric site. Direct interaction between its aminothiazole group of EAI001 (50) and T790M mutant, and hydrogen bond formation between NH group of the carboxamide and Asp855 of EGFR, were observed (Fig. 25a).69 Zhao et al. reported the crystal structure of EGFRT790M/C797S/V948R in complex with EAI045 (PDB: 5ZWJ) and the complex EGFRT790M/V948R with EAI001.98 As per the data analysis, EAI045 formed a tighter complex to mutant EGFR than EAI001 (Fig. 25b).

Fig. 25. (a) EGFR kinase in complex with the allosteric inhibitor, EAI001 (PDB ID: 5D41);69 (b) EGFRT790M/C797S/V948R in complex with EAI045 (PDB ID: 5ZWJ).98.

Fig. 25

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5. Clinical trials of fourth-generation EGFR TKIs

The development of fourth-generation EGFR TKIs brings great hope to address the C797S mutations, and a number of clinical trials are in progress. The clinical studies are summarized in Table 9. Most of the inhibitors are in phase 1/2 clinical trials and their efficacies are not well understood. At present, no drug has been approved by the FDA for treating the C797S mutation.

Clinical trials of next-generation EGFR TKIs in NSCLC.

Drug/inhibitor Company Disease Clinical trial identification number Status Ref.
BLU-945 (9) Blueprint NSCLC NCT04862780 Phase I/II 30, 99
BLU-701 Blueprint EGFR-mutant NSCLC NCT05153408 Phase I/II 100
WJ13405 Junjing Advanced or metastatic NSCLC NCT05662670
TBQ3804 (23) Advanced malignant tumor NCT04128085 Phase I
BBT-176 Bridge Advanced NSCLC NCT04820023 Phase I/II 101, 102
BBT-207 Bridge EGFR mutant advanced NSCLC NCT05920135 103, 104
BPI-361175 Betta Pharma Advanced solid tumor NCT05329298 Phase I 105
BDTX-1535 Black Diamond NSCLC or glioblastoma NCT05256290 Phase I
HS-10375 Hansoh Advanced or metastatic NSCLC NCT05435248 Phase I/II
TAS3351 Taiho NSCLC NCT05765734 Phase I/II 106
QLH11811 Qilu Advanced NSCLC NCT05555212 Phase I 107
BAY2927088 Bayer Advanced NSCLC with EGFR or HER2 mutations NCT05099172 Phase I 108
JIN-A02 JIB EGFR mutant NSCLC NCT05394831 Phase I/II 109, 110
H002 R&G Pharma Advanced or metastatic NSCLC NCT05552781 Phase I/II 111
TRX-221 Therapex EGFR mutant NSCLC NCT06186076 Phase I/II 112
THE-349 Theseus Planned 113
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The PK, safety and efficacy from the phase 1 study of BBT-176, a fourth-generation EGFR TKI for advanced NSCLC after EGFR TKI therapy, has been published.101 The drug was well-tolerated and no dose-limiting toxicity of the treatment was reported. An open-label phase 1/2 study in patients with advanced NSCLC was started to evaluate efficacy, response rate and progression-free survival.102 BBT-207 is a 4th-generation EGFR TKI with broad spectrum activity for the treatment of NSCLC. BBT-207 showed IC50 values <10 nM against EGFR triple mutants.103 BBT-207 significantly inhibited tumor growth in an in vivo model with the EGFRDel19/T790M/C797S mutation. Lee et al. reported the preclinical efficacy of BI-4732 as a novel fourth-generation EGFR TKI using patient-derived preclinical models.114 They demonstrated good antitumor efficacy of BI-4732 as a single agent in various PDX models with EGFRC797S-mediated osimertinib resistance. TRX-221 is a potent 4th-generation EGFR TKI. TRX-221 showed incisory activity of cell and mutant EGFR. In CDX tumor models, TRX-221 demonstrated antitumor efficacy in osimertinib-resistance tumor models, and the initiation of a first-in-human clinical trial is expected.112 JIN-A02 is a novel fourth-generation EGFR TKI against EGFRDel19/T790M/C797S. It showed high efficacy in mutant cell lines, BaF3-EGFRDel19/T790M/C797S (IC50 = 4.7 nM) and NCI-H1975-EGFRL858R/T790M/C797S (IC50 = 12.8 nM). In a PDX mouse model (YHIM-1094, EGFRDel19/T790M/C797S), JIN-A02 delayed tumor growth at a dose of 30 mg kg−1. An open-label, multi-center study in phase 1/2 is ongoing to evaluate the safety, tolerability, and pharmacokinetics of JIN-A02 (NCT05394831). TBQ3804 is currently entered into phase I clinical trials to assess its tolerability and PK in patients with advanced malignant tumors (NCT04128085). The phase 1/2 open-label trial of BLU-945, both as monopathy and in combination with osimertinib (NCT04862780), aimed to evaluate the safety, tolerability and PK/PD, and efficacy. Another 4th-generation EGFR TKI BPI361175 inhibited many EGFR mutations, including triple mutants (EGFRDel19 or EGFRL858R/T790M/C797S).105 TAS3351 is a 4th-generation EGFR TKI overcoming T790M and C797S-mediated resistance in NSCLC.106 TAS3351 exhibited almost comparable inhibitory potency against EGFR harboring ex19del or L858R with or without C797S and/or T790M, while sparing wild type EGFR activity. TAS3351 demonstrated favorable anti-tumor activity in NIH/3T3 allografts transformed by human EGFR harboring ex19Del/T790M/C797S. Huang et al. reported on H002 as a wide spectrum, highly selective 4th-generation EGFR inhibitor overcoming resistance from EGFR harboring the C797S mutation in NSCLC.111

6. Conclusion

In the past two decades, many first-, second- and third-generation EGFR TKIs have been discovered and demonstrated good in vivo efficacy. Drug resistances in lung cancer patients after TKIs treatment due to acquired mutations on EGFR kinase remain a challenging topic of research. The C797S mutation is one of the major causes for drug resistances against the third-generation EGFR TKIs, including osimertinib. These C797S mutations includes double mutations (19Del/C797S or L858R/C797S) and or triple mutations (19Del/T790M/C797S or L858R/T790M/C797S).

Conceptually, allosteric inhibitors were discovered to overcome drug resistance against EGFRL858R/T790M/C797S, sparing EGFRwt. However, these inhibitors were not effective against Del19 bearing the triple mutation, and a combination of drugs was required to achieve better efficacy.

Inhibitors that occupy both ATP-binding pocket and allosteric pocket are shown to be efficacious against EGFRL858R/T790M/C797S. These inhibitors are designed to bind the mutant EGFR tightly. For spreading over a larger space, from the ATP binding site to the allosteric pocket, large size inhibitors are required. Consequently, the drugability of these inhibitors become relatively poor.

At present, various fourth-generation EGFR TKIs are known to overcome triple mutant EGFR with C797S mutations, and demonstrated effectiveness in preclinical models. However, no fourth-generation EGFR TKI has been approved by the FDA. Several inhibitors have been pushed into phase I/II clinical trials, and some of the TKIs already demonstrated their efficacies in human patients. These promising results indicate that development of fourth-generation EGFR TKIs will solve the third-generation EGFR-TKIs drug resistance problems. Discovery efforts for developing next-generation EGFR-TKIs targeting the EGFR C797S mutation will bring a better solution to overcome osimertinib resistance in NSCLC. At present, a number of clinical trials are in progress, and the study data will prove the efficacy and superiority of these fourth-generation EGFR-TKIs in human. There is a huge possibility that some of the fourth-generation EGFR-TKIs will be approved by the FDA in the near future.

Abbreviations

EGFR

Epidermal growth factor receptor

FDA

The US Food & Drug Administration

HER

Human epidermal growth factor receptor

MAPK

Mitogen-activated protein kinase

PI3K

Phosphatidylinositol-3-kinase

NSCLC

Non-small cell lung cancer

ORR

Overall response rate

PFS

Progression free survival

RTK

Receptor tyrosine kinase

SCLC

Small cell lung cancer

TKIs

Tyrosine kinase inhibitors

TGI

Tumor growth inhibition

Data availability

No primary research results, software or code have been included and no new data were generated or analyzed as part of this review.

Conflicts of interest

The authors declare no conflict or competing financial interest.

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