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Drug discovery targeting anaplastic lymphoma kinase (ALK) Xiaotian Kong, Peichen Pan, Huiyong Sun, Hongguang Xia, Xuwen Wang, Youyong Li, and Tingjun Hou J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00446 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Journal of Medicinal Chemistry

Drug discovery targeting anaplastic lymphoma kinase (ALK) Xiaotian Kong,1,3,ǂ Peichen Pan,1,ǂ Huiyong Sun1, Hongguang Xia,2 Xuwen Wang,1 Youyong Li,3 Tingjun Hou1*

1Hangzhou

Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China

2Department

of Biochemistry & Research Center of Clinical Pharmacy of the First

Affiliated Hospital, Zhejiang University, Hangzhou 310058, China 3Institute

of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123, China

ǂEquivalent

authors

Corresponding author: Tingjun Hou E-mail: [email protected]

1

ACS Paragon Plus Environment

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Abstract: As a receptor tyrosine kinase of insulin receptor (IR) subfamily, anaplastic lymphoma kinase (ALK) has been validated to play important roles in various cancers, especially anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastomas. Currently, five small-molecule inhibitors of ALK, including Crizotinib, Ceritinib, Alectinib, Brigatinib and Lorlatinib, have been approved by the U.S. Food and Drug Administration (FDA) against ALK-positive NSCLCs. Novel Type-I1/2 and Type-II ALK inhibitors with improved kinase selectivity and enhanced capability to combat drug resistance have also been reported. Moreover, the “proteolysis targeting chimera” (PROTAC) technique has been successfully applied in developing ALK degraders, which opened a new avenue for targeted ALK therapies. This review provides an overview of the physiological and biological functions of ALK, the discovery and development of drugs targeting ALK by focusing on their chemotypes, activity, selectivity, and resistance as well as potential therapeutic strategies to overcome drug resistance.

Keywords: Anaplastic lymphoma kinase, ALK inhibitors, drug resistance, non-small cell lung cancer.

2


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1. Introduction Anaplastic lymphoma kinase (ALK), a member of the insulin receptor (IR) protein-tyrosine kinase superfamily, was originally identified as a nucleophosmin (NPM)-ALK fusion form in an anaplastic large cell lymphoma (ALCL) cell line with a t(2;5) chromosomal rearrangement1. The physiological functions of ALK have not been fully elucidated, but some evidences have validated the regulatory roles of ALK in the normal development and function of central and peripheral nervous systems2. As shown in Figures 1 and 2, chromosomal rearrangements, as the most prevalent genetic aberrations of ALK, result in the generation of a wide variety of ALK fusions, in which the kinase domain and the N-terminal portion of protein partners are fused3-7. Different ALK fusion proteins, such as CARS-ALK in inflammatory myofibroblastic tumor (IMT)8, CLTC-ALK in diffuse large B-cell lymphoma (DLBCL)9 and EML4-ALK in non-small-cell lung cancer (NSCLC)10, can mediate different signaling cascades and enhance oncogenic potential of ALK by promoting dimerization/oligomerization of ALK fusion proteins11. Other tumorigenic forms of ALK, such as the acquisition of activating point mutations and gene amplification in the full-length ALK (shown in Figure 3), have also been reported in neuroblastoma, anaplastic thyroid cancer (ATC), rhabdomyosarcoma, ovarian cancer, etc. (Figure 1)12-15. Over the past decade, great efforts have been made to develop targeted therapies

against

ALK

in

both

pharmaceutical

industry

and

academia.

Crizotinib/PF2341066 (1, 2011, Pfizer)16 is the first FDA-approved drug for the treatment of ALK-positive NSCLC (Figure 4). Unfortunately, ALK-positive NSCLC patients rapidly developed resistance to 1, which led to the development of more effective drugs (Figure 4)17,

18,

including Ceritinib/LDK-378 (3, 2014, Novartis)19,

Alectinib/CH5424802 (4, 2015, Roche)20, Brigatinib/AP26113 (5, 2017, Ariad)21 and Lorlatinib/PF-0646392 (11, 2018, Pfizer)22. Moreover, a number of other ALK inhibitors, such as Belizatinib/TSR-011 (6)23, CEP-37440 (7)24, ASP3026 (8)25, Entrectinib/RXDX-101 (9)26, 27, Ensartinib/X-396 (10)28 and Repotrectinib/TPX-0005 (12)29, are currently being evaluated in different stages of clinical trials (Figure 4). In 3



spite of the great efforts that have been made in the past decade, drug resistance still remains a crucial problem. As shown in Figure 3, acquired secondary mutations, and gene amplification and/or increased copy number account for about one-third of drug resistance cases18, 30, 31. In addition, the activation of alternative signaling pathways and epithelial-mesenchymal transition (EMT) can also compromise therapeutic efficacy 32. In order to overcome drug resistance and improve kinase selectivity, novel Type-I1/2 and Type-II inhibitors of ALK that can probe the extended hydrophobic pocket were developed33-35. Particularly, inspired by the success of the “proteolysis targeting chimera” (PROTAC) technique in drug discovery, several ALK PROTACs have already been developed36-38. This review provides a general outline of the structure, biological functions and aberrations of ALK, the discovery and development of ALK drugs, structural rationales for drug resistance and potential anti-resistance therapeutic strategies.

2. Structure and downstream signaling pathways of ALK 2.1. Structural Biology of ALK As shown in Figure 2A, human ALK consists of a signal peptide (1-18), an extracellular ligand-binding domain (19-1038), a transmembrane domain (1039-1059), and an intracellular tyrosine kinase domain (1060-1620). Rearrangements on the 2p23 chromosomal segment can result in the fusion of ALK to various partner genes5, as exemplified by NPM-ALK and EML4-ALK (Figure 2B and 3C). As shown in Figure 5, the kinase domain of ALK comprises a small amino-terminal lobe (N-lobe) and a large carboxyl-terminal lobe (C-lobe) that are connected by a flexible hinge region. A cleft that serves as the binding pocket for ATP is formed between the two lobes. The small N-lobe is mainly composed of five stranded β-sheets (β1-β5) and an important regulatory α-helix called the αC-helix39. The N-lobe also contains a glycine-rich loop (G-loop) between the β1- and β2-strands, which participates in the coordination of the β- and γ- phosphates of ATP39. The large C-lobe contains six conserved α-helices (αD-αI) and two short conserved β-strands (β7-β8) located between the αE- and 4


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αF-helice39, 40. In Figure 5A, the unphosphorylated ALK kinase domain contains an additional helix (αEF) within the activation loop (A-loop) that follows the β8 strand. In addition, ALK contains two conserved hydrophobic non-contiguous motifs, termed the regulatory spine (R-spine) and catalytic spine (C-spine), which span both the Nand C-lobes (Figure 5A). Assembly of the R-spine is the hallmark of the insulin receptor activation, and structural changes in the A-loop and αC-helix by disassembling the R-spines regulates the activation/in-activation cycles of ALK41. As shown in Figure 5B, Lys1150, Glu1167, Asp1249 and Asp1270 (K/E/D/D signature) in the ATP binding region are critical residues involved in the catalytic activity of ALK39,

40.

Biochemical data suggests that auto-phosphorylation of the Y’RAS’YY

motif in the A-loop affects both substrate binding and catalytic efficiency of ALK39, 40. Phosphorylation of Tyr1278 in the Y’RAS’YY motif may activate ALK by relaxing ALK from inactive conformational constraints42. 2.2. Downstream signaling pathways of ALK ALK is a membrane-bound receptor, which normally receives and transfers extracellular signals by activating multiple intracellular signaling pathways. Different ALK aberrations cause diverse pathogenic signaling anomalies41, such as SRC/RAS/MEK/ERK1/2, JAK/STAT, PI3K/AKT/mTOR, and PLC-γ/DAG/PKC pathways31,

41.

The PLC-γ/DAG/PKC and SRC/RAS/ERK1/2 pathways affect cell

growth and proliferation, while the JAKs/STAT and PI3K/AKT/mTOR pathways regulate cell survival31. However, the full picture of ALK signaling has not been illuminated. The critical pathways involved in ALK downstream signaling events can be activated by the fusion proteins such as NPM-ALK and EML4-ALK31,

41.

As

shown in Figure 6, ERK1/2, PI3K/AKT/mTOR and STAT3 could be activated by the NPM-ALK fusion protein, while the transforming potential of NPM-ALK in ALCL is mainly mediated by STAT343, 44. In cellular models of NSCLC harboring EML4-ALK rearrangement, the PI3K/AKT and RAS/MAPK pathways are strongly activated6, 41. In recent years, extensive efforts have been dedicated to study the ALK-dependent signaling events in neuroblastoma characterized by point mutations/amplification in the full-length ALK, and the results illustrate that the activated ALK acts 5



synergistically

with

the

over-expressed

MYCN

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to

drive

neuroblastoma

development45-47. Besides, activation of adaptor proteins and other cellular proteins, such as NF-ƙB, SRC, SHC-GRB2, IRS-1, and JUN/AP1, has been proved to play important roles in ALK signaling transduction, suggesting potential “bypass” signaling pathways6, 41.

3. Pathological roles of ALK in related diseases 3.1. ALK translocations At present, nearly 30 fusion proteins of ALK have been identified as oncogenic drivers in many types of cancers (Figure 1), among which EML4-ALK and NPM-ALK are the two most common variants. Figure 2B and 2C illustrate that the translocations lead to the fusion of the normally expressed NPM and EML4 proteins to the ALK kinase domain. EML4-ALK is the most common fusion type in ALK-positive NSCLC patients, and about 3~7% of all NSCLC cases worldwide contain an EML4-ALK rearrangement48,

49.

EML4-ALK rearranged NSCLC often occurs in young and

lifelong non-smoking adenocarcinoma patients10,

50.

Notably, the oncogenic

EML4-ALK fusion gene exclusively exists in NSCLC cases without epidermal growth factor receptor (EGFR) or kirsten rat sarcoma virus (KRAS) mutation, which makes EML4-ALK an essential target for NSCLC51. Additionally, some less frequent ALK fusions have also been identified in NSCLC, including PTPN3-ALK, TFG-ALK, HIP1-ALK, KIF5B-ALK, KLC1-ALK, STRN-ALK and TPR-ALK17, 31, 52, 53. NPM-ALK has been detected in nearly 75%~80% of ALK-positive ALCL patients1,

43,

and about 15% of ALK-positive ALCLs have TPM3/4-ALK

translocations31, 41. Besides, the other fusion proteins occur at much lower frequency (< 2%), including RNF213-ALK54, TFG-ALK, MSN-ALK, ATIC-ALK, MYH9-ALK, CLTC-ALK and TRAF1-ALK31, 41. ALK-positive IMT is a kind of solid tumor, typically occurring in soft tissues of young patients55. TPM3/4-ALK fusion proteins were detected in up to 50% of IMTs56. 6


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The expressions of the other ALK fusion partners in IMTs, such as CLTC, CARS, ATIC, SEC31A, RRBP1, RANBP2 and ETV6, were found at much lower level (< 5%)57. DLBCL is an aggressive type of non-Hodgkin lymphoma in adults58, and shows poor response to conventional therapies. The majority of ALK-positive DLBCL possess the t(2;17)(p23;q23) chromosomal translocation, producing the CLTC-ALK fusion protein59. NPM-ALK and other fusion proteins (SQSTM1-ALK and SEC31A-ALK) are less expressed in DLBCL60, 61. Studies show that ALK-rearranged DLBCL could benefit from individually tailored therapy by targeting ALK62, 63. Besides, ALK translocations are also involved in the occurrence of esophageal squamous cell carcinoma (TPM4-ALK)64, renal cell carcinoma (VCL-ALK, TPM3-ALK and EML4-ALK)65-67, colorectal cancer (C2orf44-ALK)68, breast cancer (EML4-ALK)69, papillary thyroid cancer (EML4-ALK, GFPT1-ALK, TFG-ALK and STRN-ALK)70-72, spitz tumors (DCTN1-ALK and TPM3-ALK)73, etc. Recently, a novel fibronectin 1 (FN1)-ALK fusion was identified in serous ovarian carcinoma (SOC) and IMTs74. 3.2. Gene mutations or amplification of ALK About 7% of neuroblastoma are ALK-positive at diagnosis, and recent data indicate that ALK mutations occur in 20~25% of patients with relapsed neuroblastoma regardless of initial ALK status75. Cancer-associated ALK mutations can be roughly divided into three groups: ligand-independent mutations (such as F1174I/S/L and R1275Q), ligand-dependent mutations (such as D1091N, T1151M and A1234T), and a kinase-dead mutation (I1250T)76. In Figure 1, F1174L/S/I/C/V, R1245L/Q and R1275Q/L in the kinase domain are three hotspot mutations observed in neuroblastoma and occur in ~85% of all mutated cases4,

76, 77.

The synergy of the

F1174L mutation and MYCN overexpression aggressively potentiates the oncogenic activity of MYCN and drives the formation of neuroblastoma with enhanced lethality47. Moreover, gain-of-function mutations, such as L1198F and G1201E, have been reported in ATC14. Collectively, ALK mutations can induce the occurrence of various types of cancers, including neuroblastoma, esophageal cancer, colorectal 7



cancer, inflammatory breast cancer, rhabdomyosarcoma and NSCLC. However, the mechanisms involving cancer initiation and progression remain to be elucidated. Of note, neuroblastomas harboring both ALK mutations and amplified MYCN oncogene are more aggressive than the other cases78.

4. Development of ALK inhibitors and their therapeutic implications 4.1. Approved drugs targeting ALK Blockading of the aberrant ALK signaling pathways by small-molecule inhibitors has been validated as an effective way to inhibit the growth of ALK-positive cancer cells. Over the past decade, intensive efforts have thus been made to develop ALK inhibitors with different chemotypes18, 31, 79. At present, five ALK inhibitors, including 1, 2, 4, 5 and 11, have been approved by the FDA for the treatment of ALK-positive NSCLCs18, 30, 31, 79. Compound 1, the first FDA-approved ALK inhibitor, was initially designed for the c-Met kinase by Pfizer using an integrated drug discovery strategy16, 80. As shown in Figure 7, Pfizer’s researchers firstly identified a 3-substituted indoline-2-ones derivative, SU-11274 (13), and then optimized it to a potent inhibitor of c-Met, PHA-665752 (14). Due to its poor physicochemical properties, they re-designed the core skeleton to a novel 5-aryl-3-benzyloxy-2-aminopyridine core by structure-based drug design (SBDD)16. Further optimization based on the core structure of 15 led to the discovery of 1 as an effective dual inhibitor against c-Met and ALK. As a Type-I inhibitor, compound 1 occupies the front ATP binding pocket in the active “DFG-in” conformation and forms two H-bonds with Glu1197 and Met1199 (Figure 8A). The biochemical and cellular assays illustrate that 1 is nearly 20-fold more selective for c-Met and ALK over a panel of > 120 kinases from different families80. Compound 1 can effectively suppress NPM-ALK phosphorylation in Karpas-299 and SU-DHL-1 ALCL cell lines with IC50 values of 32 nM and 43 nM, respectively80. A series of clinical response data, including PROFILE 1001 (phase I, 2008), PROFILE 1005 (phase I/II, 2011), PROFILE 1007 (phase III, 2012) and PROFILE 1014 (phase III, 8


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2014), confirmed that 1 is superior to the standard chemotherapy (pemetrexed or docetaxel) in the treatment of advanced or metastatic EML4-ALK NSCLCs81-83, which led to the approval of 1 for the treatment of ALK-positive NSCLC in 2011. In addition, compound 1 also shows excellent efficacy in ALK-positive IMTs and Kelly neuroblastomas induced by gene amplification and/or mutations84, 85. Unfortunately, the success of 1 was overshadowed by the rapid emergence of drug resistance30, 86. The most frequently acquired mutations are L1196M, C1156Y, G1202, and G1269A/S (Figure 9B). Moreover, the 1151Tins, L1152R, I1171T/N/S, F1174V/L/C, G1202R, D1203N, S1206Y and V1180L mutations were also reported to be able to confer resistance to 1 (Figure 9A). The emergence of drug resistance and increasing need for better drugs led to the development and approval of the second- and third-generation ALK inhibitors (Figures 10, 11, 12 and 13), including 3, 4, 5 and 1128, 30, 41, 87, 88.

In 2013, Marsilje and colleagues from Novartis reported the discovery of 3 with a 2,4-diaminopyrimidine core scaffold which was optimized based on the structure of compound 2 from high-throughput screening (HTS)19, 79. TAE684 exhibited strong potency against Ba/F3 harboring NPM-ALK and two NSCLC cell lines (NCI-H2228 and NCI-H3122) harboring EML4-ALK89. However, as shown in Figure 10, compound 2 didn’t enter clinical trials due to its potential toxic effects as a result of oxidative metabolism79,

89.

Structure-activity relationship (SAR) analysis further

confirmed that generation of the reactive metabolite was primarily attributed to the solubilizing group linked by the nitrogen atom on the central aniline moiety (Figure 10). Further modifications were performed to improve kinase selectivity and to obstruct the formation of reactive metabolites, and ultimately yielded 390. In Figure 8B, compound 3 forms two H-bonds with the hinge region residue Met1199, and the reversed piperidine ring is engaged in a salt bridge with Glu1210. The isopropoxy group can form favorable interactions with Arg1120, Glu1132 and the Leu1198-Ala1200-Gly1201-Gly1202 hinge segment, which may improve the selectivity and potency of 3. As expected, compound 3 retained high potency against ALK with an IC50 value of 0.2 nM, and showed potent anti-proliferative activity for 9



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Karpass-299 and Ba/F3 cell lines carrying the NPM-ALK fusion gene with IC50 values of 22.8 and 26.0 nM, respectively88. Moreover, compound 3 could induce complete tumor regression in H2228 rat xenograft model and partially suppress tumor progression in Karpass-299 xenograft model79, 88. Three multicenter phase I/II trials (ClinicalTrials.gov identifiers: ASCEND-1, -2 and -3) and two randomized phase III clinical trials (ClinicalTrials.gov identifiers: ASCEND-4 and -5) showed that 3 exhibited favorable efficacy in patients with ALK-rearranged, Crizotinib-naïve and -resistant NSCLCs88,

91-94.

In patients with advanced NSCLC harboring ALK

rearrangements, compound 3 achieved an overall response rate (ORR) of 58% and a median progression free survival (PFS) of 8.2 months (ClinicalTrials.gov identifiers: ASCEND-1)91. Compound 3 bearing a unique benzo[b]carbazole scaffold is a potent and selective ALK inhibitor modified from the initial screening hit 18 (Figure 11)20, 91, 95.

In the co-crystal structure of ALK bound to 4, the tetracylic benzo[b]carbazolone

scaffold adopts a planar conformation where the carbonyl and cyano groups form favorable H-bonds interactions with Met1199 and Lys1150 (Figure 8C). In in vitro testing, compound 4 displayed strong inhibitory activity against different ALK-driven cell lines, especially NCI-H2228 and Karpas-29996, 97. Of note, compound 4 is more effective than 1 against tumors with brain metastasis (BM)95, which can be explained by the higher penetration rate of 4 across the blood-brain barrier (BBB)98. In two multicenter

phase

II

studies

(ClinicalTrials.gov

identifiers:

NP28761

and

NP28673)99-101, the excellent efficacy and good tolerance of 4 were confirmed in patients with advanced crizotinib-refractory ALK-positive NSCLC102, which accelerated its approval by the FDA in 2015. In 2016, Huang and colleagues at Ariad Pharmaceuticals reported a series of dimethylphosphine oxide (DMPO)-containing 2-aminopyrimidine-based ALK inhibitors with improved activity and selectivity compared with the unsubstituted analog (23, 24 vs 22)21. As shown in Figure 12, compounds 5, 25, 26 and 27 (ALK-IN-1) show favorable selectivity over InsR and IGF1R. Moreover, Huang et al. explored the SAR at the C2 position of the pyrimidine core21, and they found that seven-membered homopiperazine or five-membered pyrrolidine analogs were less active than the corresponding six-membered inhibitors 10


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(28 vs 27, 30 vs 29, and 32 vs 31)21. The improved potency of 5 and its analogues may be largely due to the enhanced interactions of the methoxy group with Leu1198, the C5-chlorine atom with Leu1196, and the dimethylphosphine oxide (DMPO) moiety with the DFG motif (Figure 8D). Besides, the unique DMPO acts as a H-bond acceptor, effectively fostering the intramolecular interactions and stabilizing its U-shaped conformation. In both enzyme-based and cellular assays, compound 5 exhibited low nanomolar IC50 against WT ALK/ROS1/EGFR and a broad panel of drug-resistant mutants of ALK18,

30, 31, 103, 104

(shown in Table 1). In addition,

compound 5 demonstrated remarkable efficacy in multiple mouse models of NSCLCs without significant side effects21. In 2016, a phase II, multicenter, randomized study (ClinicalTrials.gov identifier: ALTA) demonstrated that the disease control rate (DCR) of 5 could reach 86% in patients with advanced or metastatic ALK-positive NSCLC resistant to crizotinib105. Compound 5 also yielded substantial whole-body (ORR: 54%) and intracranial responses (ORR: 67%) with robust PFS up to 12.9 months105. Compound 5 was approved by the FDA in 2017 due to its favorable potency, selectivity and ADME (absorption, distribution, metabolism, and excretion) profiles106. Although 3, 4 and 5 were active in inhibiting several crizotinib-resistant mutants of ALK, they were not able to cover the broad range of drug-resistant cases18, 30, 31, 79. In ALK-positive Ba/F3 models, compound 3 is highly active against L1196M, G1269A, S1206Y, and I1171T EML4-ALK mutants, but less active against C1156Y, G1202R, 1151Tins, L1152P, and F1174C mutants107, 108. Patients with V1180L and I1171T/N/S mutations are sensitive to 3 but are insensitive to compound 4109. Even through 5 exhibited higher potency against ALKG1202R than 3 and 4, its potency in Ba/F3 cells harboring the G1202R mutation was unfavorable110. The ALKE1210K/S1206C and ALKE1210K/D1203N mutants were successively detected in excisional biopsies of recurrent left axillary disease after treating with 5109. The ALKG1202R/1196M double mutants conferred resistance to all first- and second-generation inhibitors of ALK110. Obviously, G1202R is a common high-level resistance mutation to 1, 3, 4, and 518, 30, 98.

Besides, “bypass” drug resistance mechanism also affects drug efficacy. An 11



MAP2K1 K57N-activating mutation was identified in 3- and 4-resistant cases103. Activation of the hepatocyte growth factor (HGF)/Met pathway was also potentially responsible for the resistance to 4, and the NRG1-HER3-EGFR axis was proposed to mediate the resistance to both 3 and 418, 30. All FDA-approved ALK drugs are ATP-competitive inhibitors that contain an ATP-adenine equivalent kinase hinge binder and an extra motif extending to the solvent area. Inevitably, these compounds are vulnerable to the solvent-front mutations (SFMs), such as ALKG1202R. Fortunately, in 2014, Johnson et al. reported the discovery of an effective ALK inhibitor with an amido-linked 12-membered macrocycles, compound 11, using a structure guided approach22. In their work, the design campaign of 11 was initiated by comparing the crystal structures of apo ALK with ALK/1. As shown in Figure 13, the final successful generation of the macrocyclic 11 is credited to a series of efforts made to improve the potency, selectivity, CNS ADME and lipophilic efficiency (33, 34, 35, and 36). In Figure 8F, the rigid macrocycle in 11 is precisely anchored in the adenine binding site with a bio-active binding posture predefined to avoid the entropy penalty. Besides, compound 11 can form two stable H-bonds with Glu1197 and Met1199, and favorable contact with DGF-Asp1270 via van der Waals interaction. As expected, compound 11 is a potent dual ALK/ROS1 inhibitor with Ki values of < 0.02 nM, < 0.07 nM and 0.7 nM for ROS1, ALK and L1196M mutated ALK, respectively, and shows favorable inhibitory activity against Ba/F3 cells expressing ALKG1202R with the IC50 value of 77 nM111. Besides, compound 11 exhibits remarkable in vivo efficacy against H3122 EML4-ALKWT, H3122 EML4-ALKL1196M, H3122 EML4-ALKG1269A and H3122 EML4-ALKG1202R tumors in mice112. Moreover, compound 11 has been reported as an effective agent against neuroblastoma carrying ALK resistant mutations (F1174L, F1245C, and R1275L), especially the aggressive cases caused by the co-overexpression of ALK and MYCN75. Of note, as a substrate of the ATP binding cassette subfamily B member 1 (ABCB1), compound 11 can effectively cross the BBB and penetrate into the central nerve system (CNS)113, 114. According to the phase I/II clinical trials launched by pfizer (ClinicalTrials.gov identifiers: 12


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NCT01970865, NCT02584634 and NCT02927340), compound 11 showed promising systemic and intracranial activity in patients with ALK- or ROS1-positive NSCLC7, 115, 116.

The results from the phase I/II trials promoted 11’s first approval in Japan on

September 2018 for the treatment of drug-resistance ALK fusion-positive advanced and/or recurrent NSCLC as the third-generation inhibitors, followed by its approval in the USA on November 2018 against ALK-positive metastatic NSCLC resistant to 1, 3 or 4117,

118.

Currently, a multinational phase III trial (ClinicalTrials.gov identifier:

NCT03052608) has been initiated to compare 11 and 1 as first-line monotherapy against ALK-positive NSCLC118. Besides, clinical trials of 11 are still ongoing in order to explore the efficacy in lymphoma (phase II, NCT03505554) and neuroblastoma (phase I, NCT03107988) 118. In a preclinical study reported by Shaw et al., the L1198F mutation conferred resistance to 11 through unfavorable steric interference with drug binding, but it could enhance the binding of 1 and negate the unfavorable effect of C1156Y119.

4.2. ALK inhibitors in clinical and preclinical trials Currently, numerous ALK inhibitors are under clinical and/or preclinical studies, which are expected to outperform the FDA-approved drugs. On the basis of the core scaffolds, the reported ALK inhibitors can be classified into several series, including 2,4-aminopyrimidine21, aminopyrazine124,

24,

79,

89,

120-122,

1,3,5-triazine123,

acyliminobenzimidazoles,

3-benzyloxyaminopyridine126,

3-aminoindazole27,

1H-pyrrolo[2,3-d]pyrimidine125,

benzo[b]carbazole

derivative127,

1H-pyrazol-5-yl-1H-pyrrolo[2,3-b]pyridine derivatives128, as well as the recently identified macrocyclic ALK inhibitors29, 129. 4.2.1. 2,4-diaminopyrimidine. Over the years, Teva Pharmaceuticals made great efforts to develop effective ALK inhibitors based on the 2-aminopyrimidine core scaffold. As shown in Figure 14, Gingrich and colleagues from Teva identified compound 37 with the bicyclo[2.2.1]hept-5-ene ring, which could achieve the balance between ALK potency and global kinase selectivity24, 120, 130. However, the moderate IR/ALK selectivity (> 100) prompted further optimization of this diaminopyrimidine 13



analog. In their work, the medicinal chemistry efforts by combining 37 and 2,3,4,5-tetrahydro-1H-benzo[d]azepine fragment of the diaminopyrimidine analogue 38 yielded 39 (CEP-28122), which exhibited enhanced ALK inhibitory activity and improved IR selectivity (> 600-fold)120. However, the development of 39 was unfortunately terminated because of the severe lung toxicity in monkey models, which advanced the generation of a novel dual ALK/FAK inhibitor 7 by several rounds of optimizations (40 and 41)24. Of particular note, compound 7 showed high potency against multiple ALK mutants, including 1151Tins ALK (Kd = 2.6 nM), C1156Y ALK (Kd = 1.6 nM), F1174L ALK (Kd = 1.1 nM), and L1196M ALK (Kd = 3.3 nM)24. Compound 7 could induce complete tumor regression without toxicity in both Sup-M2 and Karpas-299 mouse xenograft models24. Moreover, compared with 39, compound 7 possessed superior solubility and metabolic stability, improved oral bioavailability, lower clearance and toxicity and favorable brain penetration24. At present, the completed phase I clinical trials showed the striking inhibitory activity of 7 in patients with advanced or metastatic solid tumor, such as inflammatory breast cancer and NSCLC131. AZD3463 (42, Figure 14), a preclinical ALK/IGF1R inhibitor designed by AstraZeneca in 2013, showed good activity against multiple crizotinib-resistant mutations (L1196M and T115Ins), and inhibited ALK autophosphorylation in tumor cell lines containing ALK fusions, such as DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK)132. In 2016, Wang et al. reported that 42 could effectively suppress the proliferation of neuroblastoma cell lines with the WT ALK and ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway133. In 2014, Liu et al. identified a series of 2,4-diarylaminopyrimidine analogues (DAAPalogues) by repurposing a typical dopamine D1/D5 receptor agonist motif (43 and 44), C1-substituted-N3-benzazepine (A) or benzazecine (B), into the C2 position of the DAAP skeleton, which led to the generation of a 1-methyl/aryl-N3-benzazepine framework121. As shown in Figure 15, compound 45 bearing a C1-methyl substituted N3-benzazepine motif showed high potency against both the c-Met and ALK with 14


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IC50 values of 6.0 and 9.0 nM, respectively. Compound 46 bearing a larger ring system has an IC50 value of 17 nM against ALK and is 40-fold more potent than that against c-Met. In BF3 EML4-ALK xenograft mice model, compound 45 displayed significant antitumor efficacy with an inhibitory rate of 60.6% at the dose of 1 mg/kg, higher than that of 1 at the dose of 50 mg/kg. However, compound 45 needs further optimization due to its moderate inhibitory activity towards L1196M and C1156Y ALK mutants. By opening the azepanone ring of 47 (Figure 16), a series of novel DAAP analogues bearing a flexible amino acid side chain were synthesized by Song et al122. Compound 49 optimized from 48 was identified a potent and selective ALK inhibitor possessing IC50 values of 2.7 and 15.3 nM against the WT and L1196 mutated ALK, respectively. Compound 49 bearing a primary amino group showed an IC50 value of 15.3 nM against SUPM2 cell line, and was found to inhibit several resistant tumors with the L1196M, L1174M or/and R1275Q mutated ALK. By adopting a bioisosteric strategy, Zhang et al. developed a new series of potent ALK inhibitors with a unique amino pyrazole side-chain in DAAPs scaffold, in which the N atom in morpholine was removed to avoid the metabolic liability134. The SAR exploration of the scaffold delivered several potential compounds (50, 51, 52, 53, and 54) with nanomolar IC50 values against the WT and L1196M ALK, and favorable anti-proliferative activity toward Karpass-299 and H2228 cell lines (Figure 17). Among them, compound 52 showed good pharmacokinetic properties in rats and dogs and dose-dependent anti-tumor efficacy in H2228 xenograft model134. 4.2.2. 1,3,5-triazine. Compound 8, developed by Astellas, is an analog of 2 from Novartis. The only difference is the structure that binds in the hinge-binding motif (1,3,5-triazine vs 5-chloropyrimidine)25. In xenograft model of H2228, 8 could be well absorbed by tumor tissue via oral administration, where the concentration was 10-fold higher than that in plasma, and could induce tumor regression with a wide therapeutic window25. Besides, compound 8 exhibited potent antitumor activity against cells expressing the 1-resistant L1196M and 4-resistant V1180L mutants135, 136. According to the two completed phase I clinical trials (ClinicalTrials.gov identifiers: NCT01401504 and NCT01284192), compound 8 also showed efficacy in patients 15



with solid tumors and B-cell lymphoma137, 138. 4.2.3. 3-aminoindazole. In 2016, Maria et al. screened a promising 3-amino-5-substituted indazole compound (55) which showed a good biochemical potency (IC50 = 73 nM) against ALK and moderate anti-proliferative activity against ALK-positive Karpass-299 cell line (IC50 = 253 nM)27. Further optimization of the substituent of ring A led to the discovery of 9 (Figure 8). The co-crystal structure of ALK in complex with 9 (Figure 8E) indicated that the 4-aminotetrahydropyranyl moiety added to ring A optimally occupies the hydrophobic space underneath G-loop and adopts a roughly orthogonal orientation with respect to the scaffold, which can explain the improved biochemical potency with IC50 of 12 and 31 nM against ALK and Karpass-299 cell line27. Besides, compound 9 is sensitive to crizotinib-resistant mutations, such as L1196M and C1156Y. Of note, compound 9 could efficiently cross the BBB and thus could be used in the treatment of NSCLCs with brain metastasis26. In addition to stable regression in ALK-dependent ALCL and NSCLC, the novel CAD-ALK dependent colorectal cancer could also be well suppressed by 9139. In the ongoing phase I/II trials launched by Ignyta, durable objective responses (DOR) were observed in patients with solid tumors harboring TRKA/B/C, ROS1, or ALK gene fusion, such as neuroblastoma (ALKA-372-001 and STARTRK-1)140, 141. 4.2.4. Aminopyrazine. Compound 10 was synthesized based on 1 according to the procedure published in WO 2009/15476928. As shown in Figure 19, compounds 10 and 1 share the same hydrophobic 2,6-dichloro-3-fluoro-phenylethoxy groups and similar kinase hinge binding groups, but the side chains are significantly different. The cellular activity of 10 against H3122 was 10-fold higher than that of 128, and 10 exhibited improved inhibitory activity against L1196M and C1156Y mutants124. In a phase I/II, multicenter “Xalt2” study of 10, 10 out of 17 (59%) ALK-rearranged NSCLC patients achieved partial responses, and 7 out of 13 patients resistant to 1 responded to 10 due to its favorable penetration into the brain142. Currently, a phase III study (eXalt3) is in progress to compare the efficacy and safety of 10 with 1 in patients with ALK-positive NSCLCs143. 4.2.5. Acyliminobenzimidazoles. Compound 56, a 2-acyliminobenzimidazole 16


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derivative, was discovered via HTS from the Amgen compound library and used as a starting structure. A series of optimization on 56 finally led to the discovery of the 3,5-difluoro analogues 59 (Belizatinib) and 61 with improved potency and selectivity toward ALK (Figure 20). Both compounds had acceptable PK distribution and high selectivity over JAK2, SRC and IGF1R, and could also effectively inhibit R1275Q and L1196M mutants23. In particular, compound 61 achieved dose-dependent regression in an NPM-ALK driven tumor xenograft model23. A completed phase I/II study of 59 (ClinicalTrials.gov identifier: NCT02048488) showed that 59 is a well-tolerated agent against ALK-dependent and crizotinib-refractory NSCLCs144, 145. 4.2.6. Benzo[b]carbazoles. Inspired by the structural insights of 4 bound to ALK (PDB ID: 3AOX), JH-VIII-157-02 (63) was developed with the expectation to overcome resistance caused by G1202R mutations127 (Figure 21). In addition to the extremely potent inhibitory activity against the G1202R mutant, 63 exhibited improved potency against ALK-positive neuroblastoma cell lines (Kelly, SH-SY5Y (F1174L), and LAN-5 (R1275Q)) and NSCLC cell lines (H3122, DFC176 (L1152R) and DFCI114 (G1269))146, 147. 4.2.7. 1H-pyrrolo[2,3-d]pyrimidine. GSK1838705A (64), a derivative of 1H-pyrrolo[2,3-d]pyrimidine, was firstly identified as a IGF-IR inhibitor125. Later on, it was found to inhibit ALK with an IC50 of 0.5 nM (Figure 22). Compound 64 could repress the growth of ALCL and NSCLC cell lines containing ALK fusion genes in vitro and induce complete regression of NPM-ALK dependent tumors in vivo148, 149. 4.2.8. 3-benzyloxyaminopyridine. Compound 65 shown in Figure 22 was generated by modifying the 2,6-dichloro-3-fluorophenyl head and pyrazolopiperidine tail groups in 1126. The in vitro activity of 65 was significantly improved against both WT and resistant mutants of ALK (L1196M, G1269A, S1206Y, C1156Y, F1174L, L1152R and 1151Tins) compared with that of 1. In cellular assays, the activity of 65 was also improved in both the WT cell lines (H3122, H2228, and Karpas299) or engineered mutant lines (H3122-L1196M and H3122-G1269A). 4.2.9. 1Hpyrazol-5-yl-1H-pyrrolo[2,3-b]pyridine derivatives. Most recently, Fushimi et al. reported the discovery of a 1H-pyrazol-5-yl-1H-pyrrolo[2,3-b]pyridine 17



Page 18 of 83

derivative 68 as a highly selective, potent, and brain-penetrant ALK inhibitor128. Figure 23 shows that the discovery process started from a HTS hit 66, followed by scaffold hopping and SBDD-driven lead optimization128. Then, compound 68 was proved to have good pharmacokinetic and BBB profiles, and could significantly decrease the phosphorylated-ALK levels in hippocampus after intraperitoneal administration128. These results suggest that 68 can serve as a useful in vivo tool to explore the mechanism of ALK-mediated CNS disorders. 4.2.10. Macrocyclic ALK inhibitors. The great success of newly FDA-approved drug 11 intrigued the researchers’ enthusiasm to design macrocyclic ALK inhibitors. And most recently, TP Therapeutics developed another macrocyclic compound, the fourth generation ALK inhibitor 12 (Figure 1), which showed excellent efficacy for WT ALK and SRC (IC50 = 1.01 and 5.3 nM) as well as a broad spectrum of acquired resistant mutants, especially G1202R (IC50=1.26 nM) and L1196M (IC50=1.08 nM)29. Structural modeling predicted that 12 could accommodate the bulky and positively charged arginine side chain in the solvent front without any steric clashes129. Additionally, compound 12 effectively suppressed the phosphorylation of EML4-ALK and SRC substrate paxillin with IC50 values of 13 and 107 nM, respectively129. Moreover, compound 12 also could inactivate multiple downstream targets, such as ERK, AKT and STAT3, which indicated its potential ability to overcome ALK resistance due to “bypass” signaling activation150,

151.

At present, a

phase I/II, open-label and multicenter clinical trial of 12 has been initiated to evaluate its safety, tolerability, pharmacokinetics and antitumor activity in patients with advanced

solid

tumors

expressing

ALK/ROS1/TRK(A/B/C)

rearrangements

(ClinicalTrials.gov identifier: TRIDENT-1)129.

4.3 Type-I1/2 and Type-II inhibitors of ALK In 2013, Bryan and colleagues firstly reported a novel Type-I1/2 ALK inhibitor33, piperidine carboxamide 69, which occupies both the ATP active site and the less conserved back cleft in a “DFG-in” conformation (PDB ID: 4CDE) shown in Figure 24B and 24C. A series of analogues with improved activity, such as 70, 71 and 72, 18


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were subsequently synthesized by a modular and parallel-synthesis approach. As expected, these Type-I1/2 compounds exhibited selectivity over the other kinase family members, such as insulin-like growth factor-1 (IGF1R), because of the further extension into the less conserved hydrophobic back pocket (Figure 24A). However, the binding affinities of these piperidine carboxamide derivatives were relatively weak compared to Type-I ALK inhibitors in clinic. In 2017, by tethering a rigid conjugate structure, pyrrole pyrimidine, to the piperidine carboxamide structure, our group designed a scaffold containing hydrogen bond donors and/or acceptors and hydrophobic moieties, and finally synthesized 32 Type-I1/2 ALK inhibitors after several rounds of optimizations (Figure 25B and 25C)35. In enzyme-based assay, five compounds (73, 74, 75, 76 and 77) exhibited extremely potent inhibitory activity against ALK (Figure 25A). The most potent analogue 77 is > 37-fold (IC50 = 0.27 nM) more active than piperidine carboxamide 70 (IC50= 10 nM), and more potent than 1 (IC50=4.66 nM) and 3 (IC50=3.94 nM)35. Besides, compound 77 displayed superior sensitivity over the FDA-approved ALK inhibitors, 1 and 3, against L1196M, C1156Y, R1275Q, and F1174L ALK mutants with IC50 of 7.51, 4.71, 2.69 and 9.81 nM, respectively35. Moreover, compound 77 showed excellent anti-proliferation activity against NSCLC cell line NCI-H2228 and ALCL cell line Karpass-299 with the inhibition rates of 71.67% and 70.99 %, respectively, at the concentration of 8 ng/mL. In 2016, a 3-phenyl-1H-5-pyrazolylamine-based compound bearing a urea substituent (79) was identified as a Type-II ALK inhibitor with an IC50 value of 177 nM from an in-house screening34. Chih Hsiang et al. revealed that 79 induced the reallocation of the activation loop, αC-helix, and juxtamembrane domain (Figure 26B and 26C), which affected the auto-inhibition of ALK and the downstream signaling pathways34. Comparison of the co-crystal structures of the ALK/78 and ALK/79 complexes indicated that the substituted-urea tail moiety regulated the switch between the Type-I and Type-II binding modes by inducing dramatic conformational changes of the A-loop, αC-helix, and JM domain (Figure 26). This work is the first structural biology study to explore how the structure changes of a small molecule regulate the 19



switch between the “DFG-in” and “DFG-out” conformations, which provides valuable guidance to the development of novel Type-II inhibitors for ALK or other kinases. In summary, the improved kinase selectivity and enhanced capability to combat drug resistance of these Type-I1/2 and/or Type-II inhibitors against ALK can be explained by the conformational changes of the back hydrophobic cavity, such as Arg1275 and Leu1174, which is supported by the crystallographic evidence reported by Epstein et al.152. Two molecular modeling studies published in 2015 and 2018 by our group indicated that the hydrophobic interactions between Type-I1/2 inhibitors and residues surrounding the extended pocket determined the binding strength of these inhibitors153, 154. And the umbrella sampling (US) simulations suggested Type-I1/2 and Type-II inhibitors may exhibit decreased dissociation rate (koff) with prolonged residence time, which is consistent with the results of other studies on these types of inhibitors155-158. However, these types of inhibitors may also encounter several problems due to relatively high molecular weight and lipophilicity, such as limited solubility and poor pharmacokinetic (PK) properties159, suggesting rational modifications need to be made on these types of drugs.

4.4 Proteolysis Targeting Chimeras (PROTACs) of ALK Currently, “proteolysis targeting chimera” (PROTAC) technique has been widely used in the field of drug discovery160, 161. Inspired by the success of this approach in degradation of multiple protein targets, several ALK PROTACs have been developed (Figure 27). In 2018, Powell et al. developed two ALK PROTACs, 80 (TL13-12) and 81 (TL13-112) by conjugating pyrimidine-based ALK inhibitors 2 and 3 to the CRBN ligand Pomalidomide37. These bivalent small molecules led to the ubiquitination and degradation of ALK in NSCLC cells harboring EML4-ALK, ALCL cells harboring NPM-ALK, and NB cells expressing F1174L/R1275Q ALK, meanwhile sustaining the inhibition of downstream ALK signaling. In the same year, Zhang et al. reported other two ALK PROTACs, 82 (MS4077) and 83 (MS4078), by connecting 3 and Pomalidomide using two distinct linkers38. In addition to the degradation of 20


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NPM-ALK and EML4-ALK, 82 and 83 could inhibit the phosphorylation of ALK and STAT3 in a concentration- and time-dependent manner in both SU-DHL-1 cells and NCI-H2228 cells. Subsequently, Kang and colleagues reported new ALK PROTAC, 84 (TD-004), that showed excellent efficacy in cellular level and in vivo xenograft mouse model36. Even if the efficacy of ALK degraders needs further exploration, ALK PROPACs undoubtedly opened a new window for targeted ALK therapies.

5. Structural analyses of drug-resistant mutants of ALK Crizotinib-resistant mutations, such as L1196M, G1269A, G1202R, and S1206Y, may hamper drug binding and induce drug resistance of ALK (Table 1). For instance, L1196M is a typical "gate keeper" mutation, which locates just in the ATP binding pocket of ALK and can directly hinder crizotinib binding

162.

MD simulations

executed by Nagasundaram et al. suggested that L1196M and G1269A had significant impact on the dynamics and flexibility of ALK163. Although the C1156Y and L1152R mutations locate a bit far from the ATP binding pocket of ALK (~15 Å), they conferred drug resistance either by continuously activating the protein or by affecting the conformation of the binding pocket, which could attenuate the binding affinity of 1 to ALK indirectly164. Our theoretical studies showed that the L1152R and C1156Y mutations could affect the conformation of the G-rich loop (also known as P-loop) in ALK, which is flexible and susceptible to be regulated by both drugs and mutations around it165,

166.

Moreover, Kumar et al. pointed out that the F1174L mutation

enhanced the flexibility and dynamics of ALK, and affected the formation of intermolecular interactions, especially H-bonding interaction, which led to the resistance to 1166. Notably, both Gly1202 and Ser1206 are solvent-front residues located at the entrance of the ATP binding pocket and can form direct contacts with drugs such as 1, 3 and 4 (Figure 8A, 8B and 8C). Unfortunately, these residues inevitably mutate (such as G1202R and S1206Y) and confer resistance to several FDA-approved ALK drugs, where G1202R is a very harmful mutation and can confer resistance to many ALK drugs (Table 1). Mutations located at the same position as 21



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G1202R are also found in other kinases, such as G2032R in ROS1 and G595R in TRK1167, which all confer resistance to ALK inhibitors, suggesting that it is urgent to uncover the drug-resistance mechanisms for designing new inhibitors to overcome the resistance induced by the solvent-front mutations for various kinases. To reach the goal, our group has also done several theoretical works to reveal the drug resistance mechanisms induced by the solvent-front mutations168,

169.

Taking crizotinib as an

example, these solvent-front mutations (G1202R and S1206Y) can hinder drug binding to the "binding channel" outside the ATP binding pocket and thus directly attenuate the binding of crizotinib to ALK168. To overcome the G1202R-induced drug resistance, macrocyclic inhibitors, such as 11, have been designed, and they almost bind into the center of the pocket and do not form direct interaction with G1202. Thus, macrocyclic inhibitors are not quite sensitive to the G1202R mutation. Unfortunately, in 2016, the L1198F mutation was found to confer resistance in 11-resistant patients119. As shown in Figure 8F, the L1198F mutation may force 11 to rotate away from Phe1271, and attenuates its interaction with the hinge region. However, as to 1, the distance between Phe1271 and 1 in the L1198F mutant is smaller than that in the WT complex, thus possibly enhancing the interactions between 1 and ALK (Figure 8A). These findings coincide with the experimental observation that the L1198F mutation confers resistance to 11 but not 1119 and also suggest that drug combination is needed to overcome drug resistance in ALK.

6. Potential therapeutic strategies to overcome drug resistance 6.1 Sequential dosing of multiple ALK TKIs According to the accumulated basic knowledge, different ALK inhibitors have their own advantages and defects, and exhibit specific target profiles in various ALK mutants, suggesting a potential therapeutic strategy that the patients could be treated sequentially with multiple ALK TKIs. For example, patients harboring the C1156Y mutation, which occurred after the 1 treatment, were resistant to 1 but sensitive to 22


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11119. In some cases, the secondary acquired resistance to 11, such as L1198F, could be overcome again by 1119. Activation of the HGF/Met pathway and/or c-Met amplification was a validated factor inducing resistance to 4, while 1 could strongly inhibit the activity of the Met tyrosine kinase in both in vitro and in vivo assays170. Thus, compound 1 might demonstrate favorable efficacy in patients with 4-refractory NSCLC harboring EML4-ALK and MET activation. Two randomized clinical trials (ClinicalTrials.gov identifiers: J-ALEX and ALEX) are currently ongoing in order to compare the performance of 4 versus 1 in participants with treatment-naïve ALK-positive advanced NSCLC101,

171.

In view of this, clinicians should have a

precise and deep insight into ALK kinase aberration and/or its signaling transduction to put forward a tailored and personalized ALK TKI therapies.

6.2. Combinational therapy The effectiveness of combined inhibition of both ALK and another related kinase (IGF1R, MEK, cKIT, EGFR, VEGF, mTOR or SRC) is still under preclinical/clinical investigations (ClinicalTrials.gov identifiers: NCT02321501, NCT0252105, etc.), which might overcome drug resistance mediated by alternative “bypass” pathways18, 30, 31, 103, 104.

For instance, compound 4 was combined with a EGFR inhibitor Erlotinib,

and 1 was administrated together with a pan-Her inhibitor PF-0299804 for the treatment of NSCLC patients172. The combination of 11 and PI3K pathway inhibitors exhibited good efficacy in overcoming ALK resistance117. Two clinical trials for the combination of the non-kinase Hsp90 inhibitor AUY922 and 3 as well as the Hsp90 inhibitor Onalespib and 1 are also underway (ClinicalTrials.gov identifiers: NCT01772797 and NCT01712217)173. Recent preclinical data indicated that the combinational therapies of checkpoint (PD-1/PD-L1 and CTLA-4) inhibitors with ALK inhibitors might improve the therapeutic effects in ALK-positive NSCLC patients, which were illustrated in several clinical trials, including 3 with Nivolumab (ClinicalTrials.gov

identifier:

NCT02393625)174,

1

or

11

with

Avelumab

(ClinicalTrials.gov identifier: NCT02584634)175, 1 with Ipilimumab or Nivolumab (ClinicalTrials.gov identifier: NCT01998126)176. In addition, combination with 23



Page 24 of 83

conventional chemotherapy also remains an option for patients with ALK translocations (ClinicalTrials.gov identifier: NCT02134912)177,

178.

Collectively, the

selection of appropriate combination strategy should be individualized according to the mechanisms of acquired resistance and the signal transduction of ALK.

7. Concluding remarks and future perspectives Oncogenic ALK acts as a driver in a set of cancer types, such as NSCLC, ALCL, IMT, and neuroblastoma, which could be suppressed by small molecule inhibitors of ALK. The emergence of drug resistance led to the development of the first-, second-, thirdand fourth-generation ALK inhibitors. Besides, novel Type-I1/2 and Type-II inhibitors of ALK that could occupy both the ATP active site and the extended less-conserved hydrophobic pocket were developed. This kind of inhibitors is expected to improve kinase selectivity and enhance capability to combat drug resistance. Particularly, the PROTAC technique was also applied in the development of ALK drugs, and several potential ALK PROTACs have already been reported, which opened a new window for targeted ALK therapies. Dual blockade of both ALK and other kinases has been proved to be a potential anti-resistant therapeutic strategy. In addition, combination of ALK inhibitors with chemotherapy or immunotherapy is also being evaluated in both preclinical and clinical trials, but special attention should be paid to avoiding additional toxicity. Due to the heterogeneity of tumors and the constantly acquired drug resistance, the pathogenesis and signal transductions of ALK in different tumors exhibit distinct features, which brings more challenge to successful therapy. Nowadays, more sophisticated detection techniques, such as fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) of tumor tissue, and sequencing of circulating tumor cells (CTCs), serve as alternative or complementary strategies to optimize drug treatment. Besides, computational modeling methods also benefit to discovering and developing novel ALK drugs, as well as exploring the binding features and resistance mechanisms of the drugs. Together, in-depth understanding the roles of ALK in cancer biology and more precise techniques to 24


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detect ALK aberrations are of vital significance to improve the therapeutic outcomes in clinic. Besides, attempts to discover novel ALK drugs and alternative targeted ALK therapies are in urgent need to combat drug resistance. Acknowledgments This study was supported by National Science & Technology Major Project of China “Key New Drug Creation and Manufacturing Program” (2018ZX09711002-007), National Science Foundation of China (21575128, 81603031), and Zhejiang Provincial Natural Science Foundation of China (LZ19H300001).

Author Information Corresponding Authors *Phone: +86-517-88208412; E-mail: [email protected] Notes The authors declare no competing financial interest. Biographies Xiaotian Kong received her Ph.D. degree in Chemistry in 2018 under the supervision of Prof. Tingjun Hou at Zhejiang University and Prof. Youyong Li at Soochow University. Now, she is a postdoctoral fellow in College of Pharmacy at Ohio State University. She focuses on design and discovery of novel small molecules targeting cancer-related proteins, such as ALK. Peichen Pan is a Ph.D candidate in College of Pharmaceutical Sciences at Zhejiang University. His research mainly focuses on screening and optimization of small-molecule inhibitors targeting ALK. Huiyong Sun received his Ph.D. from Institute of Functional Nano & Soft Materials (FUNSOM) at Soochow University (2012~2015). Currently, he is an associated professor in School of Pharmacy at China Pharmaceutical University. His research interest is in Computer-Aided Drug Design (CADD). Up to date, He has co-authored more than 50 peer-reviewed articles. Hongguang Xia received his Ph.D. from shanghai institute of organic chemistry. He did his postdoctoral program in Junying Yuan's lab at Harvard medical school. Now, 25



Page 26 of 83

he is a Professor and deputy director of Molecular Medicine Center in School of Medicine at Zhejiang University. Prof. Xia has published over 30 peer-reviewed articles in top journals and he is one of the main inventors for a novel anti-tumor drug which is now in Phase I clinical trials. Xuwen Wang is a Ph.D candidate in college of Pharmaceutical Sciences at Zhejiang University in China. His research focuses on design and discovery of novel leading compounds towards key targets relevant to cancers. Youyong Li received his B.S. from Peking University and Ph.D. from California Institute of Technology. He is currently a full professor of the Institute of Functional Nano & Soft Materials at Soochow University. His main research interest is the development of multiscale simulation methods and their applications for different functional materials. Dr. Li has published over 200 articles. Tingjun Hou received his Ph.D. in Computational Chemistry from Peking University in 2002. His current research focuses on molecular modeling and computer-aided drug design (CADD), including development of structure-based virtual screening methodologies, theoretical predictions of ADMET and drug-likeness, discovery of small molecular inhibitors towards important drug targets, and multiscale simulations of target-ligand recognition. Currently, he is a full professor in College of Pharmaceutical Sciences at Zhejiang University. He is also the director of Drug Informatics and Computational Biology Center in Hangzhou Institute of Innovative Medicine at Zhejiang University. Prof. Hou has co-authored more than 320 publications in peer-reviewed journals with a h-index of 52, 4 book chapters and 1 multimedia courseware.

Abbreviations Used ABCB1, ATP binding cassette subfamily B member 1; AKT, protein kinase B (PKB); ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; ATC, anaplastic thyroid cancer; ATIC, bifunctional purine biosynthesis protein PURH; BBB, blood-brain barrier; BM, brain metastasis; C2orf44, 26


chromosome

2

open

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reading

frame

44;

CAD,

carbamoyl-phosphate

synthetase

transcarbamylase, and dihydroorotase; CARS, cysteinyl-tRNA

2,

aspartate

synthetase;

cKIT,

KIT proto-oncogene; CLTC, clathrin heavy chain; c-Met, tyrosine-protein kinase Met or hepatocyte growth factor receptor; CNS, central nerve system; C-spine, catalytic spine; CTCs, sequencing of circulating tumor cells; CTLA, cytotoxic T-lymphocyte associated protein 4; DAG, dystroglycan; DCR, disease control rate; DCTN1, dynactin subunit 1; DFG,Asp-Phe- Gly; DLBCL, diffuse

large

B-cell

lymphoma; DMPO, dimethylphosphine oxide; DOR, durable objective responses; EGFR, epidermal growth factor receptor; EML4, echinoderm associated protein-like 4; EMT,

epithelial-mesenchymal

microtubule

transition;

ERK1/2,

mitogen-activated protein (MAP) kinase 3/1; ETV6,transcription factor Etv6; FDA, food and drug administration; FISH,

fluorescence in situ hybridization; FN1,

fibronectin 1; GFPT1,

glucosamine

efructose

6

phosphate

isomerizing 1; GRB2,

growth factor receptor bound protein 2; HER3, human

epidermal growth factor receptor 3; HGF, hepatocyte

growth

aminotransferase

factor;

HIP1,

huntingtin-interacting protein 1; IGF1R,

insulin-like growth factor 1 receptor; IMT,

inflammatory myofibroblastic tumor; IR,

insulin receptor; IRS-1,insulin receptor

substrate 1; JAK,janus kinase; Jun/AP1,

Jun proto-oncogene/activating protein-1;

KIF5B, kinesin family member 5B; KLC1, kinesin light chain 1; KRAS, kirsten

rat

sarcoma virus; MAP2K1, mitogen-activated protein kinase kinase 1; MAPK, mitogen activated kinase-like protein; MEK, mitogen-activated protein kinase kinase; MSN, moesin; mTOR, serine/threonine-protein kinase mTOR; MYCN, N-myc proto-oncogene protein; MYH9,

myosin heavy chain 9; NF-ƙB,

nuclear

factor

kappa-light-chain-enhancer of activated B cells; NGS, next-generation sequencing; NPM,

nucleophosmin; NRG1, neuregulin 1; NSCLC, non-small cell lung cancer;

ORR, overall response rate; PD-1/PD-L1, programmed

cell

death

1/programmed death-ligand 1; PI3K, phosphoinositide 3-kinases; PKC, kinase C; PLC-γ, phosphoinositide targeting chimera; PTPN3, RANBP2,

phospholipase

C-γ;

PROTAC,

protein

protein proteolysis

tyrosine-protein phosphatase non-receptor type 3;

E3 SUMO-protein ligase RanBP2; RNF213 27


E3,

ubiquitin-protein


ligase RNF213; ROS1,

Page 28 of 83

ROS proto-oncogene 1; RRBP1,

ribosome

binding

protein 1; R-spine, regulatory spine; SAR, structure-activity relationship; SBDD, structure based drug design; SEC31A, protein transport protein Sec31A; SFMs, solvent-front mutations; SHC, SHC-adaptor protein; SOC, ovarian carcinoma; SQSTM1, sequestosome 1; SRC, SRC proto-oncogene; STAT, signal and activator of transcription; STRN, striatin; TFG, protein

transducer

TFG;

TPM3/4,

tropomyosin ¾; TPR,translocated promoter region; TRAF1, TNF

receptor

associated factor 1; TRKA/B/C, tropomyosin receptor kinase A/B/C; VCL, vinculin; VEGF, vascular endothelial growth factor A; EML4, echinoderm

microtubule-associated

epithelial-mesenchymal transition; ERK1/2, kinase 3/1; ETV6,

protein-like

4;

mitogen-activated

transcription factor Etv6; FDA,

EMT,

protein

(MAP)

food and drug administration;

FISH, fluorescence in situ hybridization; FN1, fibronectin

1;

GFPT1,

glucosamine—fructose-6-phosphate aminotransferase isomerizing 1; GRB2, growth factor receptor bound protein 2; HER3,human epidermal growth factor receptor 3; HGF,

hepatocyte growth factor; HIP1,

huntingtin-interacting protein 1; IGF1R,

insulin-like growth factor 1 receptor; IMT, inflammatory myofibroblastic tumor; IR, insulin receptor; IRS-1,

insulin receptor substrate 1; JAK, janus kinase; Jun/AP1,

Jun proto-oncogene/activating protein-1; KIF5B, KLC1, kinesin light chain 1; KRAS, kirsten

kinesin rat

sarcoma

mitogen-activated protein kinase kinase 1; MAPK, mitogen protein; MEK,

family

member

virus;

MAP2K1,

activated

kinase-like

mitogen-activated protein kinase kinase; MSN, moesin;

serine/threonine-protein kinase mTOR; MYCN, N-myc

5B;

proto-oncogene

mTOR, protein;

MYH9, myosin heavy chain 9

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15); Abstract 8063. 146. Katayama, R. Therapeutic Strategies and Mechanisms of Drug Resistance in Anaplastic Lymphoma Kinase (ALK)-rearranged Lung Cancer. Pharm. Ther. 2017, 177, 1-8. 147. Wang, H.; Wang, Y.; Guo, W.; Du, B.; Huang, X.; Wu, R.; Yang, B.; Lin, X.; Wu, Y. Insight into Resistance Mechanism of Anaplastic Lymphoma Kinase to Alectinib and JH-VIII-157-02 Caused by G1202R Solvent front Mutation. Drug Des. Dev. Ther. 2018, 12, 1183-1193. 148. Zhou, F.; Chen, X.; Fan, S.; Tai, S.; Jiang, C.; Zhang, Y.; Hao, Z.; Zhou, J.; Shi, H.; Zhang, L.; Liang C. GSK1838705A, an Insulin-like Growth Factor-1 Receptor/insulin Receptor Inhibitor, Induces Apoptosis and Reduces Viability of Docetaxel-resistant Prostate Cancer Cells Both in Vitro and in Vivo. OncoTargets Ther. 2015, 8, 753-760. 149. Zhou, X.; Shen, F.; Ma, P.; Hui, H.; Pei, S.; Chen, M.; Wang, Z.; Zhou, W.; Jin, B. GSK1838705A, an IGF-1R inhibitor, Inhibits Glioma Cell Proliferation and Suppresses Tumor Growth in Vivo. Mol. Med. Rep. 2015, 12, 5641-5646. 150. Zhai, D.; Deng, W.; Huang, Z.; Rogers, E.; Cui, J. J. The Novel, Rationally-designed, ALK/SRC Inhibitor TPX-0005 Overcomes Multiple Acquired Resistance Mechanisms to Current ALK Inhibitors. Presented at the 107th Annual Meeting of the American Association for Cancer Research (AACR), New Orleans, LA, 2016; Abstract 2132. 151. Deng, W.; Huang, J.; Zhai, D.; Rogers, E.; Cui, J. Overcoming Acquired Drug Resistance by TPX-0005, an ALK, ROS1 and Pan-TRK Inhibitor. Presented at the Annual Meeting of the American Association for Cancer Research (AACR), Washington, DC. 2017; Abstract 3168. 152. Epstein, L. F.; Chen, H.; Emkey, R.; Whittington, D. A. The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of 51



Anaplastic Lymphoma Kinase. J. Biol. Chem. 2012, 287, 37447-37457. 153. Kong, X.; Pan, P.; Li, D.; Tian, S.; Li, Y.; Hou, T. Importance of Protein Flexibility in Ranking Inhibitor Affinities: Modeling the Binding Mechanisms of Piperidine Carboxamides as Type I1/2 ALK Inhibitors. Phys. Chem. Chem. Phys. 2015, 17, 6098-6113. 154. Kong, X.; Sun, H.; Pan, P.; Zhu, F.; Chang, S.; Xu, L.; Li, Y.; Hou, T. Importance of Protein Flexibility in Molecular Recognition: a Case Study on Type-I1/2 Inhibitors of ALK. Phys. Chem. Chem. Phys. 2018, 20, 4851-4863. 155. Time, O. T. Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine Wentsch. Heike K. 5363-5367. 156. Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan, J. Biology, M. Inhibition of p38 MAP Kinase by Utilizing a Novel Allosteric Binding Site. Nat. Struct.Mol. Biol. 2002, 9, 268-268. 157. Walter, N. M.; Wentsch, H. K.; Bührmann, M.; Bauer, S. M.; Döring, E.; Mayer-Wrangowski, S.; Sievers-Engler, A.; Willemsen-Seegers, N.; Zaman, G.; Buijsman, R.; Lämmerhofer M. Design, Synthesis, and Biological Evaluation of Novel Type I1/2 p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R-spine. J. Med. Chem. 2017, 60, 8027-8054. 158. Zhang, D.; Huang, S.; Mei, H.; Kevin, M.; Shi, T.; Chen, L. Protein–ligand Interaction Fingerprints for Accurate Prediction of Dissociation Rates of p38 MAPK Type II Inhibitors. Integr. Biol. 2019, 11, 53-60. 159. Blanc, J.; Geney, R.; Menet, C. Type II Kinase Inhibitors: an Opportunity in Cancer for Rational Design. Anti-Cancer Agent. Med. Chem. 2013, 13, 731-747. 52


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160. Paiva, S.-L.; Crews, C. M. Targeted Protein Degradation: Elements of PROTAC Design. Curr. Opin. Chem. Biol. 2019, 50, 111-119. 161. Zou, Y.; Ma, D.; Wang, Y. The PROTAC Technology in Drug Development. Cell Biochem. Funct. 2019, 37, 21-30. 162. Choi, Y. L.; Soda, M.; Yamashita, Y.; Ueno, T.; Takashima, J.; Nakajima, T.; Yatabe, Y.; Takeuchi, K.; Hamada, T.; Haruta, H.; Ishikawa, Y. EML4-ALK Mutations in Lung Cancer that Confer Resistance to ALK Inhibitors. New Engl. J. Med. 2010, 363, 1734-1739. 163. Nagasundaram, N.; Wilson Alphonse, C. R.; Samuel Gnana, P. V.; Rajaretinam, R. K. Molecular Dynamics Validation of Crizotinib Resistance to ALK Mutations (L1196M and G1269A) and Identification of Specific Inhibitors. J. Cell. Biochem. 2017, 118, 3462-3471. 164. Doebele, R. C.; Pilling, A. B.; Aisner, D. L.; Kutateladze, T. G.; Le, A. T.; Weickhardt, A. J.; Kondo, K. L.; Linderman, D. J.; Heasley, L. E.; Franklin, W. A.; Varella-Garcia, M. Mechanisms of Resistance to Crizotinib in Patients with ALK Gene Rearranged Non–small Cell Lung Cancer. Clin. Cancer Res. 2012, 18, 1472-1482. 165. Sun, H. Y.; Ji, F. Q. A molecular Dynamics Investigation on the Crizotinib Resistance Mechanism of C1156Y Mutation in ALK. Biochem. Bioph. Res. Co. 2012, 423, 319-324. 166. Kumar, A.; Ramanathan, K. Exploring the Structural and Functional Impact of the ALK F1174L Mutation Using Bioinformatics Approach. J. Mol. Mod. 2014, 20, 2324. 167. Russo, M.; Misale, S.; Wei, G.; Siravegna, G.; Crisafulli, G.; Lazzari, L.; Corti, G.; Rospo, G.; Novara, L.; Mussolin, B.; Bartolini, A. Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer. Cancer Discov. 2016, 6, 36-44. 168. Sun, H.; Li, Y.; Li, D.; Hou, T. Insight into Crizotinib Resistance Mechanisms Caused by 53



Three Mutations in ALK Tyrosine Kinase Using Free Energy Calculation Approaches. J. Chem. Inf. Model. 2013, 53, 2376-2389. 169. Sun, H.; Li, Y.; Tian, S.; Wang, J.; Hou, T. P-loop Conformation Governed Crizotinib Resistance in G2032R-mutated ROS1 Tyrosine Kinase: Clues from Free Energy Landscape. PLoS Comput. Biol. 2014, 10, e1003729. 170. Isozaki, H.; Takigawa, N.; Kiura, K. Mechanisms of Acquired Resistance to ALK Inhibitors and the Rationale for Treating ALK-positive Lung Cancer. Cancers 2015, 7, 763-783. 171. Gadgeel, S. M.; Gandhi, L.; Riely, G. J.; Chiappori, A. A.; West, H. L.; Azada, M. C.; Morcos, P. N.; Lee, R.-M.; Garcia, L.; Yu, L.; Boisserie, F. Safety and Activity of Alectinib against Systemic Disease and Brain Metastases in Patients with Crizotinib-resistant ALK-rearranged Non-small-cell Lung Cancer (AF-002JG): Results from the Dose-finding Portion of a Phase 1/2 Study. Lancet Oncol. 2014, 15, 1119-1128. 172. Courtin, A.; Smyth, T.; Hearn, K.; Saini, H. K.; Thompson, N. T.; Lyons, J. F.; Wallis, N. G. Emergence of Resistance to Tyrosine Kinase Inhibitors in Non-small-cell Lung Cancer can be Delayed by an upfront Combination with the HSP90 Inhibitor Onalespib. Br. J. Cancer 2016, 115, 1069. 173. Gainor, J. F.; Niederst, M. J.; Lennerz, J. K.; Dagogo-Jack, I.; Stevens, S.; Shaw, A. T.; Sequist, L. V.; Engelman, J. A. Dramatic Response to Combination Erlotinib and Crizotinib in a Patient with Advanced, EGFR-mutant Lung Cancer Harboring De Novo MET Amplification. J. Thorac. Oncol. 2016, 11, e83-e85. 174. Felip, E.; De Braud, F. G.; Maur, M.; Loong, H. H.; Shaw, A. T.; Vansteenkiste, J. F.; John, T.; Liu, G.; Lolkema, M. P.; Scott, J. W.; Yu, R. Ceritinib plus Nivolumab (NIVO) in Patients 54


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(pts) with Anaplastic Lymphoma Kinase Positive (ALK+) Advanced Non-small Cell Lung Cancer (NSCLC). J. Clin. Oncol. 2017, 35(Suppl 15); Abstract 2502. 175. Shaw, A. T.; Lee, S.-H.; Ramalingam, S. S.; Bauer, T. M.; Boyer, M. J.; Carcereny Costa, E.; Felip, E.; Han, J. Y.; Hida, T.; Hughes, B. G. M.; Kim, S.W. Avelumab (anti–PD-L1) in Combination with Crizotinib or Lorlatinib in Patients with Previously Treated Advanced NSCLC: Phase 1b Results from JAVELIN Lung 101. J. Clin. Oncol. 2018, 36; Abstract 9008. 176. Hellmann, M. D.; Rizvi, N. A.; Goldman, J. W.; Gettinger, S. N.; Borghaei, H.; Brahmer, J. R.; Ready, N. E.; Gerber, D. E.; Chow, L. Q.; Juergens, R. A.; Shepherd, F. A. Nivolumab plus Ipilimumab as First-line Treatment for Advanced Non-small-cell Lung Cancer (CheckMate 012): Results of an Open-label, Phase 1, Multicohort Study. Lancet Oncol. 2017, 18, 31-41. 177. Aggarwal, C.; Borghaei, H. Treatment Paradigms for Advanced Non‐small Cell Lung Cancer at Academic Medical Centers: Involvement in Clinical Trial Endpoint Design. Oncologist 2017, 22, 700-708. 178. Hainsworth, J. D.; Waterhouse, D. M.; Shih, K. C.; Boccia, R. V.; Priego, V. M.; McCleod, M. J.; Kudrik, F. J.; Mitchell, R. B.; Burris, H. A.; Greco, F. A.; Spigel, D. R. Phase II Trial of Preoperative Pemetrexed plus Carboplatin in Patients with Stage IB-III Nonsquamous Non-small Cell Lung Cancer (NSCLC). Lung Cancer 2018, 118, 6-12.

55



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Table 1. Acquired secondary mutations conferring resistance to ALK inhibitors.

Drugs

Tumor Type

Activity against L1196M

Activity against C1156Y

Activity against G1202R

Other Crizotinib resistant mutations (Sensitive)

No

No

No

L1198F

NSCLC Crizotinib

IMT

Resistant mutations

1151insT, L1152R,

Neuroblastoma

F1174V, S1206Y, G1269A, D1203N EML4-ALK/1151Tin

Ceritinib

Alectinib

NSCLC Thyroid

Neuroblastoma NSCLC

G1269A, Yes

No

No

I1171T/N/S, S1206Y, V1180L

G1269A, S1206Y, Yes

Yes

No

L1152R, F1174L, 1151T-ins

s EML4-ALK/L1152R EML4-ALK/C1156Y EML4-ALK/F1174C EML4-ALK/I1171Ti ns EML4-ALK/V1180L

G1269A, A1206Y, Brigatinib

NSCLC

Yes

Yes

No

1151T-ins, F1174C, I1171T, D1203N,

NA

E1210K, F1245C Entrectinb X-396 ASP3026

Colorectal Lung Neuroblastoma ALCL NSCLC

Yes Yes Yes

Yes Yes NA

NA

NA

NA

NA

F1174L, R1275Q

NA

NA

F1174L

NA

G1269A, F1174L, Lorlatinib

ALCL Neuroblastoma

S1206Y, I1171T, Yes

Yes

Yes

E1210K, L1152R, 1151Tins, V1180L, D1203N

56


L1198F

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Legend of Figures Figure 1. Schematic summary of different categories of ALK alterations (gene amplification, mutations and rearrangement) and corresponding cancers. Figure 2. (A) Overview of the native human anaplastic lymphoma kinase (ALK). The extracellular segment of ALK (residues 19-1038) contains an LDLa domain (453-471) sandwiched between the two MAM domains (264-427 and 480-626) along with a glycine-rich component (UniprotKB ID: Q9UM73). Architectures of (B) the anaplastic large cell lymphoma kinase (ALCL) NPM-ALK fusion protein, and (C) non-small cell lung cancer (NSCLC) EML4-ALK fusion protein (variant 1). Figure 3. The mechanisms of acquired resistance to ALK inhibitor therapy in ALK-positive cancers, including acquired secondary mutations of ALK and/or fused ALK, gene amplification, bypass signaling pathway activation and other mechanism. Figure 4. Representative ALK inhibitors in clinical and preclinical stages. Figure 5. (A) Crystal structure of the ALK domain (PDB ID: 4Z55), and (B) 2D-diagram of the inferred interactions between the human ALK catalytic core residue, ATP, and a protein substrate. Figure 6. ALK signaling pathways and the important downstream targets of ALK. ALK, as a membrane receptor, receives and transfers extracellular signals by activating

multiple

intracellular

signaling

pathways,

such

as

the

SRC/RAS/MEK/ERK1/2, JAK/STAT, PI3K/AKT/mTOR, and PLC-γ/DAG/PKC pathways. The PLC-γ/DAG/PKC and SRC/RAS/ERK1/2 pathways. Figure 7. The discovery and design procedure of the dual c-Met/ALK inhibitor, Crizotinib, by combing SBDD strategy and medicinal chemistry. The procedure started from a moderate c-Met inhibitor (compound 13, SU-11274) and optimized it to a potent inhibitor for c-Met (PHA-665752), and then generated compounds 15, 16, 17 57



and Crizotinib after a series of optimizations. Figure 8. The binding modes of the selected representative ALK inhibitors: (A) Crizotinib (PDB ID: 2XP2), (B) Ceritinib (PDB ID: 4MKE), (C) Alectinib (PDB ID: 3AOX), (D) Brigatinib (PDB ID: 5J7H), (E) Entrectinib (PDB ID: 5FTO), and (F) Lorlatinib (PDB ID: 4CLI). The key residues involved in the interactions between ALK kinase and ALK inhibitors, such as Leu1122, Glu1167, Ile1171, Phe1174, Glu1197, Met1199, and Phe1271. Figure 9. (A) Reported Crizotinib resistant mutations in the ALK kinase domain (PDB: 2XP2), and (B) the frequency (reported cases) of each mutation detected directly from the Crizotinib-resistant patients. Figure 10. The evolution of Ceritinib (3) from the initial hit TAE684 (2) due to its toxicity as a result of oxidative metabolism. Figure 11. The design strategy used from the initial hit 18 to the FDA-approved Alectinib (4) through a series of optimizations (compounds 19, 20 and 21). Figure 12. The design and optimization strategy used to develop Brigatinib (5) and ALK-IN-1 (27), and key Brigatinib analogues during the discovery process (compounds 25, 26, 28, 29, 30, 31, and 32). Figure 13. Macrocyclic ALK inhibitor TPX-0005 (12) and the design procedure of Loratinib (11) by the cyclization (compounds 33, 34, and 35) and optimization (compound 36) of Crizotinib. Figure 14. The development strategy for CEP-28122 (39) and CEP-37440. The combination of compound 37 and the 2,3,4,5-tetrahydro-1H-benzo[d]azepine fragment of the diaminopyrimidine analogue 38 yielded CEP-28122. The toxicity of CEP-28122 advanced the generation of CEP-37440 by optimizations (compounds 40 and 41). Figure 15 Design of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) by repurposing a typical dopamine D1/D5 (compounds 43 and 44), which led to the generation of compounds 45 and 46. Figure 16. The development strategy for the new DAAPalogues with a flexible amino acid side chain by ring opening (compound 48) and SBDD (compound 49). 58


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Figure 17. Representative ALK inhibitors (compounds 50, 51, 52, 53, and 54) with the DAAPs scaffold by adopting a bioisosteric strategy. Figure 18. The optimization of the substituent of ring A in hit 55 led to the discovery of Entrectinib (9). Figure 19. X-396 was generated by optimizing the side chain of Crizotinib. Figure 20. Representative ALK inhibitors with the acyliminobenzimidazole scaffold. The optimizations of the HTS hit 56 led to the generation of the 3,5-difluoro analogues 59 (Belizatinib) and 61 with improved potency and selectivity toward ALK. Figure 21. The optimization process (compound 62) of Alectinib leading to the development of JH-VII-157-02 (63). Figure

22.

Compound

65

was

generated

by

the

optimization

of

the

2,6-dichloro-3-fluorophenyl head and pyrazolopiperidine tail groups in Crizotinib (1). Figure 23. The discovery of a 1H-pyrazol-5-yl-1H-pyrrolo[2,3-b]pyridine derivative 68 as a highly selective, potent, and brain-penetrant ALK inhibitor. The discovery process started from a HTS hit 66, followed by scaffold hopping and SBDD-driven lead optimization. Figure 24. (A) Representative Type-I½ ALK inhibitors with the piperidine carboxamide scaffold (compounds 69, 70, 71, and 72), (B) co-crystal structure of compound 69 in complex with the ALK kinase, and (C) the pharmacophore modeling of compound 69 in the ALK kinase. Figure

25.

(A)

Representative

Type-I½

ALK

inhibitors

with

the

1-purine-3-piperidinecarboxamide scaffold (compounds 73, 74, 75, 76, and 77), (B) co-crystal structure of compound 77 in complex with the ALK kinase, and (C) the pharmacophore modeling of compound 77 in the ALK kinase. Figure 26. (A) The transition from Type-I (compound 78) to Type-II (compound 79) ALK inhibitors, (B) co-crystal structure of compound 79 in complex with the ALK kinase; and (C) the pharmacophore modeling of compound 79 in the ALK kinase. Figure 27. Structures of the reported ALK PROTACs, and the degradation process of ALK by PROTACs. 59



Figure 1

60


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Figure 2

61



Figure 3

62


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Figure 4

63



Figure 5

64


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Figure 6

65



Figure 7

66


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Figure 8

67



Figure 9

68


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Figure 10

69



Figure 11

70


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Figure 12

71



Figure 13

72


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Figure 14

73



Figure 15

Figure 16

74


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Figure 17

Figure 18

75



Figure 19

Figure 20

76


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Figure 21

Figure 22

77



Figure 23

78


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Figure 24

79



Figure 25

80


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Figure 26

81



Figure 27

82


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Table of Contents Graphic

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Drug discovery targeting anaplastic lymphoma ... - ACS Publications

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