Medicinal Chemistry

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Aeterna Zentaris, Inc. developed and characterized an oral, highly selective and active ERKi AEZS-131 (struc- ture undisclosed) [135]. About 15–20% of patients ...

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Medicinal Chemistry

The RAF-MEK-ERK pathway: targeting ERK to overcome obstacles of effective cancer therapy

Currently, dozens of BRAF inhibitors and MEK inhibitors targeting RAF-MEK-ERK pathway have been introduced into clinical trials for cancer therapy. However, after 6–8 months of initial response, acquired drug resistance among the majority of those treated patients sharply diminished their clinical efficacy. Important mechanisms responsible for acquired resistance of BRAF inhibitors and MEK inhibitors have been elucidated. Continually, ERK1/2 locates in the critical position and features unique characteristics, such as activating hundreds of substrates, participating in feedback regulation, being catalyzed by MEK specifically and no acquired resistant mutation. Taking in account the inspiring outcomes of ERK inhibitors in preclinical research, ERK1/2 might be the optimal target to overcome acquired drug resistance in RAF-MEK-ERK pathway.

Why targeting RAF-MEK-ERK pathway? Cancer has become a major human health threat throughout the world, with 25% of deaths caused by carcinoma [1] . Among all of the hallmarks of cancer during the multistep development, sustaining proliferative signaling has been extensively implicated to promote carcinogenesis [2,3] . Being widely researched in the near decades, mitogen activated protein kinase (MAPK) pathways transduce a large variety of signals, resulting in a wide range of cellular responses, including proliferation, differentiation, survival, migration, neuronal function and immune responses [4–6] . MAPK signaling adopt a conservative three-tiered kinase cascade, usually initiated by ligands that bind a variety of membrane receptors, such as receptor tyrosine kinases (RTKs), GPCRs, toll-like receptors and cytokine receptors [7,8] . After the initial activation, membrane receptor engages adaptor protein and exchange factor to induce activation of the small G protein, RAS [9] . GTPbound active RAS then recruits and activates the first kinase RAF in MAPK cascade. Subsequently, RAF phosphorylates MEK, and

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the cascade is fully activated [10,11] . The dualphosphorylation and activation of ERK1/2 by MEK on a threonine and a tyrosine residue locate in its activation loop. Recent research conferred that nearly 200 substrates could be phosphorylated by activated ERK1/2, including nuclear substrates (e.g., Elk1, c-Fos) and cytoplasmic substrates (e.g., RSK, paxillin, scaffold proteins), which in turn resulted in gene expression changes and alterations in cell proliferation, differentiation and migration [12] . JNK, p38 and ERK5 cascades, however, which should be noted here, can function in parallel to RAF-MEK-ERK – all of which are members of MAPK signaling system [13–16] . Considerable evidence has identified mutations and aberrations in elements of RAFMEK-ERK cascade as the prominent role responsible for the development and progression of various human cancers [17–19] . For example, RAS mutations (including H-, K- and NRAS), observed in about 30% of tumors, can lead to remarkable phosphorylation and activation of RAF, and then activate the full pathway [20–22] . Continually, activated mutations of RAF, with BRAF studied exten-

Future Med. Chem. (2015) 7(3), 269–289

Zutao Yu1, Shiqi Ye2, Gaoyun Hu1, Meng Lv1, Zhijun Tu1, Kun Zhou1 & Qianbin Li*,1 1 Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South University, 410013, Changsha, Hunan, China 2 School of Medicine, Shenzhen University, 518060, Shenzhen, Guangdong, China *Author for correspondence: Tel.: +86 731 8265 0370 Fax.: +86 731 8265 0370 [email protected]

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Key terms Receptor tyrosine kinases: High-affinity cell surface receptors for many growth factors, cytokines and hormones. They have a significant function in MAPK pathway. Scaffold proteins: Regulate signal transduction and help localize pathway components to specific areas of the cell, such as kinase suppressor of Ras1 (KSR) and IQ motifcontaining GTPase activating protein 1 (IQGAP1).

sively, exist in approximately 70% of melanoma, nearly 100% of hairy cell leukemia and 41% of hepatocellular carcinoma [23] . Furthermore, in many other malignant tumors, such as thyroid cancer, colorectal cancer and mammary cancer, RAF-MEK-ERK pathway is also overactivated to various degrees [24–26] . Extensive statistics demonstrates that overactivated Raf-MEK-ERK pathway is responsible for the oncogenesis of notorious human cancers, making it an appealing target for the research of anticancer therapeutics [19,27–28] . A number of agents targeting BRAF and MEK have entered the clinic and have achieved great successes [19,29–30] . Both BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) have shown significant biological activity in MAPK-dependent cancers harboring BRAF or RAS aberrations. Unfortunately, they failed to sustain results due to the emergence of acquired resistance  [31–33] . As such, there is a pressing need to develop potential therapeutic strategies that could prevent the onset of resistance or overcome diverse resistance mechanisms once they have arisen. Extracellular signal regulated kinase 1/2 (ERK1/2) is a central point in the signaling network and is firmly established as an attractive target for pharmacological intervention in cancer therapy. Moreover, acquired resistance of BRAFi and MEKi are associated with mutations, feedback regulation and other pathway alterations, all of which result in ERK1/2 reactivation  [34] . Therefore, inhibition of RAF-MEKERK signaling through ERK1/2 could be achieved directly  [27,35–37] . In this review, we first outline the progression BRAFi and MEKi in clinics, including clinical benefits and acquired resistance. Resistance mechanisms of upstream inhibitors have also been precisely categorized. Confronted with these urgent situations, we then shift to the main focus of this review toward targeting ERK1/2 directly, in which ERK1/2 regulation and isoforms will be introduced. The critical content in this section is why we should be targeting ERK1/2 directly, and the main advantages of doing this, which we have categorized into three points. Lead generation and optimization of the major chemical series of ERK inhibitors (ERKi) will also be listed, with an emphasis on the most recent progression.

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Clinical experiences with upstream regulator inhibitors Being hyperactivated in various tumor cells, both RAF and MEK as the upstream essential elements of RAF-MEK-ERK pathway are actively pursued soon after being discovered. A number of de novo, potent and specific chemical entries are investigated to target either wild-type or mutant type of RAF/MEK, and several of them are undergoing Phase II and/or III studies [38] . After initial dramatic responses, however, all patients treated with BRAFi or MEKi eventually developed significant resistance, which could not meet the primary clinical efficacy end point [39] . Actually, the first-generation inhibitors in RAF-MEK-ERK signaling targeted RAS, in particular KRAS [40,41] . Driven by the poor clinical efficacy as this pathway could self-sufficiently overactivate in the absence of any extracellular stimuli, the decision was quickly made to terminate further research of KRAS inhibitors, which is beyond the scope of this section [42] . In this part, chemical agents of BRAFi and MEKi progressing toward clinical research will be discussed, with an emphasis on their clinical responses and emerging resistance. BRAF inhibitors

Three RAF isoforms (ARAF, BRAF and CRAF) discovered in 1983 function as homo- or heterodimers and relate to retroviral oncogenes [43] . While ARAF and CRAF mutations are rarely observed in carcinoma, BRAF mutations are ubiquitous in a variety of notorious tumors [23,44] . A substitution for valine at residue 600, namely BRAF V600, accounts for 90% of BRAF mutations observed in various human cancers, such as malignant melanoma, colorectal cancer, ovarian tumor and papillary thyroid carcinomas [17] . BRAF V600E can gain 500-fold increased activation and stimulate the persistent activation of MEK in the absence of extracellular stimuli, which allow the cell to become self-sufficient within this pathway. Great breakthroughs in studying BRAFi have changed traditional treatment upon various oncological diseases. Since the first-generation multitarget RAF inhibitor sorafenib initiated preclinical research, a certain number of agents have been approved for clinical study (Figure 1) . The first studied RAF inhibitor, sorafenib, in addition to inhibiting both mutant BRAF V600E and CRAF, also displays potent inhibition of FLT3, VEGFR, CKIT and PDGFR, unselectively. In further clinical research of the multitarget features of sorafenib targeting overactivated RAF-MEK-ERK pathway, it showed disappointing clinical efficacy in survival in both monotherapy and combined therapy. At present,

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The RAF-MEK-ERK pathway: targeting ERK to overcome obstacles of effective cancer therapy 

it is used for the treatment of renal and hepatocellular tumor mainly because of antiangiogenesis effect rather than targeting RAF [45,46] . Lessons from the unselectivity of sorafenib, the second generation of BRAFi focused on selectivity and activity. The first success in finding BRAFi is vemurafenib (PLX4032, trade name Zelboraf®), which binds the ATP-binding domain of mutant BRAF monomer to prevent phosphorylation of MEK and subsequent ERK1/2. The Phase I study of vemurafenib demonstrated near-complete inhibition of ERK1/2 signaling and a remarkable 81% response rate in an expansion cohort of patients with tumors harboring BRAF mutation [33,47] . Inspired by the great news from the Phase I study, Phase II/III studies were quickly initiated and approved by US FDA in August 2011 [48] . Actually, the clinical results are not so impressive and there are some points that should be noted here. First, data collected from Phase I study showed that none of 16 patients with BRAF wild-type tumors responded, suggesting that vemurafenib markedly suppressed BRAF mutation but enhanced ERK1/2 signaling in wild-type BRAF cells. Second, Phase II and III studies confirmed that the response rate existed in only 50% of patients and all of the patients relapsed after 6–8 months. After rounds of investigation, resistance to BRAFi therapy was found to be common and appeared to be rapidly acquired in the majority of patients [49] . Furthermore, potent anticancer effects are balanced against side-effects including skin rash and the development of squamous cell carcinoma in O O HN S

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nearly 30% of participants. The appearances of secondary mutation reported in separate researches confer acquired resistance, such as NRASQ61, MEK1C121S, MEK1Q56P and MEK1E203K [50,51] . All of those mutations could constitutively activate RAF-MEK-ERK pathway and elevate phospho-ERK (pERK) level. A secondary highly potent and specific BRAFi, dabrafenib (GSK436, trade name Tafinlar®) selectively inhibits BRAF V600E mutation and has recently been completed Phase III clinical trials. In the Phase I/II studies, selected participants were given at 150 mg twice daily. The results showed that squamous cell carcinoma was observed in approximately 11% of patients [52] . Other clinical criterions for detecting drug therapy were virtually identical with vemurafenib, such as response rate (50%) and median progression-free survival (PFS) (6.7 months) [53] . In a randomized, multicenter, open-label Phase III study among patients with metastatic melanoma harboring BRAF V600E, 187 patients were treated with dabrafenib. And for comparison, 63 patients received with dacarbazine. This study indicated significant benefits of the dabrafenib treatment. Compared with dacarbazine, PFS and response rate of which were 2.7 months and 6%, dabrafenib-treated group reached 5.1 month and achieved 50% response rate. In the dabrafenib group, however, treatment-related side effect occurred in 53% of patients and 12 patients (about 6%) developed more severe skin carcinoma [54] . Several other BRAFi have also been reported to generate significant outcomes in preclinical and

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Figure 1. Clinical status of representative BRAF inhibitors.

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Key terms Dacarbazine: Member of alkylation agents for chemotherapy, which destroy cancer cells by adding an alkyl group to its DNA. ATP noncompetitive inhibitor: Also called type II and type III inhibitors as ATP-competitive inhibitor called type I kinase inhibitor. It binds DFG out site but not the ATP site and features high specificity and activity.

clinical studies, and further clinical therapy for BRAFi resistances are still in progress, such as RAF265 [55] , GDC-0879 [56] , LGX818 [57] and PLX3603 [23] . Whether such more potent and selective BRAFi can generate better therapeutic potential will be a question under further clinical research. MEK inhibitors

MEK1 and MEK2, with molecular weights ranging between 43 and 50 kDa, show high homology. Several upstream regulators, including three isoforms of RAF, Mos and MEKK, can markedly activate serine/threonine dual-specificity kinases MEK, which subsequently phosphorylates ERK1/2 [58] . Of interest, MEK was thought to be the only activator of ERK1/2 before the discovery of MEK mutations [19,59] . Based on whether MEKi directly competes for the ATPbinding site, MEKi can be subdivided into two distinct categories, namely, ATP competitive and ATP noncompetitive inhibitors, the latter of which has been studied exhaustively (Figure 2) . ATP noncompetitive inhibitors bind to the unique allosteric site adjacent to, but not overlapping with, the ATP site, in agreement with its high specificity and activity [60] . Compared with BRAFi, MEKi can potently suppress cell proliferation in normal and tumor cells harboring both mutant and wild-type BRAF [25] . As such, directly inhibiting MEK offers a promising strategy in BRAF mutant melanoma to delay or overcome acquired resistant to BRAFi [61] . Through compound library screening, PD98059 and U0126 were identified first as selective MEKi to be developed [62] . In preclinical study, both of them feature potency (IC50 value 10 μM and 5 nM, respectively) and high specificity, with no or little inhibitory effects on other kinases. However, their poor pharmacological profiles led them to be deemed unsuitable for further clinical research [63] . CI-1040, which was the first one to reach the clinic, stopped in Phase II owing to the similar reason discussed above [64] . With several modifications of CI-1040, PD-0325901 features significant elevation in its in vitro (IC50 = 1 nM) and in vivo potency. Unexpectedly, the Phase I evaluation showed severe toxicities. A high incidence (10% of patients) of reversible visual effects and neurotoxicity were observed at doses of 15 mg [65] . In a Phase II

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study in patients with advanced NSCLC, PD-0325901 caused more severe adverse effects and did not meet its primary efficacy end point. The lack of responses, coupled with the safety issues, prompted a decision to terminate clinical development of PD-0325901 [66] . Several groups are investigating other novel MEKi. To date, several chemical structures are undergoing Phase II or III evaluation, alone or in combination with other agents, such as trametinib (GSK1120212), selumetinib (AZD6244), pimasertib (AS703026) [67] , MEK-162 and GDC-0973 [68] . Trametinib (Trade name Mekinist®) is a second generation, ATP noncompetitive inhibitor and causes a dose-dependent inhibition of pERK, with IC50 value of 0.7 and 0.9 nM against purified MEK1 and MEK2 respectively. Compared with previously published MEKi, trametinib has a unique pharmacological characteristic, which aids in promoting and initiating the first-in-human trial [69,70] . In a Phase I study, patients with BRAF mutant melanoma had a response rate of 33%, despite low-dose limiting toxicity occurred. These encouraging results led to several Phase II/III clinical trials of trametinib alone or in combination with other agents [71] . In an open-label, two-stage Phase II study, significant clinical activity has been observed in BRAFi-naïve patients who had been previously treated with chemotherapy and/ or immunotherapy (28% patients with stable disease; the median PFS was 1.8 months). However, minimal clinical effects were observed as sequential therapy in patients previously treated with a BRAFi (2% complete response, 23% partial responses and 51% patients with stable disease; the median PFS was 4.0 months). Those results implied that BRAFi resistance likely conferred resistance to MEKi monotherapy [29] . In the randomized Phase III trial, 322 patients with advanced melanoma harboring BRAF V600E or BRAF V600K mutations, were deliberately assigned in two groups, receiving trametinib orally or intravenous chemotherapy (either dacarbazine or paclitaxel). Clinical outcomes showed that trametinib delayed the resistance progression, with a PFS of 4.8 and 1.5 months for patients who received trametinib and chemotherapy respectively, but clinical significance remain minimal [72] . MEK2Q60P mutation along with BRAF amplification in a xenograft tumor derived from a melanoma patient confer resistance to the concurrent therapy of dabrafenib and trametinib [31] . Following closely at the heels of the clinical development of trametinib was that of selumetinib (AZD6244), an orally available, highly selective and ATP noncompetitive inhibitor of MEK. Selumetinib has an IC50 of 14 nM in vitro and actively against tumor cell lines grown in culture or xenografts [73] .

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The RAF-MEK-ERK pathway: targeting ERK to overcome obstacles of effective cancer therapy 

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Figure 2. Clinical status of representative MEK inhibitors.

In preclinical cell viability inhibition screening in a panel of tumor cell lines, the data confirmed the tendency that cell lines containing BRAF or RAS mutations were more likely to be sensitive to selumetinib than BRAF and RAS wild-type cell lines. Furthermore, most BRAF mutant cell lines seem to be largely dependent on MEK activity and sensitive to MEK inhibition, whereas cell lines harboring KRAS mutation do not seem to be as predictive of sensitivity to MEK inhibition, at least in vitro [74,75] . By using both immunohistochemistry and western blot, pERK has been validated as a distinct biomarker of selumetinib inhibitory activity in human tumor xenografts growing in nude mice [74] . Using microarray analysis in colorectal cancer cells, amplification of the driving oncogenes (BRAF600E or KRA S13D) can drive acquired resistance to MEKi by increasing signaling through the ERK1/2 pathway, suggesting that MEKi acquired resistance dependent on ERK1/2 pathway [76,77] . In a Phase I trial of advanced cancers, besides the grade 1/2 side effects at the recommended dose, the best response was only stable disease and it achieved lower than 33% response rates [78] . The outcomes obtained from multiple Phase II clinical trial, including colorectal cancer [79] , pancreatic cancer [80] , non-small-cell lung cancer [81] , papillary thyroid carcinoma, advanced melanoma [82] and advanced

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hepatocellular carcinoma [83] , were still disappointing, with none of those trials acquired satisfied results except for a small percentage of patients harboring RAS/RAF mutations. Acquired resistance mechanism of BRAFi & MEKi Resistance will invariably emerge as a critical issue in clinical development of BRAFi and MEKi in RAFMEK-ERK cascade, including primary and acquired resistance. First, patients could not benefit from BRAFi and MEKi in cases of primary resistance, raising the issue of patient selection or personalized therapeutics [84] . Thus, cancer samples from pretreated patients should be investigated to determine whether they are primary resistant to BRAFi and MEKi. A series of identifiable hallmarks are responsible for primary resistance at the commencement of therapy, such as cyclin D, phosphatase tensin homolog and HGF. Second, in acquired resistance, the initial treatment is effective but patients subsequently followed by tumor progression. The appreciable but limited survival advantages imply acquired resistance. Once those treated patients obtained acquired resistance, new therapeutics should be applied [85] . The primary resistance is beyond the scope of this review [86] .

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Review  Yu, Ye, Lv, Tu, Zhou & Li Based on whether other signal pathways participate in acquired resistance, acquired resistance has three simplified conditions. In the first condition, acquired resistance is caused by hyperactivation of RAF-MEKERK cascade alone. For the second situation, both RAFMEK-ERK pathway and other parallel signal pathways confer robust acquired resistance to BRAFi and MEKi. At the third place, acquired resistance is only led by alternative signal overexpression. We subdivide the first and second situation as ERK-dependent and the third one as ERK-independent resistance (Figure 3) . In this review, we will focus on the mechanism of ERK-dependent acquired resistance, which means we mainly pay attention to the mechanism of hyperactivation of RAF-MEK-ERK cascade and how to knockdown it efficiently. Elucidating the resistance mechanisms will clearly lead to a profound understanding of the mechanisms of BRAFi and MEKi function and ultimately promote the optimization of metastatic carcinoma therapy. Those issues about cross talk with other signaling and ERK-independent acquired resistance will be accomplished gradually by series of studies, for example, how to tackle cross talk

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between RAF-MEK-ERK pathway with other signaling, such as PI3K-AKT-mTOR pathway and what kinds of combination therapy should be applied in clinics [77,87] . Mutation of key kinases in RAF-MEK-ERK pathway RAS mutation

Compared with BRAF V600E mutation, RAS mutation have more options for survival signaling since they could activate various effectors in RAS-dependent survival signaling notably, such as PI3K-AKT-mTOR, which was usually necessary for RAS-induced tumorigenesis [88,89] . Overactivated PI3K pathway will be not included in this review [90] . Substantial evidences demonstrate that RAS and BRAF mutation are usually mutually exclusive, except in rare cases (