Cancer Metastasis Rev DOI 10.1007/s10555-015-9567-z

Allosteric therapies for lung cancer Ye Ling 1 & Meiling Jing 1 & Xiang-dong Wang 1

# Springer Science+Business Media New York 2015

Abstract Allostery is a regulation at a distance by conveying information from one site to another and an intrinsic property of dynamic proteins. Allostery plays an essential role in receptor trafficking, signal transmission, controlled catalysis, gene turn on/off, or cell apoptosis. Allosteric mutations are considered as one of causes responsible for cancer development, leading to Ballosteric diseases^ by stabilizing an active or inactive conformation or changing the dynamic distribution of preexisting propagation pathways. The present article mainly focuses on the potential of allosteric therapies for lung cancer. Allosteric drugs may have several advantages over traditional drugs. The epidermal growth factor receptor mutations and signaling pathways downstream (such as PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways) were suggested to play a key role in lung cancer and considered as targets of allosteric therapy. Some allosteric inhibitors for lung cancer-specific targets and a series of preclinical trials of allosteric inhibitors for lung cancer have been developed and reported. We expect that allosteric therapies will gain more attentions to develop combinatorial strategies for lung cancer and metastasis.

Ye Ling and Meiling Jing authors contribute to the article equally as the first authorship Ye Ling and Meiling Jing contributed equally to this work. * Xiang-dong Wang [email protected] 1

Zhongshan Hospital, Shanghai Institute of Clinical Bioinformatics, Fudan University Center for Clinical Bioinformatics, Biomedical Research Center of Fudan University Zhongshan Hospital, Shanghai, China

Keywords Allostery . Allosteric drug . Lung cancer . Epidermal growth factor receptor . Signal transduction pathway

1 Introduction Allostery is a process of distant regulations to convey information from one site on the protein, RNA, DNA, or lipid to another. A perturbation by an effector at one site of the molecule leads to a functional change at another through alteration of shape and/or dynamics [1]. Allostery was initially described in the hemoglobin as cooperativity of oxygen binding with one subunit to enhance the affinity of a second oxygen binding to a neighboring subunit and extended to multiareas as an intrinsic property of dynamic proteins. BAllosteric disease^ is resulted from a combination of events, of which one is the outcome of an allosteric effect [2]. Mutations can lead to allosteric diseases by changing the dynamic distribution of preexisting propagation pathways or influencing the binding of ligands, signal transmission, and gene expression. Mutations act as one of the cancer Bpathogens,^ by which the allosteric modulation can play an important role in cancer pathogenesis. Malignant tumors can be considered as an allosteric disease on basis of such understanding. The present review initially explains the concept of allostery and clarifies allosteric modulations and drugs, with a special focus on lung cancer which is the leading cause of cancer-associated death in the world. We furthermore elaborate mutations of epidermal growth factor receptor (EGFR) that activate the kinase domain by allosteric modulation and introduce allosteric therapy for lung cancer.

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2 Understanding of allostery

3 Allosteric modulation of receptors

Allostery was previously proposed to explain how the activity of a protein is regulated, by which the effector binding to one site induces a conformational switch of another [3, 4]. Results from nuclear magnetic resonance spectroscopy, computer simulations, or multiple crystal states indicate that proteins can adopt different conformations in a dynamic equilibrium [5, 6], and conformational states preexist in an ensemble [7]. An energy landscape can be described as a mapping of possible conformations of the protein or spatial positions of interacting molecules in a system and as a function of corresponding energy levels on two- or three-dimensional systems. Such energy can act as the physical basis for portraying the ensemble around the native state of the protein [2]. The conformations are separated by barriers, whose heights can be used to define the timescale for conformational exchanges. The higher barriers are, slower conformational change will be. Allosteric perturbation can result from environmental factors which can be influenced by drugs, post-translational modifications, mutations, changes in pH, or ionic strength (Fig. 1a) [8–11]. The perturbation may change in speed, frequency, and depth [12]. Structural disturbance may form new atomic interactions to create a local strain energy. The perturbation propagates and reaches another site through multiple and preexisting pathways and changes the conformation and/or dynamics [13]. Allostery is often associated with a conformational change (Fig. 1b), while it is sometimes subtle and only results in a change of protein dynamics (Fig. 1c) [14]. The perturbation at any allosteric site in the protein leads to a shift in the distribution of the preexisting conformational states across the entire population named Bpopulation shift^ [15, 16]. Allosteric perturbation changes relative distributions of states within the ensemble, rather than creates new conformational states. Mutations lead to allosteric diseases, such as cancers, by influencing the binding of ligands, signal transmission, and gene expression (Fig. 1d). Allostery occurs through a thermodynamic link between an allosteric site and another distant site in communication and can arise from changes in enthalpy or entropy alone or combination. Allostery may hardly induce observable conformational changes because enthalpy changes do not reverse the free-energy change due to the change in entropy mainly responsible for binding [14]. Catabolite activator protein (CAP) is a homodimeric transcription regulator with each subunit consisting of a cyclic AMP (cAMP)-binding domain and a DNA-binding domain. cAMP binds to CAP with strong negative cooperativity, of which the first binding to one subunit has no effect on the conformation of the other subunit, while drastically alters protein motions of the second subunit [17]. This provides the strong support for the existence of purely dynamics-driven allostery.

A series of theoretical models are used to clarify potential mechanisms of receptor interaction with corresponding ligands. G-protein-coupled receptors (GPCRs) are suggested as one of the most important drug targets, of which about 40–50 % of marketed drugs may act through different GPCRs [18]. The binding of an allosteric ligand to its site can change the conformation of a distant site, such as an orthosteric or a substrate site, and can be applied to binding. The cooperativity occurs between two ligands in a Bternary complex model^ and interacts with different sites on the same receptor [19]. In the model, Bcooperativity factor^ α is used to measure the magnitude and direction of the allosteric interaction between two conformationally linked sites. Values of α> 1, α0, or α=1 denote the allosteric ligand to promote the binding of the orthosteric ligand (positive modulation), inhibit the binding of the orthosteric ligand (negative modulation), or hardly change in the binding affinity at equilibrium (neutral cooperativity), respectively. The allosteric interaction between the allosteric and orthosteric ligands is reciprocal, since two sites are conformationally linked. The extent and direction of an allosteric interaction can vary with the nature of the orthosteric ligand, which is referred to as Bprobe dependence^ [20]. For example, LY2033298, as an allosteric modulator, causes robust potentiation of agonist actions (e.g., acetylcholine and oxotremorine at M4 muscarinic acetylcholine receptor), while it is only weakly positive when combined with the agonist (xanomeline) and neutral when tested against the antagonist ([3H] quinuclidinyl benzylate), and weakly negative when combined with the antagonist ([ 3 H] Nmethylscopolamine) [21–23]. However, the model concentrates on effects of the allosteric ligand on orthosteric ligand binding and ignores potential effects of those compounds on the ability of orthosteric ligands to cause the receptor activation. For instance, the majority of allosteric modulators of metabotropic glutamate receptors influence orthosteric ligand efficacy in the absence of effects on affinity [24]. Furthermore, an Ballosteric two-state model^ includes effects of the allosteric ligand on receptor activation [25]. The model was subsequently extended into a Bquaternary complex mode^ with the effect of G protein [26]. Those theories are used to elaborate on allosteric modulation of receptors and mechanisms of ligand-receptor interactions, as shown in Fig. 2a, b.

4 The role of allostery Allostery results in the communication between distinct sites in the protein structure, to encode specific effects on cellular pathways [27]. Proteins exist in inactive or active conformational states presented in the equilibrium [28]. On a singleprotein level, different perturbations at the allosteric site can

Cancer Metastasis Rev Fig. 1 The understanding of allosteric regulation. a Allosteric perturbation can be influenced by drugs, post-translational modifications, mutations, changes in pH, or ionic strength. b Proteins adopt active or inactive conformations in dynamic equilibrium. Binding the allosteric ligand to an allosteric site induces the conformational switch of another distant binding site by allosteric perturbation. c The change of dynamics can increase the affinity for the other ligand. d Mutations lead to allosteric diseases by stabilizing an active or inactive conformation and influencing the binding of ligands, signal transmission, and gene expression

lead to different conformations of orthosteric or substrate sites to regulate the activity of a protein. CAP is allosterically activated with the DNA binding by cAMP, switching the protein from an inactive conformation which weakly and nonspecifically interacts with DNA, to an active conformation that increases the binding affinity and specificity for the DNA substrate [29]. The cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation, while strongly activates the protein for DNA binding. The DNA binding as the wild-type CAP can be driven by a large conformational entropy, even though CAP-S62F-cAMP2 may adopt the inactive conformation [30]. It indicates that changes in protein motions may activate allosteric proteins that may by structurally inactive. Mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase to control cellular metabolism, growth, proliferation, or survival. It

forms two distinct multiprotein complexes, mTORC1 and mTORC2 [31, 32]. Rapamycin and its derivatives temsirolimus and everolimus are allosteric inhibitors of mTORC1. Rapamycin binds to the cytosolic protein FKBP12 with subsequent binding of the complex to the FKrapamycin-binding domain of mTOR and selective disruption of mTORC1 assembly, which decreases phosphorylation of mTORC1 substrates (Fig. 2c). Those allosteric inhibitors can abate cell survival, while the effect can be dampened by additional changes, including Akt activation, through negative feedback loops in other cells. The phenomenon underscores the complexity of allosteric modulation in cells. Multiple proteins, such as transcription factors and RNA or DNA polymerases, bind closely to each other on genomic DNA to carry out cellular functions. Studies demonstrated that DNA might act as an allosteric ligand to determine the affinity

Cancer Metastasis Rev Fig. 2 Biological and functional roles of allostery. a Allostery is essential for receptor trafficking, signal transmission, controlled catalysis, and also for gene turn on/off or cell apoptosis. b Allosteric and orthosteric ligands bind to the corresponding sites on a receptor and then influence the substrate site, to participate in signal transduction (one-way arrow) or modulate the affinity and/or efficacy between (doublesided arrow). c Rapamycin binds to the cytosolic protein FKBP12 with subsequent binding of the complex to the FK-rapamycinbinding domain of mTOR and selective disruption of mTORC1 assembly

of the bound transcription factor, more than simply as a docking site for transcription factors [33–35]. Recently, a single-molecule study showed that the binding affinity of a second protein separated from the first protein was altered, when a DNA molecule was deformed by specific binding of a protein [36]. Experimental observations together with molecular dynamics simulations suggest that the origin of the DNA allostery is related to the observed deformation of the DNA structure.

5 Allosteric drugs Allosteric drugs have advantages over orthosteric drugs [18, 37], mainly due to the high specificity, because drugs tend to be highly conserved in protein families without the binding with active sites. Allosteric drugs may have lower chances of side effects than orthosteric drugs. Positive allosteric modulators can strengthen the function of the protein activated by endogenous agonist and are expected to have no or only low propensity for receptor desensitization, since persistent effects

of the agonist leads to receptor downregulation. Allosteric modulators can also activate a target protein by directly binding or indirect allosteric effects. For instance, drug binding to one receptor molecule can allosterically modulate the response of another to a ligand, when two or more receptors are integrated into one signaling unit. It creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. Interactions between the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor exclusively increased when binding of GLP-1 that can be reversed with GIP addition [38]. The heteromer displays specific pharmacological characteristics with respect to GLP-1-induced β-arrestin recruitment and calcium flux as a form of allosteric regulation between receptors. Benzodiazepines, positive allosteric modulators of gammaaminobutyric acid receptors, were proven as an effective and safe treatment for anxiety and sleep disorders [39]. A series of new allosteric drugs have been approved for use in the clinic. The calcium-sensing receptor, a member of GPCRs, is a key regulator of calcium homeostasis and regulates cellular proliferation as well as synthesis and release of parathyroid

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hormone in the parathyroid gland. The deficiency of calciumsensing receptor was observed in patients with hyperparathyroidism. Cinacalcet, a positive allosteric modulator of the calcium-sensing receptor, has been approved for treatment of hyperparathyroidism [40]. Maraviroc as a negative allosteric drug of the chemokine receptor CCR5 can stabilize CCR5 receptor conformations and block CCR5-dependent entry of HIV-1 into cells for treatment of HIV-1 infections [41]. Rapalogs, as allosteric inhibitors of mTORC1, were approved for advanced renal cell cancer, pancreatic neuroendocrine tumors, and subependymal giant-cell astrocytoma associated with tuberous sclerosis [31, 42]. There is an extreme increase in the discovery and development of allosteric drugs [43].

6 Allosteric therapies for lung cancer Non-small cell lung carcinoma (NSCLC) accounts for about 85 % of death in patients with lung cancer [44], probably due to EGFR mutations [45, 46]. EGFR and its relatives, human EGF receptors 2, 3, and 4 (Her2, Her3, and Her4), play important roles in cancer development and progression [47]. Mutations induce the kinase domains of EGFR to allosterically adopt an active conformation, resulting in increased malignant cell survival, proliferation, invasion, or metastasis. 6.1 Allosteric mechanism for EGFR EGFR consists of extracellular domain, transmembrance segment, juxtamembrance segment, kinase domain, and sites of tyrosine phosphorylation. The binding of growth factors such as EGF to receptors induces the dimerization and activation of corresponding cytosplasmic kinase domains, resulting in the phosphorylation of tyrosine residues in C-terminal tails and recruitment of downstream effectors (Fig. 3) [48]. The interaction between the C-lobe of one kinase subunit (activator) and the N-lobe of a second kinase subunit (receiver) could result in the allosteric activation of the receiver through the formation of an asymmetric dimer of kinase domains [49]. The active conformation is characterized by placement of the αC-helix where the catalytically important salt bridge is maintained, while the kinase domain also adopts an inactive conformation where the αC-helix is displaced and the salt bridge is broken. The conformation is also referred to as the BCDK/Src-like^ inactive conformation and normally found in the absence of ligand binding. The juxtamembrane segment of the EGFR is necessary for ligand-dependent activation and downstream signaling [50]. The crystal structures of EGFR and Her4 kinase domains demonstrated that the conserved C-terminal segment of the juxtamembrane domain might play an important role in the stabilization of the active kinase dimer. The additional interaction between receiver kinases could extend through the C-

terminal juxtamembrane latch and the C-lobe of the activator kinase in the asymmetric dimer [51]. A segment of the EGFR inhibitor, Mig6, contains a sequence motif in the juxtamembrane latch within the EGF receptor and may prevent from the juxtamembrane latch formation by blocking the asymmetric dimer [52]. The N-terminal portion of the juxtamembrane is also a limiting factor in the activation of the kinase and forms α helix, to repeat juxtamembrane latch interactions and stabilize a daisy chain of kinase domains. The model for kinase activation is that the juxtamembrane latch of EGFR forms on the activator kinase and the N-terminal juxtamembrane segment dimerizes by forming an antiparallel coiled coil [48, 50]. Mutations are identified in exons 18–21 of the kinase domain of EGFR [53, 54] and structurally clusters around active sites cleft of kinase domains. The predominant single-point mutation is in exon 21 with the substitute of arginine for a leucine at codon 858 (L858R). The L858R substitution comprises up to about 40 % of activating EGFR mutations [55, 56]. The frequency of mutation within the phosphate-binding loop that replaces glycine 719 with serine (G719S) is less. Comparison of mutant structures with the structure of the wild-type kinase domain in an inactive conformation shows that those mutations cause a constitutive activation of the kinase by destabilizing the inactive conformation [52, 56–58]. For instance, the αC-helix is displaced from the active site, and the N-terminal portion of the activation loop forms a helical turn to lock the αC-helix in the inactive position. Leu858 is within such helical turn and forms key hydrophobic interactions with other residues in the N-lobe. The L858R substitution is readily accommodated in the active conformation, and the L858R mutation locks the kinase domain in a constitutively active conformation [56]. The threonine 790 to methionine (T790M) point mutation is often found in patients at the time of acquired resistance to EGFR tyrosine kinase inhibitor (TKI) therapy [59]. Thr790 is the gatekeeper residue in EGFR, because the key location at the entrance to a hydrophobic pocket in the back of the ATP binding cleft became an important determinant of inhibitor specificity in protein kinases [60]. Structural analysis revealed that the T790M mutation activates the kinase by stabilizing the Bhydrophobic spine,^ which is the characteristic of the active kinase conformation [61]. Moreover, the T790M mutation may allosterically enhance protein mobility in the inactive state and then strengthen structural integrity of the active conformation [58]. 6.2 Allosteric inhibitors for lung cancer PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways represent important signal transduction mechanisms by which receptor tyrosine kinase EGFR facilitates the proliferation and survival of lung cancer (Fig. 4) [62–64]. Theoretically,

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Fig. 3 Mechanism by which the EGFR can activate the catalytic domain. The binding of a ligand to EGFR induces the dimerization and activation of kinase domains, resulting in the phosphorylation of tyrosine residues in the C-terminal tails. The interactions between C-lobe of the activator and N-lobe of the receiver result in the allosteric activation of the receiver

Fig. 4 The role of EGFR mutation in activation of the PI3K/AKT/mTOR and RAS/ RAF /MEK/ERK pathways to facilitate the proliferation and survival of lung cancer. Solid lines represent the activation of downstream, and dotted lines represent the inhibitory action. EGFR and related signal pathways are targets of allosteric therapy for lung cancer. The negative feedback loop in PI3K/AKT/mTOR signal pathway influences the effect of related allosteric inhibitors

through the formation of an asymmetric dimer of the kinase domains. The juxtamembrane latch plays an important role in stabilizing the active kinase dimer. The receiver kinase extends the juxtamembrane latch to interact with the C-lobe of the activator kinase in the asymmetric dimer

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those receptors and signal transduction pathways are the targets of allosteric therapy for lung cancer. Allosteric modulators can alter the activation of those target proteins by shifting the conformations or changing the dynamic distribution of the propagation pathways. For example, the activation of the kinase domains would be suppressed if an allosteric inhibition of EGFR can disrupt interactions that stabilize the active conformation. Currently, some allosteric inhibitors for those targets have been used in clinic or are being tested in clinical trials (Table 1) [65–67]. MK-2206 is a dose-dependent allosteric Akt inhibitor to inhibit Thr308 and Ser473 phosphorylation since the active conformation of Akt can be stabilized at the S473 residue. The interaction between Akt and PDK1 requires phosphorylation at the T308 residue. Recently, MK-2206 was found to inhibit NSCLC cell proliferation with acquired resistance to cetuximab, and the combination of cetuximab and MK-2206 decrease the proliferation more than either alone [68]. The combination of MK-2206 and erlotinib had synergistic inhibitions independent of EGFR mutation status in cells where HGF blocked antiproliferative and cytotoxic effects of erlotinib, while MK-2206 could restore cell cycle arrest [69]. A number of clinical trials about MK-2206 are ongoing for patients with TKI-resistant NSCLC [70]. Rapamycin and its rapalogs are allosteric inhibitors of mTORC1 and have been approved for treatment of several tumors by the US Food and Drug Administration. For instance, temsirolimus was approved for treatment of advanced renal cell cancer and everolimus for neoplastic diseases including advanced renal cell cancer, pancreatic neuroendocrine tumors, or subependymal giant-cell astrocytoma associated with tuberous sclerosis [31, 42]. Clinical trials of allosteric mTORC1 modulators were carried out as second- or thirdline therapies for NSCLC. Soria et al. reported that 85 patients with advanced NSCLC were treated with everolimus in a phase II clinical study [71]. In the particular study, overall response rates to therapies in patients receiving chemotherapy Table 1

alone or combination of chemotherapy with TKI were about 7 or 2 %, respectively. However, a phase II study on everolimus plus gefitinib in patients with stage IIIB/IV NSCLC demonstrated that the response rate failed to meet the expected values 25 % [72]. A phase II study showed that the combination of docetaxel with everolimus increased disease control rate by 61 % and the median overall survival by 9.6 months in patients with advanced-stage NSCLC [73]. However, the combination of everolimus and erlotinib failed to show sufficient efficacy according to predefined study criteria and increased the severity of toxicity in patients with advanced NSCLC during phase II clinical study [74]. Such results of allosteric inhibitors against mTORC1 in NSCLC may be, at least partly, due to the reactivation of the PI3K/Akt/mTOR pathway through a mechanism of feedback loop or continued cellular signaling through the parallel RAS/RAF/MEK/ERK pathway. Selumetinib as an allosteric inhibitor of MEK1/MEK2 can bind to and stabilize MEK in an inactive conformation that enables binding of ATP and substrate, while preventing from molecular interactions required for catalysis and access to the ERK activation loop. To assess the efficacy and safety of selumetinib versus pemetrexed in patients with NSCLC who had failed one or two prior chemotherapeutic regimens, a phase II, open-label, randomized study demonstrated disease progression events were about by 70 or 59 %, respectively, while there was no difference to improve the survival rate [75]. A phase II study showed that selumetinib + docetaxel prolonged the survival time 4 months longer than docetaxel in patients with stage IIIB–IV KRAS-mutant advanced NSCLC [76]. Trametinib, RO4987655, and TAK-733 are also allosteric inhibitors against MEK, and RO5126766 is a first-in-class dual Raf/MEK allosteric inhibitor against B-Raf, Raf-1, and MEK. RO5126766 binds MEK and suppresses the phosphorylation of MEK by Raf via the formation of a stable Raf:MEK complex. Phase I/II studies of those allosteric modulators in patients with advanced solid tumors were performed [77–80], while the efficacy and potential mechanisms of those inhibitors against lung cancer need to be furthermore explored.

Allosteric inhibitors in signal transduction pathyways

Allosteric inhibitor

Company

Target

7 Conclusions

MK-2206 Sirolimus (rapamycin) Temsirolimus Ridaforolimus Everolimus Selumetinib

Merck Wyeth-Pfzer Wyeth-Pfzer Ariad-Merck Novartis Astra Zeneca/Array BioPharma GSK Roche/Chiron Takeda San Diego Roche

Akt mTORC1 mTORC1 mTORC1 mTORC1 MEK

Allostery plays an essential role in processes of cell biology and function and also in pathogenesis of lung cancer. EGFR mutations and signaling pathways downstream (such as the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways) play a key role in NSCLC and are highly considered as targets of allosteric therapy. A few allosteric inhibitors targeting allosteric sites in signal transduction pathways have been explored. Several allosteric drugs have been approved to treat solid tumors. Clinical trials of allosteric modulators against NSCLS are furthermore expected, although there is still a conflicting. Additional allosteric drugs, especially dual allosteric

Trametinib RO4987655 TAK-733 RO5126766

MEK MEK MEK Raf/MEK

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modulators, should be designed and developed for lung cancer. The combination with different allosteric modulators or allosteric and orthosteric drugs to target multiple proteins can be a new therapeutic strategy for lung cancer. Acknowledgments The work was supported by Zhongshan Distinguished Professor Grant (XDW), The National Nature Science Foundation of China (91230204, 81270099, 81320108001, 81270131, 81300010), The Shanghai Committee of Science and Technology (12JC1402200, 12431900207, 11410708600, 14431905100), Operation funding of Shanghai Institute of Clinical Bioinformatics, and Ministry of Education, Academic Special Science and Research Foundation for PhD Education (20130071110043).

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