Development of Heat Shock Protein (Hsp90) Inhibitors To Combat

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Development of Heat Shock Protein (Hsp90) Inhibitors To Combat Resistance to Tyrosine Kinase Inhibitors through Hsp90−Kinase Interactions Meining Wang,†,∥ Aijun Shen,‡,∥ Chi Zhang,§,∥ Zilan Song,† Jing Ai,‡ Hongchun Liu,‡ Liping Sun,*,§ Jian Ding,‡ Meiyu Geng,*,‡ and Ao Zhang*,†

J. Med. Chem. 2016.59:5563-5586. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/29/19. For personal use only.

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CAS Key Laboratory of Receptor Research, Synthetic Organic & Medicinal Chemistry Laboratory, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, 555 Zuchongzhi Lu, Building 3, Room 426, Pudong, Shanghai 201203, China ‡ Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China § Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China ABSTRACT: Heat shock protein 90 (Hsp90) is a ubiquitous chaperone of all of the oncogenic tyrosine kinases. Many Hsp90 inhibitors, alone or in combination, have shown ̈ significant antitumor efficacy against the kinase-positive naive and mutant models. However, clinical trials of these inhibitors are unsuccessful due to insufficient clinical benefits and nonoptimal safety profiles. Recently, much progress has been reported on the Hsp90−cochaperone−client complex, which will undoubtedly assist in the understanding of the interactions between Hsp90 and its clients. Meanwhile, Hsp90 inhibitors have shown promise against patients’ resistance caused by early generation tyrosine kinase inhibitors (TKIs), and at least 13 Hsp90 inhibitors are being reevaluated in the clinic. In this regard, the objectives of the current perspective are to summarize the structure and function of the Hsp90−cochaperone−client complex, to analyze the structural and functional insights into the Hsp90−client interactions to address several existing unresolved problems with Hsp90 inhibitors, and to highlight the preclinical and clinical studies of Hsp90 inhibitors as an effective treatment against resistance to tyrosine kinase inhibitors.

1. HSP90: AN EMERGING DRUG TARGET FOR CANCERS Heat shock protein 90 (Hsp90) is an abundant and evolutionally conserved molecular chaperone that plays vital roles in protein homeostasis through directing the folding and conformational maturation of its bona fide clients in cells under normal conditions as well as in adapting to hostile stress insults.1,2 Hsp90 is distinguished from other cellular chaperones because it specifically binds to intermediate conformations of partially folded polypeptides at the late stage of protein assembly.3 It interacts with diverse cochaperones to construct fully active multichaperone complexes and serves as a network hub to coordinate cellular functions of a restricted set of oncogenic signal-transducing proteins.4−6 It has been reported that the expression of Hsp90 in cancer cells is generally 2−10-fold higher than that in normal cells.4 The small molecule geldanamycin analogue 24 (17-AAG, Figure 1), the first Hsp90 inhibitor entering clinical trials, showed nearly 100-fold higher binding affinity in cancer cells than in normal cells (Chart 1).7 The high sensitivity of the inhibitor in cancer cells is proposed to be due to the formation of the Hsp90−cochaperone−client supercomplex that is highly unstable and possesses high ATPase activity, whereas in normal cells, Hsp90 either does not form such highly susceptible © 2016 American Chemical Society

supercomplexes or it forms different complexes that are independent of cancer cells. All of these results provide a solid rationale for targeting Hsp90 as an attractive moleculartargeted therapy (MTT) in cancer.8−13 Indeed, the disruption of Hsp90 chaperone activity has been confirmed to induce simultaneous proteasomal degradation of many deregulated oncoproteins that are believed to be critical for all fundamental hallmarks of cancer, including cell development, proliferation, survival, and motility.2,14−17 Therefore, Hsp90 has long been regarded as an emerging drug target for a wide spectrum of cancers, and at least 13 small molecule inhibitors targeting Hsp90 have been or are being investigated in various clinical settings.16−20

2. PROMISE AND REMAINING QUESTIONS OF HSP90-TARGETING INHIBITORS AS AN ANTICANCER TREATMENT 2.1. The Structural Types and Interaction Features of Hsp90-Selective Inhibitors. Tremendous efforts have been made during the past several decades to develop selective inhibitors that directly target Hsp90 or cancer-relevant Hsp90− Received: July 15, 2015 Published: February 4, 2016 5563

DOI: 10.1021/acs.jmedchem.5b01106 J. Med. Chem. 2016, 59, 5563−5586

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Figure 1. Four major classes of clinically well studied N-terminal Hsp90 inhibitors.

C-terminal Hsp90 inhibitors, and (3) non-ATP competitive Hsp90 inhibitors. Derivatives of geldanamycin (GMs), resorcinol (RDs), purine (PUs), and 2-aminobenzamide (ABZs) represent the four major classes of N-terminal Hsp90 inhibitors (Figure 1). The GMs include the prototypic natural product geldanamycin (GA, 1)23 and its 17-substituted analogues tanespimycin (17AAG, 2),25 alvespimycin (17-DMAG, 3),24 and the hydroquinone analogue retaspimycin (IPI-504, 4).26 Compounds 1− 3 showed IC50 values of 70, 33, and 24 nM, respectively, against SKBR3 breast cancer cells, while compound 4 was reported to have an IC50 value of 307 nM against MM1.S human multiple myeloma cells. These compounds interact with Hsp90 through critical H-bonds between the pharmacophoric carbamoyl moiety and the highly conserved residue Asp93 as well as between the central quinone moiety and Asp40 and Lys44 in the ATP-binding domain of Hsp90 (Figure 2A). The resorcinol derivatives (RDs)27 include luminespib (NVP-AUY922, 7),28,30 ganetespib (STA9090, 6),29 and onalespib (AT-13387, 8).31 The cellular potency of these radicicol analogues 6−8 was 63 nM (MG63), 16 nM (HCT116), and 48 nM (HCT116), respectively. These compounds bind to the Asp93 residue in the ATP-binding pocket of the N-terminus of Hsp90 through the resorcinol moiety (Figure 2B). The purine analogues (PUs)32 include 9 (PU-H71),33 10 (PU3), and 11 (Debio0932)34 showing IC50 values of 42, 15, and 38 nM, respectively, against Hsp90. They share a “purine-linker-aryl” template, among which the 6-aminopyrimidine group in the adenine part forms one H-bond with the Asp93 residue and two water-

Chart 1. Key Events for Hsp90 Inhibitors

client complexes. Although the crystal structure of the Nterminal domain of Hsp90 was reported in 1997,21,22 the majority of the reported Hsp90 inhibitors, especially those tested in the clinic, originated from modifications of natural products rather than from rational drug design. Structurally, these Hsp90 inhibitors can be roughly classified into three chemotypes,18 including (1) N-terminal Hsp90 inhibitors, (2) 5564



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expected to exert antitumor effects with more clinical benefits than the traditional chemotherapy, none of them has successfully reached the market (Table 1, in pink). One of the major reasons for the failure of current Hsp90 inhibitors as monotherapy is partially due to chemical structure-related toxicities that limit their clinical applicable dosages. For example, geldanamycin analogues generally suffer from hepatotoxicity likely due to the existence of a quinone moiety, whereas the resorcinol fragment in resorcinol analogues is generally relevant to multiple adverse effects, especially ocular toxicity, diarrhea, and others. Another major concern for Hsp90-targeting therapy lies in the lack of knowledge to stratify the patients who would most likely benefit from the therapy. Meanwhile, the intrinsic supportive “non-oncogene addiction” features of Hsp90 make it difficult to identify therapeutic indicators based on its genetic status, such as mutation or amplification.23,32,46 Compared to the failure of Hsp90 inhibitors as a traditional chemotherapy, fortunately, significant progress has been achieved in several tumor subtypes bearing the “highly sensitive oncogenic tyrosine kinase clients”. Among these, clinical results on Hsp90 inhibitors against human epidermal growth factor receptor-2 (HER2)-positive trastuzumab refractory metastatic breast cancer (MBC) and the fusion protein from echinoderm microtubule-associated protein-like 4 and the anaplastic lymphoma kinase (EML4-ALK)-positive crizotinib-resistant nonsmall cell lung cancers (NSCLCs) are even more promising. Therefore, Hsp90 inhibitors have been reignited as an alternative strategy to combat the endless resistance surrounding tyrosine kinase inhibitors (TKIs) (Table 1, in green). Although the underlying mechanisms are still unknown, significant progress has been achieved recently in the structure and function study of the Hsp90−cochaperone−client complex, which may be useful to rationalize the antiresistance effects of Hsp90 inhibitors and to provide some instructive information to guide the design of new Hsp90 inhibitors as well. In this regard, the rest of this perspective will focus on recent studies on the interactions of Hsp90 with its oncogenic kinase clients followed by the preclinical and clinical uses of Hsp90 inhibitors to fight against the resistance of TKIs.

Figure 2. Interaction modes of Hsp90 Inhibitors with the N-terminal domain of Hsp90. A critical H-bond is generally formed between Asp93 of Hsp90 and the inhibitors. (A) Geldanamycin and its derivatives. The carbamate moiety forms a H-bond with Asp93. (B) The resorcinol derivatives. The resorcinol moiety forms an H-bond with Asp93. (C) The purine analogues. The 6-aminopyrimidine moiety forms an H-bond with Asp93. (D) The 2-aminobenzamide derivatives. The amide moiety forms an H-bond with Asp93.

mediated H-bonds with Leu48, Asn51, Ile91, Gly97, and Thr184 (Figure 2C).35 The 2-aminobenzamide analogues (ABZs)36 include 12 (SNX-2112) and its prodrug 13 (SNX5422), having IC50 values of 3 and 32 nM, respectively, against HT-29 colonic adenocarcinoma cells. They form key interactions through the typical benzamide functionality with the Asp93 residue of Hsp90 (Figure 2D).37,38 The coumarin antibiotic novobiocin was the first natural product identified as a C-terminal Hsp90 inhibitor. It binds at a second ATP-binding site in the C-terminus of the chaperone. In SKBR3 breast cancer cells, novobiocin dose-dependently reduced protein levels of p185erbB2, mutated p53, and Raf-1 with maximal activity occurring at concentrations of 500−800 uM.39 Recent efforts have delivered new analogues, showing much improved potency and selectivity against the C-terminal Hsp90, but further validation of these inhibitors in vivo is needed.39 In addition to the N- or C-terminal Hsp90 inhibitors that bind to the ATP-binding pocket, the non-ATP competitive Hsp90 inhibitors have attracted more and more interest recently and are expected to arrest the chaperone cycle by different mechanisms. It has been proposed that inhibitors targeting the Hsp90−cochaperone (e.g., celastrol, 40,41 (3E,5R,7S,8S,11E,13R,15S,16S)-3,5,7,11,13,15-hexamethyl8,16-bis(5-oxazolylmethyl)-1,9-dioxacyclohexadeca-3,11-diene2,10-dione (FW-04-806)42) or the Hsp90−client complex (e.g., gambogic acid,43 San A-amide44,45) may be more effective in regulating the function of Hsp90. Indeed, some agents have been reported to be capable of inhibiting Hsp90 by disrupting the interactions between Hsp90 and its cochaperones or/and clients. Yet, because of the lack of direct experimental evidence, the exact mechanisms of these inhibitors are elusive. 2.2. Questions Surrounding Hsp90 Inhibitors As a Monotherapy in the Treatment of Hsp90-Overexpressing Tumors. To date, more than 20 Hsp90 inhibitors have successively entered clinical trials, all of which are ubiquitous Nterminal Hsp90 inhibitors.46 Although these inhibitors were

3. RECENT ADVANCES IN THE STRUCTURE AND FUNCTION OF COMPLEXES BETWEEN HSP90 AND ITS ONCOGENIC CLIENTS 3.1. The Thermal and Conformational Stability of the Client Proteins Determines the Hsp90−Client Interaction. In contrast to the smaller chaperones Hsp70 and Hsp60, the mechanism by which Hsp90 recognizes and discriminates its clients as well as the corresponding tumors is not known. A large number of oncogenic proteins have been reported to be the clients of Hsp90, many of which have unrelated amino acid sequences, diverse functions, and metastable folding characteristics. Therefore, many cochaperones have been reported to play important roles to assist Hsp90 functioning by involving in the rather complex Hsp90−client interactions.1 In 2012, Lindquist and co-workers systematically and quantitatively studied the interactions of the cochaperone cell division cycle 37 (CDC37) with many of the Hsp90 clients, including most human kinases, transcription factors (TFs), and E3 ligases.47 They found that most kinases interacted with CDC37 similar to how they interacted with Hsp90. Furthermore, although the interactions between Hsp90 and kinases varied in a range of 100-fold, all of these interactions 5565



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Table 1. Clinical Status of Representative Hsp90 Inhibitorsa

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a

Data were from the Thomson Reuters Cortellis at http://lifesciences.thomsonreuters.com/products/cortellis (accessed on August 1, 2015). ORR, objective response rate; PR, partial response; CR, complete response; SD, stable disease; PFS, progression-free survival; DCR, disease control rate; NSCLC, nonsmall cell lung cancer; MM, multiple myeloma; MPC, metastatic pancreatic cancer; MBC, metastatic breast cancer; HRPC, hormonerefractory metastatic prostate cancer; TNBC, triple negative breast cancer; GIST, gastrointestinal stromal tumors; CRPC, castration-resistant prostate cancer.

were reduced after CDC37 knockdown. These results indicated that CDC37 is a highly specialized cochaperone adaptor that assists Hsp90 in recognizing its kinase clients. Considering the role of Hsp90 in the evolutionary process, the Lindquist group proposed that the amino acid sequences of the client kinases may have evolved more quickly than those of nonclients. After analyzing the protein expression data and comparing the thermal stability of 56 known kinase domains with the interaction profiles of Hsp90, they found that the quantitative interaction scores of the Hsp90−clients were highly correlated with the thermal instability of the kinase. As an example, the same group studied the effects of different inhibitors of the fusion oncoprotein BCR-ABL, formed from the breakpoint cluster region protein (BCR) and the Abl protein, on the thermodynamic parameters of the Hsp90− client interactions. The ATP-competitive BCR-ABL inhibitors imatinib and dasatinib, which bind to the inactive and active conformations, respectively, were found to stabilize the BCRABL kinase. In the contrast, the T315I gatekeeper mutant prevented the corresponding binding and stabilization of BCRABL. As a result, both imatinib and dasatinib decreased the Hsp90−BCR-ABL interactions, whereas the Hsp90−BCRABLT315I interactions were not affected.47 More evidence was obtained recently from Bayliss’ study on the variations of another fusion oncoprotein EML4-ALK.48,49 EML4-ALK is a highly sensitive client of Hsp90, and the fusion of EML4 to ALK in NSCLCs by gene translocation causes the expression of several oncoprotein fusion variants. Bayliss’ group successfully obtained a 2.6 Å crystal structure of the representative ∼70 kDa core of EML1 and named as the tandem atypical propeller in the EML domain (TAPE) that contains two tightly interconnected β-propellers. Subsequent

mapping of the characteristic breakpoints of the EML4-ALK variants onto the structure indicated that the EML4 TAPE domain is truncated in many variants (1, 2, 3a, and 5a), likely causing the fusion protein to be structurally unstable. After testing the sensitivity of EML4-ALK variants to the Hsp90 inhibitor 6, variant 1, with the breakpoint falling within the N-terminal β-propeller, and variant 2, with the breakpoint falling within the C-terminal β-propeller, were less stable than the other variants. Subsequent treatment with 6 led to a significant reduction of both variants 1 and 2, whereas the expression of variants 3a and 5a remained stable. Meanwhile, 6 showed an IC50 value of 11 nM against Ba/F3 cells overexpressing EML4-ALK variant 1, which was more potent than that against cells overexpressing the other variants.48,49 3.2. Advances in the Study of Structures of the Hsp90−Client Complex. 3.2.1. Structural Studies of the Hsp90−Cochaperone (CDC37, Aha1, p23) Complexes. Hsp90 function is dependent on the assistance of a number of cochaperones, which are indispensable for ATPase regulation, conformational changes, the selection of and binding with specific substrates, and the subsequent dynamics and activation of Hsp90. For example, CDC37, as mentioned earlier, is an important cochaperone for the recognition of kinase clients; the cochaperone Hop/Sti1 is believed to be involved in the early stages of the Hsp90 functional cycle and to help in recruiting Hsp70-bound client proteins, such as steroid hormone receptors, and for Hsp90 binding.50 1. Structural Study of the Hsp90−CDC37 Complex. In 2004, Prodromou’s group obtained several diffraction-quality crystals of a complex between the C-terminus of human CDC37 (148−347) and the N-terminus of yeast Hsp90 (1− 208) and found that CDC37 binds to the lid segment of the N5570



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terminus of Hsp90 (100−121) through helices 2, 3, and 5.51 The interaction center involves a hydrophobic interface formed by Ala103, Ala107, Ala110, Ala112, Met116, and Phe120 of Hsp90, together with Met164, Leu165, and Ala204 of CDC37. The interface is reinforced by several polar interactions formed between Gln119 of Hsp90 and Gln208 of CDC37 and between Ser109 of Hsp90 and Lys202 of CDC37 (Figure 3).

Figure 4. Crystal structure of the Hsp90−p23−ATP complex. The complex structure is shown as cartoon in green (Hsp90) and purple (p23/Sba1). Two p23/Sba1 molecules bind to the N-terminal domains of the Hsp90 dimer in a symmetrically closed conformation (taken from PDB ID code 2CG9).

move together and interact with the N-domain of the other monomer. All of these changes produce a reinforced ATPbound conformation and extend the lifetime of the state required for client activation (Figure 5).53

Figure 3. Interface of the Hsp90 and CDC37 complex. The interface between the lid segment (residues 100−121, yellow cartoon) of NHsp90 and the helices (residues Met164, Leu165, and Ala204, blue cartoon) of C-CDC37 is reinforced by several polar interactions highlighted by magenta dot lines (taken from PDB ID code: 1US7).

After the amino acid residues in the interface were mutated, the ATPase activity of Hsp90 was found no longer sensitive to a CDC37 inhibitor, indicating that the interface observed in the crystal structure is indeed the functional interface of the Hsp90−CDC37 complex. To reveal the mechanism by which CDC37 arrests the ATPase cycle of Hsp90, the same group identified an important H-bond formed between Arg167 of CDC37 and Glu33 of Hsp90, which likely induces alterations of the electronic and chemical environment at the top of the ATP binding pocket. On the basis of this analysis, it can be proposed that CDC37 regulates the ATPase activity of Hsp90 by first inserting the Arg167 into the mouth of the nucleotide binding pocket of Hsp90 and subsequently forming an H-bond with Glu33.51 In 2009, Schwalbe and co-workers studied the complex between the middle domain of human CDC37 and the Nterminal domain of human Hsp90 by heteronuclear magnetic resonance (NMR) spectroscopy. The data suggested a large interface between these two proteins, and Leu205 in CDC37 was crucial for the protein−protein interaction.52 2. Structural Study of the Hsp90−p23 Complex. In 2006, Pearl and co-workers disclosed a crystal structure of the fulllength yeast Hsp90 in a complex with β-γ-imidoadenosine 5′phosphate (AMP-PNP, an ATP analogue) and the cochaperone Sba1 (the yeast homologue of p23). As shown in Figure 4, two p23/Sba1 molecules are symmetrically opposed on each side of the Hsp90 dimer in the “closed” state. Conformational alternations in N- and M-domains of Hsp90 were identified from the Hsp90−p23−ATP complex. The two N-terminal βstrands (residues 1−9) from each monomer swap and bind to the β-sheet in the other monomer. The “lid” segment (residues 94−125) folds over the ATP-binding pocket by swinging nearly 180° from its “open” position. The movement of the lid segment pushes the exposed hydrophobic patch to form a substantial interface with the other monomer. The M-segments

Figure 5. Conformational changes in the N- and M-domains of Hsp90 (green) upon interacting with an ATP analogue (red) and p23/Sba1. The “lid” segment (residues 94−125), colored in blue, folds over the ATP-binding pocket by nearly 180° from its “open” position, resulting in the exposure of a hydrophobic patch. The M-segments move together and interact with the N-domain of the other monomer (taken from PDB ID codes 2IOQ and 2CG9).

Because the human chaperone system is far more complex than that of the yeast, cocrystals of full-length human Hsp90 with cochaperones or clients would be more informative. In 2011, Rüdiger and co-workers characterized the Apo and ATP states of the soluble, full-length human Hsp90 protein by NMR spectroscopy.54 By mapping the chemical shift perturbations of the full-length Hsp90, they proposed a mechanism for the Hsp90−p23 interaction. First, ATP binds to both N-terminal domains of Hsp90, followed by the dimerization of the Ndomains and the subsequent conformational change of Hsp90 to a closed state. One p23 molecule then binds to both Nterminal domains of the Hsp90 dimer and interacts with the Mdomain as well.54 3. Structural Study of the Hsp90−Aha1 Complex. In contrast to the cochaperones CDC37 and p23 that inhibit the ATPase cycle of Hsp90, Aha1, the activator of Hsp90 ATPase, is the only cochaperone of Hsp90 that stimulates the ATPase activity. Hch1, the high copy Hsp90 suppressor, is an isoform of Aha1 and shares high sequence homology to the N-terminal 5571



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its natural, folded client in a mature state. Although the structural details of the complex are still unavailable due to the low-resolution of the negative stained EM, Pearl’s work provides preliminary evidence on the Hsp90−cochaperone− client supercomplex that may be critical for Hsp90s recognition of both its wild-type and mutant oncogenic kinase clients. In addition to tyrosine kinases, many nonkinase proteins are also clients of Hsp90. Therefore, there might be some similarities in the binding modes of Hsp90 with these diverse classes of clients, which will in turn enlighten our understanding of Hsp90−kinase interactions. For example, Rüdiger and co-workers58 recently developed a specific isotope labeling strategy and obtained a structural model of Hsp90 with the microtubule-associated protein Tau in the Tau-bound state. Meanwhile, structural study on the interaction between Hsp90 and hormone-bound glucocorticoid receptor (GR) was also reported recently.59,60

part of Aha1. These two cofactors can directly bind through the N-terminal domains to the central segment of Hsp90 that is essential for Hsp90-dependent activation of its clients. To investigate the Aha1-induced regulation mechanism, Pearl and co-workers obtained a crystal of the N-terminal domain (1− 153) of Aha1 in complex with the middle segment of Hsp90 (273−530).55 A structural analysis and mutagenesis study showed that the binding to the N-terminus of Aha1 induced a conformational switch in the catalytic loop (370−390) in the middle segment of Hsp90. As a result, the catalytic Arg380 residue was released to interact with the ATP binding site in the N-terminal domain of Hsp90. Later, Buchner’s group identified a larger interface and more complex interactions between the full-length Hsp90 and Aha1 proteins using multidimensional NMR techniques than those revealed by crystal structures.56 On the basis of this result, it is likely that one subunit of Aha1 is used for the ATPase activityrelated conformational change and the other is responsible for substrate protein processing.56 3.2.2. Structural Study of the Hsp90−CDC37−CDK4 Complex. In 2006, Pearl’s group obtained an Hsp90− CDC37−CDK4 (cyclin-dependent kinase 4) complex, defined its stoichiometry, and determined its three-dimensional structure by single-particle electron microscopy (SPEM).57 They first fit the known crystal structure of Hsp90 into the electron microscopy (EM) reconstruction by adjusting the relative orientations of Hsp90 domains through small rotations in the hinge regions and placing the M-terminal domain of CDC37 into the EM reconstruction. The final docking results revealed that the C-terminal domain of CDC37 binds to the Nterminal domain of Hsp90, while the N-terminal domain of CDC37 interacts with the kinase domain of CDK4. In addition, the larger C-terminal lobe of the CDK4 client is believed to associate with the M-domain of Hsp90, while the smaller Nterminal domain of CDK4 is associated with N-terminal domain of Hsp90, CDC37, or both proteins (Figure 6). This robust system is the first structural model of Hsp90 bound to

4. TARGETING HSP90 AS A PROMISING STRATEGY TO COMBAT TYROSINE KINASE INHIBITOR RESISTANCE 4.1. Drug Resistance: An Unmet Challenge of Tyrosine Kinase Inhibitors (TKIs). Drugs targeting the mutations in or rearrangements of receptor tyrosine kinases (RTK) (e.g., mutated EGFR, amplified HER2, or fused EML4ALK) have become the most successful approach in the paradigm of molecular-targeted cancer therapy in the past few decades.61,62 Nearly 30 small molecule inhibitors targeting one or more different RTKs have been successfully launched in the market to treat the corresponding kinase-addictive patients. Despite the remarkable success, most patients eventually suffer from relapse and tumor regrowth due to the development of acquired resistance.63−67 Therefore, the development of nextgeneration TKIs that tackle the resistance to the early generation inhibitors has become a major challenge in this field.63,64,68−70 Fortunately, more and more studies have shown that the intrinsic feature of the Hsp90 chaperone in complex with multiple oncogenic kinase clients has provided the rationale for targeting Hsp90 as a novel solution to treat both the client kinase-positive and kinase inhibitor-resistant patients. 4.2. Application of Hsp90 Inhibitors in the Battle against TKI Resistance. Within the last 10 years, a number of preclinical or clinical studies of Hsp90 inhibitors have been conducted in various TKI-resistant tumor cells or patients, and the overall results are promising, particularly for studies investigating the highly sensitive client RTKs, including EML4-ALK, HER2, EGFR, BRAF, c-Met, and others (Table 1, in green). 4.2.1. Hsp90 Inhibitors Overcome Drug Resistance to EML4−ALK TKIs. 1. ALK Represents One of the Highly Sensitive Clients of Hsp90. EML4-ALK is an oncogenic protein discovered in NSCLC, and its first-generation ALK inhibitor crizotinib was discovered in 2011, showing over 60% of objective response rates (ORR) and impressive rate of progression-free survival (PFS) in EML4-ALK-positive NSCLC patients. However, resistance to this drug was developed rapidly, within one year, leading to patient relapse and tumor regrowth. The recently approved second-generation ALK inhibitors ceritinib and alectinib were found to effectively tackle the major secondary mutations observed in resistant patients, particularly the L1196M gatekeeper mutation. However, because of the highly complex mechanisms of crizotinib resistance, new inhibitors capable of overcoming a

Figure 6. EM reconstruction of the Hsp90−CDC37−CDK4 complex. The structure was revised from the EM reconstruction and the crystal structures of Hsp90, CDC37, and CDK4 (PDB ID codes 2CG9, 1US7, and 2W99). The C-terminal lobe of CDK4 (red cartoon) interacts with the M-domain of the Hsp90 monomer (green cartoon), and the small N-domain interacts with either the N-terminal domain of Hsp90 or the N-terminal domain of CDC37, while the C-terminal domain of CDC37 (blue cartoon) interacts with the other Hsp90 monomer, forming an asymmetrical complex. 5572



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was nearly 30-fold more potent than that of crizotinib (IC50: 300 nM). More encouragingly, at the weekly dose of 50 mg/kg, 6 markedly suppressed tumor growth in the ALK-positive NSCLC xenografts and showed extended overall survival in the H3122 xenograft model.75 3. Hsp90 Inhibitors Are Highly Effective against Acquired Resistance to ALK Inhibitors Driven by Various Resistant Mechanisms. Hsp90 inhibitors can ef fectively overcome crizotinib resistance caused by various secondary mutations: By treating nucleophosmin (NPM)-ALK-expressing BaF3 cells with various concentrations of crizotinib, Proia’s group recently successfully obtained 15 different mutations associated with crizotinib resistance in the ALK kinase domain, including six that had been clinically observed in NSCLC patients. Compared to its potency against the parental cells, crizotinib was approximately 1.6−5-fold less potent against these mutants. However, the Hsp90 inhibitor 6 showed a nearly identical high potency against cells expressing all of these secondary mutations and the corresponding parental cells, indicating that all of these NPM-ALK mutants were sensitive clients of Hsp90.75 Hsp90 inhibitors can effectively overcome crizotinib resistance caused by both ALK gene amplification and kinase mutations: Gene amplification is another mechanism of crizotinib resistance in NSCLC patients. To test whether an Hsp90 inhibitor is effective against ALK gene amplification-related resistance, Proia and co-workers treated NB-39-nu neuroblastoma cells that express 30−40 copies of the ALK gene per cell with 6 and crizotinib, respectively. They found that the Hsp90 inhibitor 6 showed increased sensitivity in the ALK gene amplified cells, with an IC50 of 10 nM, whereas the ALK inhibitor crizotinib was at least 20-fold less potent (IC50 = 240 nM) in the same cell lines.75 Similarly, Shaw and colleagues found that treatment of 2 at the dose of 10 nM for 72 h significantly suppressed the growth of H3122 CR cells bearing both EML4-ALK gene amplification and the L1196 M gatekeeper mutation.76 Hsp90 inhibitors can ef fectively overcome ALK inhibitor resistance driven by the activation of alternative signaling pathways: In 2014, Yano and co-workers found that addition of the EGFR ligand EGF and the MET ligand HGF (hepatocyte growth factor) to the EML4-ALK-dependent NSCLC cells induced resistance to the second-generation ALK inhibitor alectinib. However, treating these cells with 0.3 μM of the Hsp90 inhibitor 3 for 24 h significantly inhibited the viability of the resistant cells.77 Hsp90 inhibitors can effectively overcome crizotinib resistance caused by unknown mechanisms: Recently, Normant’s group incubated H3122 cells with crizotinib and obtained resistant cells (H3122R) with unknown mechanisms other than secondary mutations or gene amplification. Crizotinib was found at least 12-fold less sensitive against the resistant cells than the parental cells. Surprisingly, these resistant cells were sensitive to the Hsp90 inhibitor 4, with GI50 values of 76 nM.74 Similarly, Proia’s group also obtained a resistant H3122 CR1 cell line through an unknown mechanism by incubating the H3122 cells with crizotinib. The cell line was insensitive to either the first-generation ALK inhibitor crizotinib or the second-generation ALK inhibitor alectinib; however, it was much more sensitive to the Hsp90 inhibitor 6.75 4. Clinical Advances of Hsp90 Inhibitors in the Battle against Crizotinib Resistance. On the basis of the promising preclinical results of the Hsp90 inhibitor against the resistance

wider spectrum of resistant mutants and reducing the activation of multiple bypass signals are needed (Figure 7).71,72

Figure 7. ALK signaling pathway, mechanisms of resistance to ALKTKIs, and application of Hsp90 inhibitors to overcome the resistance. The EML4-ALK fusion protein (dark gray) is aberrantly expressed in cancer. ALK-TKIs (dark violet), such as crizotinib, could bind to the ATP-binding pocket of EML4-ALK, inhibit the kinase activity, and block the downstream signaling pathways. The resistance mechanisms of ALK-TKIs are rather complicated, and Hsp90 inhibitors (red) are effective against the acquired resistance through diverse mechanisms.

Earlier in 2010, Wong and co-workers found that the Hsp90 inhibitor 2 had a dramatic inhibitory effect against EML4-ALKdriven tumor cells. When ALK-dependent H3122 cells were treated with 2 at concentrations as low as 25 nM for 1 h, a marked degradation of EML4-ALK was observed, indicating that EML4-ALK is a sensitive Hsp90 client.73 The Normant group further demonstrated that EML4-ALK may be one of the most sensitive clients of Hsp90. They incubated EML4-ALKpositive, HER2-positive, and both wild-type and mutant EGFRdependent cells with the Hsp90 inhibitor 4 (1 μM) and found that EML4-ALK was completely depleted within 3 h, while most of the HER2 and mutant EGFR were degraded after 24 h, suggesting that EML4-ALK is a more sensitive client than either mutant EGFR or HER2.74 2. Hsp90 Inhibitors Are Even More Potent Than ALK Inhibitors in Both ALK-Dependent Cells and Xenograft Models. In 2011, Normant’s group incubated the Hsp90 inhibitor 4 with two types of HEK293FT cells for 72 h, one expressing ALK with a kinase dead mutant (293FTALK‑KD) and the other expressing the active EML4-ALK fusion protein (293FTALK). They found that 4 (either 100 nM or 1000 nM) had little effect on the 293FTALK‑KD cells, while the growth of the 293FTALK cells was significantly suppressed. Further, the same group studied the antitumor effect of 4 in the NSCLC xenograft model expressing EML4-ALK-addicted H3122 cells. Encouragingly, 4 showed tumor regression at the dose of 75 mg/kg twice weekly, which was comparable to that of a daily dose of 50 mg/kg of the ALK inhibitor crizotinib.74 Proia and co-workers recently investigated the antitumor effects of Hsp90 inhibitor 6 against ALK-rearranged NSCLC cells as well as ALK-positive NSCLC xenograft models. It was found that 6 was nearly 20-fold more potent than crizotinib (IC50: 13 vs 202 nM) in EML4-ALK-dependent H2228 cells. Similarly, 6 also showed a significant antiproliferative effect, with an IC50 of 10 nM against ALK-driven H3122 cells, which 5573



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were unexpectedly activated following suppression of the EGFR-TKIs, including c-Met, AXL, NF-κB (nuclear factor kappa B), and others (Figure 8).71

caused by crizotinib treatment, many clinical trials of these inhibitors have been or are being conducted. In a phase II study of 2 as monotherapy in advanced NSCLC patients, eight ALK+ patients were identified. Among which, four achieved PR, three showed SD, and one experienced disease progression after 16 weeks. Further compelling evidence was obtained from a 24year-old male with ALK+ NSCLC and had progressed on crizotinib after 12 month-treatment. The computed tomography (CT) imaging revealed significant tumor shrinkage following one cycle monotherapy with 2.78 In 2010, a phase I/II trial with 4 was reported in 76 NSCLC patients who progressed on EGFR inhibitor treatment. Among the three patients with an ALK gene rearrangement, two had PR and the third had prolonged stable disease (SD, 7.2 months, 24% reduction in tumor size). Grades 1 and 2 fatigue, nausea, and diarrhea were the most common adverse events observed. This result indicated the Hsp90 inhibitor 4 might be a more efficient treatment for NSCLC patients bearing ALK rearrangements.79 In a phase II trial of 6 in patients with stage IIIB or IV NSCLC who had received prior TKI treatment or chemotherapy, significant tumor shrinkage was observed in one patient who had ALK+ cancer progression and was previously treated with crizotinib.80 In 2013, another phase II trial of 6 with 99 NSCLC patients was disclosed. The objective response rate (ORR) was 4%, and all of the four responders showed PR and were ALK+ crizotinib-naiv̈ e.81 More trials of 6 in combination with crizotinib in ALK-rearranged NSCLC patients are ongoing (Table 1). In a phase II trial of the Hsp90 inhibitor 7 in the ALKpositive stratum patients (n = 22), the ORR, disease control rate (DCR), and the rate of progression-free survival (PFS) at 18 weeks were 32, 59, and 36%, respectively.80 Meanwhile, in ̈ to crizotinib (n = 8), the ALK-positive patients who were naive ORR, DCR, and the rate of PFS at 18 weeks were 50, 100, and 62.5%, respectively. In a similar phase II trial of 7 with 121 advanced NSCLC patients, 21 patients were ALK-positive. Six of the patients achieved PR, and the estimated PFS at 18 weeks was 42%.82 More trials of 7 in combination with ceritinib are ongoing (Table 1). Because the Hsp90 inhibitor 8 has showed synergistic tumor growth inhibition in combination with crizotinib in preclinical studies, a phase I trial has been conducted. The maximum tolerated dose (MTD) has been determined to be 260 mg/m2 iv weekly for three weeks on a four-week cycle. The doselimiting toxicities (DLTs) include infusion-related symptoms, gastrointestinal effects, and fatigue.83 More clinical trials of 8 in NSCLC patients with or without crizotinib are ongoing. 4.2.2. Hsp90 Inhibitors Overcome Drug Resistance to EGFR TKIs. 1. Application of Hsp90 Inhibitors to Fight Resistance to EGFR TKIs Driven by the T790 M Gatekeeper Mutation or T790M/L858R Double Mutations. The identification of activating EGFR mutations in the kinase domain, which contain small in-frame deletions in exon 19 and single point mutations, most commonly L858R (EGFRL858R), has made EGFR one of the earliest MTT in NSCLC. Small molecule EGFR inhibitors (e.g., gefitinib, erlotinib) were found to deliver double ORR and promising PFS for EGFR mutationpositive patients compared with the traditional chemotherapy.71 Acquired resistance against these TKIs generally occurred within one year of treatment, and the T790M gatekeeper mutation (EGFRT790M) was found to be the major cause of the acquired resistance. Meanwhile, a number of alternative kinases

Figure 8. EGFR signaling pathway, mechanisms of EGFR TKIs resistance, and application of Hsp90 inhibitors to overcome EGFR TKIs resistance. EGFR dimerization mediates phosphorylation of this receptor and initiates downstream signaling pathways, such as the PI3K/AKT and RAS/RAF/MEK/ERK pathways (dark gray), which are essential for cancer cell survival and proliferation. Several mechanisms of EGFR TKI resistance have been identified, and Hsp90 inhibitors (red) are effective against the acquired resistance through diverse mechanisms.

It has been shown that several EGFR mutants, particularly EGFRL858R and EGFR L585R/T790M, as well as MET kinase are all client proteins of Hsp90. Therefore, Hsp90 inhibition was proposed as a mechanism to overcome the EGFR-TKI resistance induced by EGFR mutations or MET amplification (Figure 8).84,85 In 2008, Solit and co-workers reported that treatment with 0.1 μM of the Hsp90 inhibitor 2 degraded the mutant EGFR (L858R and A750P) much more quickly than the wild-type EGFR in NIH-3T3 cells. Meanwhile, degradation of the mutant EGFR (L858R, T790M) as well as Raf-1, pMAPK, p-AKT, and AKT was also observed in human lung adenocarcinoma H1975 cells upon treatment with 0.1 μM of 2 for 24 h. At the maximum tolerated dose of 75 mg/kg three times per week, 2 was found to cause a significant growth delay in H3255 (L858R EGFR), H1650 (ΔE746-A750 EGFR), and H1975 xenografts, all of which showed intermediately high levels of resistance to gefitinib treatment. In addition, promising data from a combination of 2 with paclitaxel suggested that Hsp90 inhibitors may be most effective in combination with cytotoxic compounds in lung adenocarcinoma patients with EGFR mutations, both with de novo and acquired resistance.86 In addition, 2 was reported to suppress EGFR-AKT-mTORp70S6K-S6 signaling at a low concentration of 0.01 μM in cells, regardless of the presence of T790M.87 In 2011, Ying and co-workers compared the antiproliferative effects of the Hsp90 inhibitor 6 and the EGFR inhibitor erlotinib against the NCI-H1975 drug-resistant cell line that is driven by the EGFRL585R/T790M double mutation. They found that 6 exhibited full potency at the concentration of 25 nM, 5574



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Figure 9. Representative Hsp90 Inhibitors in preclinical or clinical studies.

(100 nM) was used in combination with ionizing radiation.95 Meanwhile, the Yano and Ying groups studied the effect of the Hsp90 inhibitor 3 on HGF-triggered resistance and found that in the presence of HGF, both PC-9 and Ma-1 cells expressing EGFR mutants were insensitive to erlotinib. However, 3 markedly inhibited the cellular growth, with IC50 values ranging from 0.01 to 0.03 μM. 3 also showed growth inhibition against the erlotinib-resistant human HGF-transfected Ma-1/HGF cells and in the severe combined immunodeficient (SCID) mice. A study of the molecular mechanism revealed that the growth inhibition of 3 was associated with the degradation of EGFR, MET, and their downstream signaling intermediates.88,96 3. Clinical Advances of Hsp90 Inhibitors in the Battle against EGFR TKI Resistance. Because Hsp90 inhibitors have provided a solid in vitro and in vivo basis in the battle against EGFR-TKI resistance, several of these inhibitors have been further evaluated in the clinic. In 2010, a phase I/II trial with 4 was reported in 76 NSCLC patients who progressed on EGFR inhibitor treatment. An ORR of 7% (5 of 76) was observed in the overall study population, 10% (4 of 40) in patients who were EGFR wild-type, and 4% (1 of 28) in those with EGFR mutations. Both EGFR groups were below the target ORR (20%), and grade 3 or higher liver function abnormalities were observed in nine patients (11.8%).79 Early in 2009, Synta Pharmaceuticals initiated a phase II trial of 6 plus docetaxel in stage IIIB or IV NSCLC patients. The data presented in June 2011 showed that a clinical response was generally observed in patients with advanced NSCLC tumors harboring wild-type EGFR and KRAS.97 Of the 76 evaluated patients, the overall disease control rate (DCR; complete response/partial response or stable disease) at 8 weeks was 54% and the overall ORR was 5.3%. In 2010, Novartis reported a phase II trial of 7 in patients with advanced NSCLC. In patients with EGFR mutations (n = 35), the ORR was 20% and the DCR was 57% and the rate of PFS at 18 weeks was 35.2%. In patients with EGFR mutations who had an EGFR-TKI as part of their last treatment regimen (n = 19), the ORR, DCR, and the rate of PFS at 18 weeks were 26, 68, and 46.6%, respectively.80 In 2013, a phase II trial of 7 plus erlotinib in lung cancer patients with EGFR mutations who had acquired resistance to erlotinib met its primary end point (defined as CR + PR at 8 weeks).98 Recently, a phase I/II study of 7 and erlotinib for EGFR mutant lung cancer with acquired resistance to erlotinib was reported. In phase I, 18

whereas erlotinib did not exhibit any effects in concentrations ranging from 0.1 to 50 nM.88 Meanwhile, the Proia group reported that in the EGFRL585R/T790M double mutant erlotinib-resistant NCIH1975 cells, treatment with 6 at the concentration of 50 nM for 24 h resulted in destabilization of EGFRL585R/T790M.89 Tumor growth inhibition was significantly improved in mice bearing NCI-H1975 tumors. A similar result was observed when 6 (50 mg/kg) was used in combination with another EGFR TKI, alatinib (5 mg/kg).90 Prolonged animal survival was also observed after treatment with the Hsp90 inhibitor 13 alone or in combination with erlotinib in the resistant NCI-H1975 xenografts.91 In addition, Ono and colleagues reported that a combination of aminopyrimidine 14 (CH5164840) (Figure 9), another Hsp90 inhibitor (12.5 mg/kg daily for 11 days), with erlotinib (25 mg/kg) markedly enhanced the antitumor efficacy in the T790 M gatekeeper mutant NCI-H1975 xenografts.92 In 2014, Jeong’s group investigated the antitumor activity of 15 (WK88-1) (Figure 9), a new GA-derived Hsp90 inhibitor lacking the benzoquinone moiety, and found that treating the gefitinib-resistant H1975 cells with 15 resulted in the overall degradation of EGFR, ErbB2, and ErbB3, which then induced growth arrest and apoptosis. Meanwhile, the tumor growth in the corresponding xenografts was also significantly suppressed.93 In 2012, Toyooka’s group examined the antiproliferative effects of 3 in various cells expressing 13 different EGFR mutants. They found that all thirteen EGFR mutant cells were significantly more sensitive than cells expressing the wild-type EGFR. The subsequent Western blotting analysis showed that the expression levels of EGFR, p-EGFR, p-AKT, p-MAPK, CDK4, and CD1 were depleted in most of the EGFR mutant cells after treatment with 0.2 μM of 3, while the proteins in EGFR wild-type cells were only partially depleted after treatment with 1 μM of 3. In the mouse xenograft models, 3 (25 mg/kg) also showed greater growth inhibition in the EGFR mutant tumors (PC-9 and RPC9).94 2. Application of Hsp90 Inhibitors to Fight the Resistance of EGFR TKIs Driven by Mechanisms Other than EGFR Mutations. Recently, Toyooka’s group found that the Hsp90 inhibitor 7 could exert antiproliferative effects in gefitinibresistant HCC827 cells driven by MET amplification (IC50 = 7.0 nM), and this effect could be significantly enhanced when 7 5575



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patients were treated with 7 once per week and erlotinib once per day in 28-day cycles using a 3 + 3 dose-escalation design. The PR was 16% (4 of 25 patients) that was independent of tumor T790 M status. In phase II, 19 additional patients were treated at the maximum tolerated dose (MTD), however, no CR or PR was observed.99 In another study of 7 in combination with erlotinib to overcome the resistance from EGFR TKI, 16 patients were enrolled and two achieved a PR, both with T790 M gatekeeper mutation. Subsequently, 25 more patients were enrolled. An ORR of 16% and SD of 25% were achieved. Three of the four patients with PR have EGFR T790 M mutation. The result indicated that combination of 7 with erlotinib can overcome resistance from EGFR TKI treatment.100,101 4.2.3. Hsp90 Inhibitors Overcome Drug Resistance to BRAF TKIs. 1. Application of Hsp90 Inhibitors to Overcome the Acquired Resistance of BRAF Inhibitors. Melanoma is an aggressive form of skin cancer, and the activated tyrosine kinase BRAF mutant (over 90% BRAFV600E) is an oncogenic driver in more than half of the melanoma cases. Selective ATPcompetitive BRAFV600E inhibitors, such as vemurafenib and dabrafenib, typically induce initial profound tumor regression, but unfortunately, long-lasting responses have been limited due to the emergence of drug resistance.71 The gatekeeper mutation has not been observed in BRAF TKI relapsed patients, which is different from the resistance to EGFR TKIs. Instead, two different mechanisms underlying the acquired resistance of BRAF inhibitors have been identified, including mitogenactivated protein kinase (MAPK)-dependent and MAPKindependent mechanisms. The MAPK-dependent resistance is primarily caused by incomplete inhibition of ERK signaling. Therefore, dual inhibition of MEK and BRAF appears to be a promising approach to overcome the acquired resistance to BRAF inhibitors. MAPK-independent resistance is caused by the loss of BRAF dependence that may be driven by activation of the phosphoinositide-3 kinase (PI3K)/AKT pathway due to the loss of phosphatase and tensin homologue (PTEN) or the upregulation of platelet-derived growth factor receptor-β (PDGFR-β) and insulin-like growth factor receptor 1 (IGF1R).71,102 In 2012, Smalley’s group tested the inhibitory effect of the Hsp90 inhibitor 16 (XL-888, Figure 9) in vemurafenib-resistant melanoma cells. They found that in the four tested resistant cells (WM164R, M229R, M249R, and 1205LuR), treatment with 300 nM of 16 for 48 h degraded IGF-1R, PDGFR-β, ARAF, CRAF, pERK, pS6, and cyclin D1 and inhibited pAKT, pERK, and pS6 signaling as well. Furthermore, 16 significantly inhibited tumor growth in vemurafenib-resistant M229R and 1205LuR xenografts at the dose of 100 mg/kg. In addition, 16 induced significantly higher levels of apoptosis in resistant cells containing COT amplification (a member of the MEK family) and PDGFR-β overexpression compared to MEK/PI3K inhibitors.103 In 2013, Wallis and co-workers found that the Hsp90 inhibitor 8 could delay the emergence of BRAF inhibitor resistance. First, they treated vemurafenib-sensitive A537 and SK-MEL-28 cells, respectively, with vemurafenib alone or in combination with 8. It was found that the cells treated with the BRAF inhibitor alone generated resistant colonies, while the combination with 8 did not. In vemurafenib-sensitive SK-MEL28 xenografts, the combination of 8 with vemurafenib prevented tumor regrowth over 5 months, whereas under the same conditions, treatment with the BRAF inhibitor vemurafenib alone induced tumor relapse.104

Meanwhile, Proia’s group recently found that 6 (IC50, 15 nM) was 4−13-fold more potent than either the selective BRAFV600E inhibitor vemurafenib (IC50, 81 nM) or the MEK inhibitor selumetinib (IC50, 255 nM) in A375 melanoma cell lines, and the combination of 6 with vemurafenib or selumetinib synergistically enhanced tumor growth inhibition. As overexpression of COT is another mechanism of intrinsic resistance to BRAF inhibitors, a study was conducted by treating the vemurafenib-resistant, COT-overexpressing, and selumetinib-insensitive RPMI-7951 melanoma cells with 6. The result showed that 6 (50 nM for 24 h) potently abrogated MAPK and AKT signaling and induced apoptosis. In addition, in the vemurafenib-resistant A375 (A375-VR) xenografts, weekly treatment with 6 (150 mg/kg) in combination with a MEK inhibitor inhibited 84% of tumor growth, which was much higher than that from treatment with MEK inhibitor alone (34%).105 All of these results suggest that inhibition of Hsp90 is a practical and effective strategy to overcome diverse mechanisms of resistance to BRAF inhibitors. 2. Clinical Advance of Hsp90 Inhibitors in the Fight against Resistance to BRAF TKIs. An open label phase I trial was conducted in 2014 to evaluate the safety and efficacy of the Hsp90 inhibitor 8 in combination with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib in melanoma patients (expected n = 38) with BRAF mutations.80 Exelixis Inc. started a phase I trial of the Hsp90 inhibitor 16 in combination with vemurafenib in patients with unresectable BRAF mutated stage III/IV melanoma. The results showed that the combination was well tolerated and an overall ORR of 92% was achieved. Tumor regression was observed in 11 of the 12 responded patients with two CR and nine PR. The estimated PFS at 6 and 12 months was 63% and 39%, respectively.106 4.2.4. Hsp90 Inhibitors Overcome Drug Resistance to BCRABL TKIs. Chronic myeloid leukemia (CML) is a bone marrow clonal myeloproliferative disorder characterized by the presence of the fusion oncogene BCR-ABL.107 Imatinib mesylate (gleevec) is the first ATP competitive small molecule BCRABL inhibitor and showed dramatically improved long-term survival rates in CML patients. However, acquired drug resistance occurs shortly after the treatment, and patients treated with the drug failed to reach sustained remission. The predominant mechanism of imatinib resistance is the occurrence of point mutations in the ABL kinase domain, particularly the T315I gatekeeper mutation, and a number of other mutations, such as Y253H, E255K, M351T, and G250E. In addition, BCR-ABL overexpression also holds great importance during the resistance of BCR-ABL TKIs.107,108 BCR-ABL is also a typical client of the Hsp90 chaperone, and its mutants were reported to be more dependent on Hsp90. Early in 2002, Sawyer’s group tested the antiproliferative effect of Hsp90 inhibitors 1 and 2 against hematopoietic cells expressing wild-type BCR-ABL and two imatinib-resistant BCR-ABL mutants. They found that both 1 and 2 were 2−5fold more potent in these cells (∼1−2 μM) than that in cells expressing the wild-type BCR-ABL (∼5 μM).109 Similarly, Li’s group transplanted bone marrow cells expressing both wild-type BCR-ABL and the T315I gatekeeper mutant into the same recipient mice and treated them with imatinib (100 mg/kg, twice a day) and the Hsp90 inhibitor 4 (50 mg/kg, once every 2 days). The result showed that cells expressing the gatekeeper mutation became dominant (81.5%) in the imatinib-treated mice after 15 days, while they were negligible (0.5%) in the 4-treated mice after 28 days, indicating 5576



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growth inhibition against all of the resistant cells and downregulation of the total AKT levels were observed. Meanwhile, 4 significantly inhibited tumor growth in both trastuzumab-sensitive BT474 and trastuzumab-resistant BT474R xenografts. These results correlate well with the observed decrease in the levels of HER2, p-AKT, and pMAPKs.118 Recently, Wainberg’s group treated 16 human gastric and 45 breast cancer cell lines with the Hsp90 inhibitor 7 at various concentrations. They found that the HER2-amplified cells were much more sensitive to the inhibitor. HER2, p-HER2, and AKT were down-regulated by 18 h with treatment of 7 at 100 nM in two gastric HER2-amplified cells. HER2 signaling through MAPK and AKT was also inhibited. Furthermore, the combination of 7 (0.58 μg/mL) with trastuzumab (2.5 μg/ mL) was synergistic against HER2-amplified, trastuzumabresistant breast and gastric cells, and in HER2-amplified, trastuzumab-resistant NCI-N87-TRC-bearing gastric cancer xenografts.119 2. Clinical Advance of Hsp90 Inhibitors in the Battle against HER2 TKI Resistance in Patients. Early in 2005, a phase II trial of the Hsp90 inhibitor 2 in combination with trastuzumab was conducted in patients with HER2-positive metastatic breast cancer (MBC). Of the 20 evaluated patients, five showed PR, two showed minor responses, and four showed extended SD. In 2012, Modi and colleagues reported an encouraging phase II study of 2 with trastuzumab in 31 patients bearing advanced trastuzumab-refractory HER2-positive breast cancer. The ORR is 22%, and the clinical benefit rate (CR + PR + SD) is 59%. The median PFS and median OS (overall survival) are 6 and 17 months, respectively. The most common toxicities, largely grade 1, were diarrhea, fatigue, nausea, and headache. This result indicated that the combination of 2 with trastuzumab possesses significant anticancer activity in HER2positive MBC previously progressing on trastuzumab.120 In a phase I trial of 3 in combination with trastuzumab in patients with HER2-positive MBC, eight (42%) of the 19 evaluated patients showed the expected clinical benefit. A similar phase I trial of 3 in combination with trastuzumab was reported in 2012.121 Of 28 HER2-positive MBC patients, 6.25% ORR and 18.75% SD were observed. Despite the observed antitumor activity, ocular toxicity emerged as a major concern. A phase II trial of 4 in combination with trastuzumab in patients with HER2-positive MBC was also reported.122 5% ORR and 70% SD were observed in heavily pretreated patients previously exposed to trastuzumab. No significant hepatic toxicity was observed that warranted this combination for further evaluation. In 2014, a phase II trial of 6 in an unselected cohort of patients with MBC was reported. Twenty-two patients were enrolled, and most of them had at least two previous lines of chemotherapy in the metastatic setting. Most common toxicities, largely grade 1/2, were diarrhea, fatigue, nausea, and hypersensitivity reaction. 15% OR was seen from the subset patients with HER2-positive MBC. The study did not meet the prespecified criteria for ORR. However, antitumor activity was observed in trastuzumab-refractory HER2-positive patients.123 In a phase II trial of 7 in combination with trastuzumab to treat advanced NSCLC patients with HER2 overexpression or amplification or mutation, seven of 31 patients had PR and 16 had SD.124 4.2.6. Application of Hsp90 Inhibitors to Overcome JAK TKI-Induced Drug Resistance. Janus kinase (JAK) is a family of

that the mutant BCR-ABL protein was more sensitive to the Hsp90 inhibitor.110,111 In 2015, Wu and co-workers reported that the curcumin derivative 17 (C086, Figure 9) could potently inhibit the proliferation of imatinib-resistant CML cells through dual suppression of BCR-ABL kinase activity and Hsp90 chaperone function. This compound was highly potent not only against wild-type and mutant BCR-ABL kinases but also against the proliferation of both imatinib-sensitive and -resistant CML cells.112 To gain a deeper insight into how the Hsp90 inhibitor precisely down-regulates the expression of BCR-ABL, Arlinghaus’s group identified a high molecular weight network complex (HMWNC) of signaling molecules in BCR-ABLpositive cells.113 This HMWNC contains Hsp90 and many of its clients, including BCR-ABL, JAK2, STAT3, and AKT, and Hsp90 was directly bound to these clients. Later, they treated BCL-ABL-positive 32Dp210 cells containing the HMWNC with 7 (0.5 μM) and found that the expression of BCR-ABL dramatically decreased within 1 h in a time-dependent manner, while the degradation of other kinases (e.g., JAK2, AKT) was much slower, indicating that the stabilization of BCR-ABL is more dependent on Hsp90. Meanwhile, after treatment with 10 nM of 7 for 16 h, the levels of mutated BCR-ABL in the resistant cells (T315I, E255K, and F359V) were dramatically decreased, while the wild-type BCR-ABL was only modestly reduced, suggesting that the mutated BCR-ABL is more sensitive to the Hsp90 inhibitors.114 Although a few Hsp90 inhibitors have been tested in the clinic in CML patients harboring BCR-ABL mutations, no objective responses have been reported. Therefore, the fate of the Hsp90 inhibitors in the battle against BCR-ABL TKIinduced drug resistance is still under investigation and requires more well-designed clinical investigations.80 4.2.5. Hsp90 Inhibitors Overcome Drug Resistance to HER2 TKIs. 1. Application of Hsp90 Inhibitors to Overcome HER2 TKI-Induced Drug Resistance. HER2 amplification has been demonstrated in up to 30% of breast cancers and is an important oncogene for tumor initiation and progression. Trastuzumab, a humanized monoclonal HER2 antibody inhibitor, is currently the main first-line therapy for HER2overexpressing breast cancer patients; however, its widespread and long-term application is limited by the intrinsic and acquired resistance. Several mechanisms have been proposed to underlie the trastuzumab-induced resistance, including the expression of p95-HER2, an N-terminally truncated form of HER2 that has lost the trastuzumab binding epitope, HER2 gene amplification, activation of parallel or/and downstream signaling pathways, and defects in the apoptosis pathway in tumor cells.115,116 In 2009, Rosen’s group treated p95-HER2-expressing T47D cells with the Hsp90 inhibitor 12 (1 μM for 3 h or 0.1 μM for 6 h) and found a marked reduction in the expression of HER2, p95-HER2, p-HER2, and p-p95-HER2. This result indicated that both HER2 and p95-HER2 are sensitive client proteins of Hsp90. Subsequent treatment with 13, an oral prodrug of 12, three times per week resulted in complete tumor growth inhibition over two weeks in the p95-HER2-addicted xenograft mice.117 In addition, Baselga and co-workers assessed the antiproliferative effects of Hsp90 inhibitor 4 in the HER2-overexpressing, trastuzumab-resistant BT474R, SKBR-3R, and BT474H1047R (BT474 cells bearing H1047R mutation) cells. Dose-dependent 5577



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KIT A822L drug-resistant mutation.132 Kiyoi’s group also found that 2 significantly inhibited the colony formation of D816V-KIT-expressing AML cells that was resistant to KIT inhibitors.133 In addition, 2 (500 nM) markedly reduced p-KIT and total KIT expression and inactivated KIT signaling intermediates such as AKT and MPK in imatinib-resistant, KIT-dependent GIST430 and GIST48 cells but not in imatinibresistant, KIT-independent GIST62 cells.134 Recently, Smyth’s group tested the antiproliferative potency of the Hsp90 inhibitor 8 against imatinib-resistant GIST430 and GIST48 cells. The corresponding IC50 values were 34 and 55 nM, respectively. The results were well correlated with the observed depletions of KIT, p-KIT, AKT, and p-AKT. At the weekly dose of 70 mg/kg, 8 exhibited tumor growth inhibition against imatinib-resistant GIST430 xenograft models in vivo.135 PDGFRΔDIM842−844 and PDGFRD842V are the two GISTrelated PDGFR mutations. Debiec-Rychter and co-workers found that the Hsp90 inhibitor 4 exhibited 10-fold higher potency against the imatinib resistant PDGFRD842V (IC50: 62 vs 642 nM).136 Despite the significant preclinical effects of Hsp90 inhibitors in the treatment of drug resistance caused by KIT or PDGFR inhibitors, no clinical settings have been reported yet.

intracellular, nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. Genetic alternations of JAK2 (e.g., V617F and R683G mutations) have been identified in several diseases, especially in cancer and rheumatoid arthritis. The first JAK1/2 inhibitor, ruxolitinib, received the FDA’s approval in 2011 for the treatment of intermediate to high-risk myelofibrosis (MF) patients. Acquired resistance to ruxolitinib has also been identified, and the underlying mechanisms include stabilization of activated JAK2, increased JAK2 mRNA expression, and heterodimerization of activated JAK2 with JAK1 or TYK2.125 Early in 2007, Albitar’s group reported that the Hsp90 inhibitor 2 resulted in a reduction of p-JAK2, total JAK2, pSTAT5, total STAT5, and total AKT in HEL cells with a homozygous V617F JAK2 mutation. The cell viability was reduced to a much greater extent than that in cells treated with the JAK2-specific inhibitor or the pan-JAK inhibitor.126 A study on the treatment of the Hsp90 inhibitor 7 with the JAK2 inhibitor N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yl amino)benzenesulfonamide (TG101209) in the JAK2 TKI-resistant cells (HEL/TGR and UKE-1/TGR) indicated that 7 was synergistic with the JAK2 inhibitor and the combination treatment effectively suppressed the proliferation of JAK2resistant cells. This result correlates well with the observed reductions of p-JAK2, p-STAT5, p-AKT, and Bcl-xl in these two resistant cell lines after treatment with 7 (50 nM, 24 h) alone.127 Similar to the resistance to other TKIs, secondary mutation is an important mechanism underlying the resistance to JAK2 inhibitors. To this end, the Weigert group generated three synthetic mutations in the JAK2 kinase domain (G935R, Y931C, and E864K) together with clinically relevant JAK2 mutations (V617F or R683G). They found that binding of JAK2 inhibitors in the ATP binding site was interrupted in these mutants, leading to much reduced potency.128 However, the Hsp90 inhibitors 2 and 7 both showed high cytotoxicity in Ba/F3EpoR cells with a JAK2 V617F mutation and in Ba/ F3CRLF2 cells with a JAK2 R683G mutation. Compared to cells with no secondary mutations, 7 showed greater potency against cells harboring Y931C or E864K mutations.129 In addition, both the JAK2-inhibitor-persistent (JAK2Per) and parental cells were reported equally sensitive to the Hsp90 inhibitor 9, and treatment with this inhibitor led to degradation of JAK2 and blockade of the subsequent signaling in the JAK2Per cells.128 4.2.7. Hsp90 Inhibitors Overcome Acquired Resistance to KIT and PDGFR TKIs. The tyrosine kinases KIT and PDGFR (platelet-derived growth factor receptor) are constitutively activated in over 90% of gastrointestinal stromal tumors (GISTs) due to gain-of-function mutations. Therapeutic inhibition of KIT and/or PDGFR by small molecules such as imatinib has achieved objective responses in most GIST patients. However, nearly 10−20% GIST patients have been reported to exhibit primary resistance to imatinib, and patients who initially responded to imatinib often manifested resistant secondary mutations within ∼2−5 years. Because both KIT and PDGFR are oncogenic clients of the Hsp90 chaperone, Hsp90 inhibition may provide an alternative therapeutic strategy to treat imatinib-resistant GIST patients.130,131 In 2005, Wang and co-workers revealed that the Hsp90 inhibitor 2 (5 μM, 72 h) induced apoptosis and a significant decrease of the KIT protein level in Kasumi-1 cells harboring a

5. CONCLUSION AND PERSPECTIVES Hsp90 is an ATP-dependent molecular chaperone that comprises 1−2% of the total cellular protein content and regulates the correct folding, activation, stability, and functions of numerous regulatory and signaling proteins. In cancer cells, Hsp90 is constitutively expressed at much higher levels and shows elevated ATPase activity compared to normal cells. Under the stress of cancer, Hsp90 forms highly susceptible complexes with its clients under the assistance of cochaperones. Hsp90 inhibitors have been reported, with over 100-fold higher aggregation in cancer cells than in normal cells. Therefore, Hsp90 has been a viable molecular target for cancer drugs and at least 13 selective Hsp90 inhibitors have been or are being exploited in a large number of clinical trials to combat a wide spectrum of tumor types. All these inhibitors belong to the Nterminal Hsp90 inhibitors, including geldanamycin analogues, resorcinol derivatives, and 2-aminobenzamide analogues. Unfortunately, this rapid growth and widely advancing approach has not successfully brought one Hsp90 inhibitor to the market. The major hurdles for the failure of current Hsp90 inhibitors are likely due to the nonoptimal safety profiles and dose-limited insufficient clinical efficacy. Hsp90 exerts its therapeutical effects through formation of supercomplexes with its clients and cochaperones. Recently, much progress has been achieved in the study of the structure and function of the Hsp90−cochaperone−client complex. The recognition of Hsp90 by its kinase clients is dependent on the cochaperone CDC37, and the interactions of Hsp90 with its kinase clients are determined by the thermal and conformational stability of the client proteins. From the available X-ray structures of Hsp90−cochaperone (CDC37, Aha1, p23) complexes, it is proposed that the binding of the cochaperone to Hsp90 often causes conformational changes in Hsp90, and the binding interface of the complex is the functional interface. Unfortunately, no crystal structures of Hsp90 with its kinase clients have been reported, which largely restricts the study of the relationship between the Hsp90−kinase interactions and the anticancer efficacy of Hsp90 inhibitors. From the limited reports on the electron microscopy and NMR studies on the Hsp90−CDC37−CDK4 complexes, it is believed that Hsp90 5578



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would benefit most from a specific Hsp90 inhibitor. Innovative strategies (e.g., biomarker, therapeutic indicators, et al.) to stratify patients are highly needed. For a subportion of patients with a specific overexpressed kinase who are resistant to the corresponding kinase inhibitors, Hsp90 inhibitors have been proposed to exert antiresistant efficacies through the Hsp90− cochaperone−kinase complex, but the exact mechanism is not clear. New Hsp90 inhibitors that are designed based on the interaction complex and selectively targeting the interface of the Hsp90−client complex will be extremely useful. Second, to what extent Hsp90 should be inhibited because complete inhibition of Hsp90 eventually leads to the degradation of many client kinases that may cause toxicity. Meanwhile, is there any information related to the level of kinase sensitivity to Hsp90 (EML4-ALKMut > EML4-ALK > HER2 > EGFRMut > Raf-1 > Akt > BRAFmut > EGFR)? Finally, Hsp90 isoform selectivity has recently been reported to correlate with the toxicity of Hsp90 inhibitors.162 Is this also an essential issue for Hsp90 inhibitors to overcome TKI’s resistance? Therefore, there is still a long way to go to develop safe and effective Hsp90 inhibitors as a therapeutic treatment to overcome the TKI-induced acquired resistance.

binds to a broad region of the client through a large binding interface, which may allow more specific recognition by many kinases, including their mutants. This result may guide the computer-aided rational drug design of new inhibitors to target the highly susceptive Hsp90−cochaperone−client supercomplexes by selectively binding to the interaction interface. Although the final fate of Hsp90 inhibitors as monotherapy require more well-designed multidimensional clinical trials, many new attempts have been conducted on the application of Hsp90 inhibitors to combat drug resistance resulting from treatment with tyrosine kinase inhibitors (TKIs), either alone or in combination. This approach has not only renewed and redirected the interest in targeting Hsp90 for cancer therapy but has also opened a new avenue for design and development of Hsp90 inhibitors to fight acquired resistance, the major challenge surrounding TKIs. The application of Hsp90 inhibitors as a new strategy to combat drug resistance of TKIs is based on the fact that all of the oncogenic tyrosine kinases (TK), including their natural forms, resistant mutants, or other activated forms (fusion, translocations, rearrangements), are the clients of Hsp90. These clients form Hsp90−cochaperone−client supercomplexes that are highly sensitive to Hsp90 inhibitors. Hsp90 inhibition prevents Hsp90 from chaperoning these kinase clients, thus leading to destabilization of the kinases and their eventual degradation by proteasomes. More importantly, secondary mutations in the TKs (kinasemut) generally underlie the TKIinduced resistance of tumor cells, and the Hsp90−cochaperone−kinasemut complexes are even more sensitive to and dependent on Hsp90. Therefore, numerous preclinical and clinical studies have been conducted and the results showed that Hsp90 inhibitors are more efficacious against mutated EGFR, amplified HER2, or fused EML4-ALK than the corresponding specific TKIs, and also highly effective against the TKI-induced acquired resistance. The most promising results were obtained from studies using Hsp90 inhibitors to treat the acquired resistance after treatment with crizotinib, an inhibitor of EML4-ALK. The mechanism of crizotinib resistance is rather complicated, including secondary mutations, such as the L1196M gatekeeper mutation and many other point mutations, alternative kinase activations, such as cMet, EGFR, and c-Kit, as well as many unknown reasons. Although two next-generation ALK inhibitors (alectinib and ceritinib) have recently been launched to address the mechanism of crizotinib resistance driven by secondary mutations (particularly L1196M), they are ineffective to address the large portion of drug-resistant patients with gene amplifications, alternative pathway activations, and other mechanisms of resistance. Appreciably, Hsp90 inhibitors, both geldanamycin analogues 2−4 and resorcinol derivatives 6−8, were ubiquitously effective against nearly all cases of crizotinibinduced resistance. In a phase II trial with the Hsp90 inhibitor 6, significant tumor shrinkage was observed after treatment of 6 in NSCLC patients who had ALK-positive cancer progression when treated with crizotinib. This drug is now under more clinical trials in combination with either crizotinib or the new inhibitor ceritinib in resistant patients. Despite of the above-mentioned progress in clinical applications of Hsp90 inhibitors to fight TKI resistance and in the study of the Hsp90−cochaperone−client complex, we must be aware that several critical questions on Hsp90 inhibitors still need to be addressed that have lingered for decades. First, there is still no clue on which cancer patients

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AUTHOR INFORMATION

Corresponding Authors

*For Ao Zhang: phone/fax, 86-21-50806035; e-mail, aozhang@ simm.ac.cn. *For Meiyu Geng: phone/fax, 86-21-50806072; e-mail, [email protected]. *For Liping Sun: phone/fax, 86-25-83301606; e-mail, lpsun@ simm.ac.cn. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Meining Wang received her B.S. in Medicine in 2010 from China Pharmaceutical University, China, and then joined Professor Ao Zhang’s research group at Shanghai Institute of Materia Medica as a doctoral student in Medicinal Chemistry. Her dissertation research focused on the synthesis of natural product-based analogues and evaluation of their pharmacological properties. She received her doctorate in 2015 and is currently doing postdoctoral research under supervision of Professor Kenner C. Rice at the National Institute on Drug Abuse in the United States. Aijun Shen received his doctorate in Pharmacology at Shanghai Institute of Materia Medica, Chinese Academy of Sciences, in 2011. After that, he joined Professor Mei-yu Geng’s research group at the same institute. He is now the Assistant Professor of Pharmacology, and his research topic focuses on the biomarker discovery for tyrosine kinase inhibitors and the exploration of molecular mechanisms of Hsp90 inhibitors. Chi Zhang received his M.S. in Medicine in 2012 from China Pharmaceutical University (CPU), China. He is currently a joint doctoral student in Medicinal Chemistry under the supervision of both Professor Li-Ping Sun at CPU and Professor Ao Zhang at the Shanghai Institute of Materia Medica. His dissertation research focuses on the synthesis of natural product-based analogues and evaluation of their pharmacological properties. Zilan Song received her B.S. degree in 2005 from Shanxi Medical University. She was awarded the M.S. degree in Medicinal Chemistry 5579



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received the Hundred Talent Project award from the Chinese Academy of Sciences and became the Professor of Medicinal Chemistry at Shanghai Institute of Materia Medica (SIMM). In 2011, he was awarded the Distinguished Young Investigator Award from Chinese Natural Science Foundation. He has coauthored more than 100 original articles and reviews. His research interests include the design and synthesis of novel small molecules as structural and functional probes for the diagnosis and treatment of brain disorders and cancers.

in 2008 from the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. After that, she joined Professor Ao Zhang’s research group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. She is now the Assistant Professor of Medicinal Chemistry, and her research topic focuses on the design and synthesis of novel small molecules targeting tyrosine kinases. Jing Ai received her doctorate from the Ocean University of China in 2009. After that, she joined Professor Meiyu Geng’s research group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. She is now the Associate Professor of Antitumor Pharmacology. Her research interest is mainly focused on the study of molecular targeting drug discovery and tumor biology, especially the mechanism of inflammatory carcinoma malignant transformation and tumor metastasis, identification, and characterization of tyrosine kinase inhibitors, as well as the development of their biomarkers.

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ACKNOWLEDGMENTS This work was supported by grants from Chinese NSF (81430080, 81125021, 81373277, 81473243, 81321092) and the Major State Basic Research Development Program (2015CB910603). Seeding grants from SIMM (CASIMM0120154002/2002) were also appreciated.

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Hongchun Liu received her B.S. degree in Pharmacy in 2004 and doctorate in Pharmaceutical Chemistry in 2010 from the Ocean University of China. After that, she joined Professor Jian Ding’s research group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. She is now the Senior Engineer of Antitumor Pharmacology, and her research topic focuses on the pharmacological evaluation of small molecules targeting HSP90 or HDACs.

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Li-Ping Sun received her doctorate in Organic Chemistry at Hongkong University of Science and Technology in 2004. After that, she did postdoctoral research at the National University of Singapore. Since 2006, she has been the Professor in Medicinal Chemistry at the China Pharmaceutical University in Jiangsu, China. She has published a number of research papers and expert reviews in the field of drug design and development. Her research interest focuses on design, synthesis, and pharmacological evaluation of small molecules. Jian Ding received his doctorate in Medicine at the National Kyushu University in Japan in 1991. Since 1995, he has been a Professor of Pharmacology at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. He was the Director of Shanghai Institute of Materia Medica between 2004 and 2014 and was elected as an Academician of the Chinese Academy of Engineering in 2009. He has published more than 240 research articles in prestigious international journals. He is a receipt of numerous prestigious national awards, including the National Natural Science Award and a few other major technology awards. Dr. Ding’s scientific research has integrated drug discovery, basic research of cancer biology, and translational research. Mei-Yu Geng received her Ph.D. in Pharmacology in 1997 from Tokyo University and set her laboratory of Molecular Pharmacology in the Ocean University of China as a Professor. She was selected as “Hundred Talents Program” of the Chinese Academy of Sciences and became a Principle Investigator of Antitumor Pharmacology of Shanghai Institute of Materia Medica in 2006. She received a number of awards including “National Science Foundation for Distinguished Young Scholars”, “National Award for Technological Invention”, and “Outstanding Academic Leader of Shanghai”. She has published more than 130 research articles and reviews in SCI-cited journals. Her interest centers on the research and development of targeted molecular inhibitors and deciphering their molecular mechanisms of cross talks in signal transduction. Ao Zhang received his doctorate in Organic Chemistry in 2000 and did postdoctoral research in Medicinal Chemistry during 2001−2004 at Georgetown University Medical Center and Harvard Medical School McLean Hospital. In 2004, he was promoted to Research Investigator and Instructor of Harvard Medical School. In 2006, he 5580



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