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The geldanamycin derivative 17-allyamino-17-demethoxygeldanamycin (17-AAG) is a clinical stage ATP-competitive HSP90 inhibitor that induces degradation of HSP90 client proteins. 17-AAG contains 1 ansamycin moiety and is highly potent in conventional cell killing assays. Since active Hsp90 exists as a dimer, we hypothesized that dimeric compounds containing 2 ansamycin pharmacophores might inhibit Hsp90 function more efficiently than 17-AAG. Here, we show that monomeric and dimeric ansamycins exert their activity in distinct ways. Under conditions of continuous exposure, 17-AAG induced client degradation and cell growth inhibition more readily than the dimeric drugs CF237 and CF483. By contrast, 24 hr treatment of various tumor cells with 17-AAG followed by drug washout caused temporary client degradation and cell cycle arrest but minimal cell death, whereas both dimers induced massive apoptosis. CF237 remained bound to Hsp90 for days after drug withdrawal and, while both monomeric and dimeric compounds caused accumulation of the inactive intermediate Hsp90 complex, this effect disappeared following washout of 17-AAG but not CF237. The dimer was also retained for longer in tumor xenografts and displayed superior antitumor activity in vivo. These results indicate that monomeric and dimeric Hsp90 inhibitors have distinct biological profiles and work differentially toward target inhibition. © 2006 Wiley-Liss, Inc.
Heat shock protein 90 (HSP90) is a conserved molecular chaperone that mediates the maturation and stability of a set of cancer-associated proteins, referred to as ‘clients’. These include steroid receptors, EGFR family members, MET, Raf-1 kinase, AKT, Bcr-abl, mutant p53, CDK4 and many other oncogenic molecules.1, 2, 3 Hsp90 functions as a super-chaperone complex in association with various cochaperone proteins. Hsp90 mainly exists in 2 types of multiprotein complexes, referred to as ‘intermediate’ and ‘mature’.3, 4 In the intermediate complex, which is the ADP-binding form, the major cochaperones are Hsp70, Hsp40, HOP and HIP. Upon ATP binding, cdc37, p23 and immunophilins replace the original cochaperones to assist the conformational maturation of the client proteins and maintain those proteins in an active state to exert their function.4 It is thought that Hsp90 inhibitors bind to and stabilize the intermediate complex, leading to recruitment of ubiquitin ligases and degradation of client proteins in the proteasome.5, 6
Ansamycin antibiotics such as geldanamycin (GM) are natural products that bind to the N-terminal ATP/ADP binding pocket of HSP90.5, 6, 7, 8 Exposure of cells to these compounds induces the degradation of a range of HSP90 clients and results in cell cycle arrest followed in some cases by apoptotic cell death.9 The GM derivative 17-allylaminogeldanamycin (17-AAG) was the first HSP90 inhibitor to enter clinical trials. The drug is well tolerated, despite the fact that it simultaneously targets many intracellular signaling proteins.10, 11
Although 17-AAG was found to be highly potent in most tumor cell lines examined in vitro, and in a variety of tumors in vivo,2, 12, 13, 14 we also noticed that 17-AAG showed limited longevity of its effects on target proteins and lost antiproliferative activity precipitously under conditions of brief cellular exposure. In a quest for ansamycin drugs with properties differing from 17-AAG, we synthesized over 400 novel semisynthetic analogues, including several dozen dimmers, and investigated their structure–activity relationships. Geldanamycin dimers were originally reported by Zheng et al.15 These early compounds were relatively weak Hsp90 inhibitors that primarily induced the degradation of the most sensitive Hsp90 client, HER-2. Since active Hsp90 is an obligate dimer and the 2 ATP binding sites are brought into close apposition during the conformationally driven ATPase cycle,16 we reasoned that 2 GM moieties separated by an optimized flexible linker might be able to engage both ATP sites simultaneously. In this case, the resulting avidity might render the binding to activated Hsp9017 operationally irreversible because both GM molecules would need to dissociate at the same instant. Here, we have characterized 2 highly active GM dimers, CF237 and CF483. As predicted from their structures, both drugs bind Hsp90 with high affinity. Furthermore, binding of CF237 to Hsp90 in cells was found to be extremely long-lived, and the compound locked Hsp90 into the nonproductive intermediate complex for days after drug withdrawal. By contrast, the biochemical and cellular effects of 17-AAG were transient. In cells, the advantages of stable binding to the target were revealed when cellular activities of monomeric and dimeric inhibitors were studied in a variety of tumor cells after continuous and brief exposure. In the continuous exposure paradigm, 17-AAG was more efficient than the dimers at causing degradation of client proteins, including HER family kinases, signal transducers involved in PI-3k/Akt and Raf/MEK signal transduction pathways and cell cycle regulators, leading ultimately to tumor cell death. In contrast, under the conditions of brief exposure CF237 and CF483 were more active in killing tumor cells. Dimer-treated cells displayed markedly reduced client protein levels for at least 48 hr after drug removal, but 17-AAG induced only transient suppression of the same clients and the pathways they control. Similarly, the potency of 17-AAG in growth inhibition and apoptosis assays was significantly reduced in a panel of tumor lines under brief exposure conditions, but that of CF237 and CF483 remained largely intact. These data indicate that the novel dimeric ansamycins CF237 and CF283 have distinct cellular properties compared to monomeric 17-AAG and may represent an alternative or complementary strategy to 17-AAG for cancer therapy. The dimer was also retained for longer in tumor xenografts and displayed superior antitumor activity in vivo.
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- Material and methods
It is becoming increasingly clear that targeting a single oncoprotein in cancer therapy may not be sufficient to obtain strong efficacy.27, 28, 29 This has brought Hsp90 center stage among experimental therapeutics because inhibition of Hsp90 causes simultaneous degradation of multiple oncogenic proteins and affects a number of signal transduction pathways that are important for cancer cell proliferation and survival.1, 2, 3 The semisynthetic geldanamycin derivative 17-AAG has completed Phase I clinical trials with encouraging results.10, 30 The compound is well tolerated and several long-lasting disease stabilizations were noted in heavily pretreated patients.10, 30 Despite this, 17-AAG has some limitations among ansamycin drugs. Although extremely potent (similar to the parent compound) in most cell lines,12, 13, 14 17-AAG, unlike geldanamycin, has limited activity against some clinically important subsets of tumor cells, including those defective in Rb signaling31, 32 and those overexpressing Bcl-2.33 Antiapoptotic Bcl-2 family proteins serve to retard commitment to apoptosis in stressed cells, raising the possibility that 17-AAG fails to inhibit Hsp90 for long enough to allow Bcl-2-overexpressing tumor cells to commit to apoptosis, even when continuously present. In that case, it would be predicted that the drug would rapidly lose apparent potency in all cells when the duration of drug exposure is limited. The data in Figures 2 and 3 show that the effects of 17-AAG on client protein expression decay rapidly after drug washout and the cytotoxic potency of the drug is markedly attenuated under brief exposure conditions relative to when the compound is present throughout the 5 day assay. One explanation for these results is that 17-AAG rapidly dissociates from its target, diffuses out of the cell and loses potency if it cannot be replenished from the extracellular environment. Since activated Hsp90 functions as dimer in the multichaperone complex,16 it seemed plausible that dimeric ansamycins might display radically reduced dissociation rates, as both arms would have to dissociate concomitantly from the binding pockets to release bound drug, assuming that either both ATP sites of a single dimer or one ATP site from 2 individual Hsp90 dimers could be bound by a single molecule of a dimeric drug. As predicted from this model, 2 dimeric Hsp90 inhibitors, CF237 and CF483, appeared to interact much more stably with Hsp90 than did 17-AAG. When cells were exposed to monomeric or dimeric ansamycins for 24 hr prior to drug washout and assays were performed 48 hr later, the dimeric compounds (but not 17-AAG) were found to (i) remain physically associated with the target, (ii) stabilize the nonproductive HOP complex, (iii) prevent reappearance of client proteins and (iv) retain potent growth inhibitory and proapoptotic activity. Client protein levels also rebounded quickly in cells treated with another ansamycin monomer, 17-DMAG, suggesting that 17-AAG is not unique in this regard. A recent study by Guo et al.34 indicates that monomeric ansamycins such as 17-AAG are reduced by the enzyme NQO1 to dihydro-17-AAG in cells and that this metabolite has increased binding affinity and intracellular retention. It is possible that NQO1-mediated activation of 17-AAG might underlie its superior activity at shorter exposure times. Although the structure of CF237 and CF483 does not preclude their similar activation, we have not observed diminished activity in NQO1-deficient lines. The effect of this metabolism may be masked by the increased affinity/avidity and consequent heightened intracellular retention of the dimeric compounds because of their extremely stable binding mode.
Not all ansamycin dimers were potent Hsp90 inhibitors. As shown in Table I, dimers with linkers of various lengths, structure and flexibility were synthesized, but no obvious features conferring potency were discernable. Among the compounds shown in Table I, CF18 and CF24 were originally described by Zheng et al.15 These dimers were quite potent against the sensitive client HER-2 (IC50 ∼ 60 nM) but had little effect on other Hsp90 clients. Since the dynamic, activated Hsp90 found in multichaperone complexes in cells cannot by crystallized by current methods, it was not possible to design an optimal linker by structural approaches or to confirm divalent binding of CF237 to a single activated Hsp90 dimer. However, the observation that the second moiety attached to a GM-linker intermediate profoundly affected potency is provocative. When smaller, cell permeable entities such as testosterone (CF124), estradiol (CF125) and raloxifene (CF691, CF692) were substituted for the second GM, activity in the HER-2 degradation assay decreased around 15-fold, indicating that the second GM most probably interacts with a high-affinity binding site on Hsp90.
Highly active dimers were superior to their monomeric counterparts under conditions of limited drug exposure, a situation that pertains in clinical cancer therapy, but 17-AAG may also possess some significant advantages in vivo. Monomers are smaller and more compact, likely increasing their ‘on’ rates when binding to Hsp90, which may explain the more rapid onset of client protein degradation and superior potency of monomers under conditions of continuous exposure (Figs. 1 and 2a). Furthermore, the greater mass of dimeric compounds (∼1,200 kDa) would likely retard their permeation through the tightly packed cells and fibrous stroma that characterize solid tumor masses.35 Similarly, the slow dissociation of dimers from their target would not favor penetration of the drug to distant extravascular sites. Thus, dimeric Hsp90 inhibitors might be most efficacious in leukemia, where limitations of drug mobility are less critical. Nonetheless, geldanamycin dimers are active against solid tumor xenografts. We have recently reported that a related dimeric ansamycin, termed EC5, shows good activity against head and neck cancer and retained potent activity in one particular cell line, JHU12, that was resistant to 17-AAG because of defects in Rb signaling.36
In this report, we have compared the biochemical, pharmacodynamic and cytotoxic properties of monomeric and dimeric ansamycin Hsp90 inhibitors in vitro and in vivo. The dimers have improved activity under conditions of brief drug exposure, probably because they form extremely stable, high avidity binding interactions with their dimeric target protein. To our knowledge, this is the first report of optimized bivalent drugs for cancer therapy, but a growing body of work suggests that bivalent and multivalent drugs can offer significant advantages over their monovalent analogues in other areas of medicine. Griffin et al. described optimization of a series of vancomycin dimers that show improved biological activity against susceptible and drug-resistant gram-positive bacteria.37 Both potency and duration of action were enhanced relative to the parent monomer.38 Although the most advanced studies with dimeric drugs involve antibiotics, bivalent modulators of mammalian targets have been reported, most notably longer-acting agonists of the dimerizing G-protein-coupled β2-adrenergic receptor.38 The binding of high-affinity dimeric Hsp90 inhibitors to their target may be essentially irreversible, but these compounds do not form covalent adducts with the Hsp90. Conventional, chemically reactive irreversible inhibitors have several inherent drawbacks, including poor solubility due to their reactivity with water,39 poor tolerability due to their reactivity with nontarget proteins and clinical management problems because their effects cannot be rapidly reversed by cessation of dosing. The approach described here, which may be applicable to other dimeric or oligomeric drug targets, does not suffer from the first 2 of these limitations. Indeed, the dimeric ansamycin was well tolerated at effective doses in xenograft studies.
In conclusion, we have shown that monomer and dimer Hsp90 inhibitors of the same structural class interact differently with the target and each possesses theoretical advantages and disadvantages for cancer treatment. The preferred approach will have to be determined clinically, but it is attractive to speculate that combination therapy with both classes of inhibitor might provide the most effective method of attacking the diversity of tumor cell populations.