Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases

Transcription

1 Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases Robert A. Casero Jr* and Laurence J. Marton Abstract The polyamines spermidine and spermine and their diamine precursor putrescine are naturally occurring, polycationic alkylamines that are essential for eukaryotic cell growth. The requirement for and the metabolism of polyamines are frequently dysregulated in cancer and other hyperproliferative diseases, thus making polyamine function and metabolism attractive targets for therapeutic intervention. Recent advances in our understanding of polyamine function, metabolic regulation, and differences between normal cells and tumour cells with respect to polyamine biology, have reinforced the interest in this target-rich pathway for drug development. DC antizyme A mammalian protein that inhibits ornithine decarboxylase (DC) and downregulates polyamine transport, the translation of which is controlled by intracellular polyamine concentrations. *The Sidney Kimmel Comprehensive Cancer Center, Johns opkins University School of Medicine, Baltimore, Maryland 21231, USA. Cellgate, Redwood City, California 94065, USA. Correspondence to R.A.C. rcasero@jhmi.edu doi: /nrd2243 Published online 27 April 2007 Although simple in structure, the polycationic polyamines spermidine and spermine and their diamine precursor putrescine are essential factors for growth in eukaryotic cells 1 3 (FIG. 1). The total intracellular concentration of the polyamines is in the millimolar range; however, free polyamine concentrations are considerably lower, as they are mostly ionically bound to various anions in the cell including DA, RA, proteins and phospholipids. The original discovery of spermine dates back to Leuwenhoek in 1678, however the tools necessary to study the specific molecular functions in which the polyamines are intimately involved have only recently become available. Understanding the molecular functions of polyamines is complicated by the fact that most of the critical interactions in which the polyamines are involved are readily reversible ionic interactions. Among the roles that polyamines have in the support of cell growth, and in some cases survival, are association with nucleic acids, maintenance of chromatin conformation, regulation of specific gene expression, ion-channel regulation, maintenance of membrane stability, provision of a precursor in the synthesis of eukaryotic translation initiation factor 5A (IF5A), and free-radical scavenging Although several recent studies have been instrumental in furthering our understanding of the molecular functions of polyamines, the interest in polyamines and polyamine metabolism as targets for antiproliferative therapy is based on several earlier observations indicating that cells synthesize more polyamines when induced to grow; the polyamine biosynthetic enzymes are coordinately regulated with growth controls; polyamine metabolism is frequently dysregulated in cancers; and polyamines are essential for eukaryotic cell growth. Data indicating that the polyamine pathway is a downstream target for known oncogenes and that inhibition of polyamine synthesis disrupts the action of those genes when taken together with data indicating that the polyamines are critical to the activity of histone deacetylase inhibitors 16 add further strength to the utility of this target. The fact that polyamines appear to be a site of intervention that is distal and common to a number of validated targets makes this target even more intriguing, and validates the active pursuit of agents that interfere with both polyamine metabolism and function as a strategy for antiproliferative intervention (TABLE 1). Polyamine metabolism Biosynthesis. The first rate-limiting step in polyamine biosynthesis is the production of putrescine by the pyridoxal, phosphate-dependent decarboxylase ornithine decarboxylase (DC). The requirement for DC activity (and putrescine production) in cell proliferation has been demonstrated in all tissues studied. DC expression is tightly regulated at several steps from transcription to post-translational modification 17. DC is active as a homodimer with a half-life that is among the shortest of any known protein (10 30 minutes in mammalian systems), and, like many short-lived proteins, DC is destroyed by the 26S proteosome. owever, unlike most proteins, DC is not first ubiquitinated, instead a specialized regulatory protein, DC antizyme 18,19, ATURE REVIEWS DRUG DISCVERY VLUME 6 MAY ature Publishing Group

2 binds to the carboxylic end of the monomeric form of DC and presents the DC to the 26S proteosome for destruction DC transcription is another important regulatory step in its expression and DC was the first transcription target of the MYC oncogene to be reported 12,23 25, which demonstrates one link between two crucial growth-promoting pathways (BX 1). The second rate-limiting step in polyamine biosynthesis is catalysed by S-adenosylmethionine decarboxylase (AdoMetDC), a pyruvoyl-containing decarboxylase 26. AdoMetDC, like DC, is highly regulated and has a short half-life 27. Unlike DC, AdoMetDC is ubiquitinated before degradation by the 26S proteosome. Two specific aminopropyl transferases, spermidine synthase and spermine synthase, act sequentially to produce spermidine from putrescine, and spermine from spermidine and 5 -deoxy-5 -(methylthio)adenosine (MTA) The aminopropyl moiety, which is necessary for the synthesis of both spermidine and spermine, comes from decarboxylated S-adenosylmethionine. Both aminopropyl transferases are constitutively 2 2 C rnithine DFM DC MDL C 2 SAM486A 2 2 Putrescine APA C Ado C 3 S + 2 C AdoMetDC Ado C 3 S + Spermidine synthase 2 2 C 3 SAM486A Ado S C 3 MTA 2 Spermidine 2 SSAT MDL APA Exported from cell Ado C 3 S + C AdoMetDC Ado C 3 S C Spermine synthase 2 SM 2 C 3 Ado S C 3 MTA SSAT 5 -deoxy-5 - (methylthio)adenosine (MTA). A product of the aminopropyl transferase reaction. The frequent loss of this salvage enzyme in tumours presents itself as an excellent target for a reverse prodrug strategy, such that only tumour cells that are unable to inactivate the cytotoxic agent are targeted. 2 Spermine 2 Enzyme Inhibitor Figure 1 Targets in the polyamine metabolic pathway. rnithine decarboxylase (DC) generates putrescine and is the first rate-limiting step in polyamine biosynthesis. It has a short half-life, is regulated at multiple steps and is inhibited by the compound 2-difluoromethylornithine (DMF). Another rate-limiting enzyme in polyamine synthesis can be S-adenosylmethionine decarboxylase (AdoMetDC), the activity of which provides the aminopropyl donor for the synthesis of both spermidine and spermine, which can be blocked by the competitive inhibitor 4-amidinoindan-1-one-2 - amidinhydrazone (SAM486A). Spermine oxidase (SM) is an inducible oxidase that is involved in the cytotoxic response of cells to specific stimuli through the production of 2 2, and 1 -acetylpolyamine oxidase (APA) is a constitutively expressed, peroxisomal oxidase that is rate limited by the availability of 1 -acetylated polyamines; both of these enzymes can be inhibited by, 1 -bis( 2,3-butadienyl)-1,4-butanediamine (MDL 72527), an active-site-directed inhibitor. Spermidine/ spermine 1 -acetyltransferase (SSAT) is an inducible enzyme that is crucial for the maintenance of polyamine homeostasis and is also implicated in the cytotoxic activity of several polyamine analogues. MTA, 5 -deoxy-5 -(methylthio)adenosine. 374 MAY 2007 VLUME ature Publishing Group

3 expressed and are primarily regulated by the availability of their substrates. It is important to note that spermine is primarily a polyamine found in eukaryotic cells, as prokaryotes do not have a spermine synthase homologue. Catabolism. The first intracellular pathway of mammalian polyamine catabolism discovered is a twostep process, the rate of which is controlled by the activity of the inducible enzyme spermidine/spermine 1 -acetyltransferase (SSAT) SSAT catalyses the formation of 1 -acetylspermine or 1 -acetylspermidine by the transfer of the acetyl group from acetyl-coenzyme A to the 1 position of either spermidine or spermine. The second step in this pathway is the peroxisomal FAD-dependent enzyme 1 -acetylpolyamine oxidase (APA) APA is a constitutively expressed enzyme that catalyses the cleavage of acetylated polyamines to produce spermidine or spermine (depending on the starting substrate), 3-aceto-aminopropanal and 2 2. Through attempts to clone APA, we identified a gene and protein product that possessed many of its expected structural properties; however, the enzyme produced by this gene was found to be both highly inducible and able to efficiently oxidize unsubstituted spermine. We originally named the gene PAh1 for the first human polyamine oxidase to be cloned 42. Vujcic et al. 43 subsequently confirmed that this gene/enzyme represented a new addition to the polyamine catabolic pathway, and they named it spermine oxidase (SM; also known as SMX) 43. SM is a highly inducible FADdependent enzyme that oxidizes spermine to produce spermidine, 3-aminopropanal and 2 2 (REFS 44 46). Importantly, tumour-necrosis factor (TF)-induces Table 1 Representative agents targeting polyamines and polyamine metabolism Agent Biochemical effects Clinical trials 2-difluoromethylornithine (DFM) (enzyme inhibitor) Methylglyoxal bis(guanylhydrazone) (MGBG) (enzyme inhibitor) 4-amidinoindan-1-one- 2 -amidinhydrazone (SAM486A/CGP48664) (enzyme inhibitor), 1 -bis(2,3-butadienyl)- 1,4-butanediamine (MDL 72527) (enzyme inhibitor) 1, 11 - di(ethyl)norspermine (DESpm) (symmetrically substituted spermine analogue) 1 -cycloheptylmethyl- 11 -ethylnorspermine (CESpm) (asymmetrically substituted spermine analogue) CGC (conformationally restricted spermine analogue) CGC (oligoamine) Inhibits DC; decreases putrescine and spermidine; cytostatic in most cases; cytotoxic to specific tumour cell types Inhibits AdoMetDC; decreases spermidine and spermine; increases putrescine; competes with polyamines for uptake; inhibits cell growth; mitochondrial toxin Inhibits AdoMetDC; decreases spermidine and spermine; increases putrescine; inhibits cell growth; low mitochondrial toxicity Active-site-directed inhibitor of APA; also inhibits SM preventing the production of 2 2 and aldehydes by these two oxidases Depletes all three polyamines; competes with polyamines for uptake; inhibits cell growth and induces apoptotic cell death in specific tumor cell types; induces SSAT and SM Competes with polyamines for uptake; cytotoxic in several tumour cell types; induces G2/M cell-cycle blockade in association with altered tubulin polymerization; induces SM but not SSAT Induces SSAT and SM; decreases all three polyamines; competes with polyamines for uptake; effective against several human tumour models in vivo; anti-angiogenic in a macular degeneration model igh affinity for DA; competes with polyamines for uptake; cytotoxic to multiple tumour cell types at sub-micromolar concentrations; active in vivo against multiple human tumour models; downregulates ERα expression; inhibits SM activity Failed as a single agent in multiple clinical trials for cancer ; welltolerated and approved treatment for African sleeping sickness ; in clinical trials as a chemopreventive agent 1,82 ; showed promising results in combination therapy for brain tumours 75 Previous clinical trials in leukaemia 83, lymphoma and solid tumours; no current trials reported Phase I and II trials in multiple cancers ; partial responses reported against non- odgkin s lymphoma; no trials pending o clinical trials so far; possible lead compound as chemopreventive agent? Phase I and II trials completed ; well tolerated when administered as single daily doses for 5 days; no objective responses as a single agent yet; ongoing clinical trial in hepatoma; considered for combination trials o clinical trials so far Phase I and II trials in multiple cancers ongoing; well tolerated o clinical trials so far AdoMetDC, S-adenosylmethionine decarboxylase; APA, 1 -acetylpolyamine oxidase; ERα, oestrogen receptor-α (also known as ESR1); DC, ornithine decarboxylase; SM, spermine oxidase (also known as SMX); SSAT, spermidine/spermine 1 -acetyltransferase. ATURE REVIEWS DRUG DISCVERY VLUME 6 MAY ature Publishing Group

4 Box 1 MYC regulation of DC in carcinogenic transformation The most direct evidence that ornithine decarboxylase (DC) has a role in tumour development comes from multiple in vitro and in vivo DC-overexpression models. DC has been implicated as an oncogene through a series of studies demonstrating that its overexpression has the potential alone, and in combination with other oncogenes, to transform various cell types The relevance of these cell-based systems gained more significance after results from transgenic mouse models in which DC overexpression was limited to the skin through the use of a K6 promoter-driven DC were reported. These experiments demonstrated that DC activity is sufficient for tumour promotion DC-haploinsufficiency models have demonstrated that reducing the DC-gene dosage by half reduces skintumour susceptibility and lymphomagenesis in the Eu-Myc transgenic model 256,267. Each of these models underscores the importance of DC and polyamines in the development of tumours, and emphasizes the validity of polyamine metabolism as a viable target for antineoplastic intervention. Cleveland and colleagues have proposed a model in which MYC-regulated DC contributes to oncogenesis (see figure). The MYC family of oncoproteins are activated in up to 70% of human cancers. MYC functions as a * MYC MAX E-box 1 E-box 2 CACGTG...CACGTG SCF Skp2 CUL1 RBX1 P ATG Polyamines CKS1 p27 Kip1 DC SKP2 SKP1 Degradation p27 Kip1 DFM DC Cancer transcription factor that dimerizes with a partner, coined MAX, and this complex binds to the E-box sequence CACGTG to activate the transcription of target genes 268. The gene for DC (DC) harbours two conserved CACGTG elements in DC intron 1, and MYC activates DC transcription by binding to these elements in cells 15,23. DC then decarboxylates ornithine to produce putrescine, which is then further converted (by respective synthases) into the polyamines spermidine and spermine. As a net result MYC-overexpressing cells express elevated levels of polyamines 272. MYC promotes carcinogenic transformation, in part, through its ability to accelerate the rates of cell-cycle traverse, which is accomplished through downregulation of the cyclin-dependent kinase (CDK) inhibitor p27 Kip1 (REF. 269). p27 Kip1 normally binds to and inactivates cyclin E/CDK2 and cyclina/cdk2 complexes that direct entry and progression through S phase 270. Degradation of p27 Kip1 is accomplished through the SCF Skp2 E3 ubiquitin ligase complex, which is composed of the p27 Kip1 -binding and -specificity factors SKP2 (S-phase kinase-associated protein 2 (p45)) and CKS1 (cyclin-dependent kinase subunit 1; also known as CKS1B), as well as RBX1 (ring-box 1), SKP1, and the cullin CUL1 (REF. 271). MYC induces the expression of CKS1 and SKP2 to promote p27 Kip1 degradation (U. Keller, J. B. ld and J. L. Cleveland, personal communication). Surprisingly, these effects of MYC on CKS1 and SKP2 expression, and on p27 Kip1 degradation, require the functions of DC as treatment of cells with the DC suicide inhibitor 2-difluoromethylornithine (DFM), or DC heterozygosity, abolished the effects of MYC on the expression of CKS1, SKP2, and p27 Kip1 (REF. 267). Therefore, polyamines play a role in the expression of key components of the SCF Skp2 complex. SM and the resulting 2 2 has been implicated as a common pathway through which inflammation from multiple sources can lead to the mutagenic changes necessary for the development and progression of multiple epithelial cancers 47. These results indicate that SM might represent an excellent target for chemoprevention strategies. Polyamine transport. Mammalian cells possess an energy-dependent and selective polyamine transport system, which has yet to be molecularly defined. As the human diet is rich in polyamines, and because normal human intestinal flora produce and excrete significant quantities of polyamines, the transport capacity has the potential to significantly affect the cellular response to treatment-induced alterations in intracellular polyamines. The best characterization of mammalian polyamine transport has been provided by Poulin and colleagues 48, who suggest that polyamines are first transported into the cell by an unidentified membrane transporter/carrier that is powered by a membrane potential, followed by rapid accumulation of polyamines into polyamine-sequestering vesicles driven by a vacuolar- ATPase p gradient and proton exchange. Such a transport and sequestration system also helps to explain the apparently high, total intracellular concentration of polyamines, even though the actual amount of free polyamines is thought to be low. This system would provide the cell with a mechanism for rapid flux of cytoplasmic polyamines through the activation of its vesicle-release mechanism. Another model to explain mammalian polyamine transport has been proposed by Belting et al. 49,50. This model implicates a role for heparin sulphate and glypican 1 (GPC1) in the transport of spermine, which suggests that recycling of GPC1 is involved in polyamine transport 49,50. The data presented supporting each of these models are not mutually exclusive, therefore, it may turn out that the actual process of polyamine transport will be a synthesis of both models. 376 MAY 2007 VLUME ature Publishing Group

5 bjective responses These are defined by the voluntary international standard known as the Response Evaluation Criteria in Solid Tumors (RECIST). They are: complete response (CR), a complete disappearance of all target lesions; partial response (PR) a 30% or greater decrease in the sum of longest diameter of target lesion; stable disease (SD) <20% increase in the sum of the longest diameter of the target lesion. Inhibition of polyamine biosynthetic enzymes DC inhibition. The recognition that polyamines are required for cell growth and that their metabolic pathway is frequently dysregulated in cancers led to the development of inhibitors for each step of the polyamine biosynthetic pathway 51,52. 2-difluoromethylornithine (DFM), an enzyme-activated irreversible inhibitor, remains the prototypical inhibitor of DC (FIG. 2). DFM initially competes with ornithine for binding to the active site of DC, it is then decarboxylated by DC to create a highly reactive intermediate that inactivates DC by forming covalent linkages to either Cys360 (predominantly) or Lys69 (REF. 56). Although other, more potent inhibitors of DC have been synthesized and tested 57,58, none has demonstrated clear advantages over DFM. In tissue culture, inhibition of DC typically produces a near complete depletion of putrescine and spermidine, but its effects on spermine concentrations are variable, with spermine concentrations actually increasing in some instances. The decrease in putrescine and spermidine is generally accompanied by a substantial decrease in growth rate; however, overt cytotoxicity is only seen in rare instances in mammalian cells Although initially counterintuitive, this is potentially a desirable result in that normal cells might be spared the most detrimental effects of polyamine depletion by DFM, whereas tumour cells that exhibit dysregulation of polyamine metabolism might be more profoundly affected. This appeared to be the case for several types of tumours and suggested a potential selectivity between tumours and normal cells 62,63. The cytotoxicity observed in human small-cell lung cancers, both in vitro and in vivo 63,64, led to a clinical trial with DFM as a single agent 65,66. DFM has subsequently been clinically evaluated for activity against several tumour models, including gliomas, melanomas, breast, prostate and cervical cancers, both as a single agent and in combination with other agents DFM was found to be well tolerated (the maximumtolerated dose (MTD) was 3 g per m 2 ; the Phase II dose was 2.25 g per m 2 ), with toxicities of thrombocytopaenia, gastrointestinal effects and reversible hearing loss observed in Phase I and II trials. The observed toxicities are considered to be minor compared with other cancer chemotherapeutic agents. Levin et al. 75 have reported some benefit of DFM in patients with glioma as a post-irradiation adjuvant in combination with the treatment regimen procarbazine 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea vincristine (PCV), demonstrating increased survival (6.3 years versus 5.1 years) compared with the PCV regimen alone. owever, results in other cancer treatment trials demonstrated little benefit. The poor results with DFM as a single agent probably relate to pharmaco kinetic and pharmacodynamic factors. DFM is poorly transported into the cell and high concentrations are necessary to maintain DC inhibition and polyamine depletion. Additionally, the compensatory mechanisms that result from polyamine depletion by DFM, including upregulation of AdoMetDC and increased uptake of circulating polyamines, contribute to the lack of clinical efficacy demonstrated so far. Importantly, DFM has been approved by the Food and Drug Administration (FDA) as an effective and non-toxic agent for African trypanosomiasis The mechanism of action in this case involves a trypanosomal DC that lacks the carboxyterminal signals, which accounts for the short half-life of the mammalian DC 79,80. Consequently, once inhibited by DFM, there is little turnover of the long-lived parasite enzyme, compared with the rapidly restored mammalian DC. Although DFM was not found to be active as a single agent in clinical trials for cancer, it should be noted that Chen et al. 81 have recently reported that the combination of DFM and the polyamine transport inhibitor MQT 1426 was significantly more effective in treating a mouse model of squamous-cell carcinoma than was either agent alone. Because DFM is well tolerated, and because polyamines have been related to the carcinogenic process, DFM is now undergoing rigorous clinical testing as a chemopreventive agent in multiple epithelial cancers, including prostate and colon cancers, both as a single agent and in combination with cyclooxygenase inhibitors 1,82. The doses of DFM used in cancer chemoprevention trials are substantially lower than those used in therapeutic trials, and evidence for the toxicities of these lower doses, particularly over the long term, remains to be established. Inhibition of AdoMetDC. In addition to DFM, efforts have been made to synthesize and develop inhibitors of the other rate-limiting enzyme in polyamine biosynthesis AdoMetDC. Among the earliest inhibitors of AdoMetDC to be discovered is methylglyoxal bis(guanylhydrazone) (MGBG; FIG. 2). MGBG was in a clinical trial as an antileukaemic agent 83 before the discovery that it is a competitive inhibitor of AdoMetDC 84. Although MGBG is a potent competitive inhibitor of AdoMetDC, its antiproliferative activity is probably related to the antimitochondrial effects that occur before any perturbations in the polyamine pools 85. MGBG also has characteristics of a spermidine analogue, which further complicate its mode of action. The systemic toxicity of MGBG has precluded it from further clinical development, but it has served as a lead structure for additional agents, as recently reviewed by Seiler 52. An interesting representative of these analogues is a ovartis compound, 4-amidinoindan-1-one-2 - amidinhydrazone (SAM486A/CGP48664), a potent competitive inhibitor of AdoMetDC that has low mitochondrial toxicity 86. SAM486A testing has been extended to multiple Phase I and II clinical trials In Phase I trials, multiple dosing schedules were examined The dose-limiting toxicities for these trials included myelosuppression, nausea, vomiting and fatigue. o objective responses were observed in Phase I trials with SAM486A as a single agent. Van Zuylen et al. 91 reported the results of a Phase I trial that combined 5-fluorouracil and SAM486A with leucovorin rescue in the treatment of metastatic colon cancer 91. This combination was well tolerated and the results suggest that further combination trials might be ATURE REVIEWS DRUG DISCVERY VLUME 6 MAY ature Publishing Group

6 2 F 2 F 2 a DFM 2 2 c SAM486A 2 b MGBG d MDL Figure 2 Structures of specific inhibitors of polyamine metabolism. a DFM (2-difluoromethyl ornithine), an enzyme-activated inhibitor of ornithine decarboxylase. b MGBG (methylglyoxal bis(guanylhydrazone)), a competitive inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). c SAM486A ( 4-amidinoindan-1- one-2 -amidinhydrazone), a potent competitive inhibitor of AdoMetDC. d MDL (, 1 -bis(2,3-butadienyl)-1,4-butanediamine), an enzyme-activated inhibitor of 1 -acetylpolyamine oxidase. owever, it should be noted that MDL also effectively inhibits spermine oxidase. warranted. Two Phase II trials with SAM486A as a single agent have been reported, one in patients with metastatic melanoma 93 and one in non-odgkin s lymphoma 92. o benefit was reported in the melanoma trial; however, in the non-odgkin s lymphoma trial a complete plus partial response rate of ~19% was observed. Again, haematological toxicities were observed. Several other inhibitors of AdoMetDC have been synthesized and evaluated in multiple systems. These include active-site-directed compounds and enzymeactivated, irreversible inhibitors 95, These agents have been demonstrated to be active in multiple tumour systems; however, none has advanced to clinical trial. Inhibitors of spermidine synthase and spermine synthase. Although much work has concentrated on inhibiting the rate-limiting steps in polyamine biosynthesis, several inhibitors of the specific aminopropyl transferases spermidine synthase and spermine synthase have also been examined in various cancer cell types, including analogues of MTA Among the most mechanistically interesting inhibitors of the aminopropyl transferases are the transitionstate analogues. S-adenosyl-3-thio-1,8-diaminooctane (AdoDAT) is a specific inhibitor of spermidine synthase 105. owever, treatment of multiple cell types revealed that although spermidine pools were reduced, putrescine and spermine pools could be increased, leading to fewer growth-inhibitory effects than were expected 106. A similar transition-state analogue that inhibits spermine synthase and reduced intracellular spermine has been synthesized 107,108. owever, it should be noted that, because of their primary amines, both AdoDAT and S-adenosyl-1,12-diamino-3-thio-9- azadodecane (AdoDATAD) are possible substrates for SSAT and various amine oxidases, thus limiting their therapeutic utility. Development of polyamine analogues Although the targeted inhibition of specific biosynthetic enzymes in the polyamine metabolic pathway has yet to have significant clinical success in the treatment of cancer, there is a large body of data that substantiate polyamine function and metabolism as rational targets for antineoplastic drug development 1,3,52,55, Therefore, a more recent strategy has been to exploit the self-regulatory nature of polyamine metabolism through the use of polyamine analogues 112,113. The goals in this strategy were to overcome the limitations of specific enzyme inhibitors, including the compensatory upregulation in biosynthesis and the uptake induced when one of the biosynthetic enzymes was blocked. The ideal analogue might use the polyamine transporter to gain entry into the cell, thus competing with the natural polyamines for uptake; downregulate multiple polyamine biosynthetic enzymes, preventing potential increases in untargeted enzymes; upregulate polyamine catabolism, resulting in greater intracellular polyamine depletion than would be afforded by simply decreasing synthesis; reduce all three natural polyamines; not substitute for the natural polyamines in growth-related functions; and exhibit tumour-selective activity (FIG. 3a). Symmetrically substituted bis(alkyl) polyamine analogues. Among the first compounds that were synthesized to meet several of these criteria were the bis(ethyl) polyamines reported by Bergeron and colleagues The elegance of these compounds is in their simplicity. Although polyamines are known to regulate their own metabolism, the addition of the natural polyamines or similar compounds with primary amines is problematic because of the existence of several amine oxidases that are capable of metabolizing the free amines to toxic compounds 37, To circumvent this problem, protective alkyl groups were symmetrically added to the primary amines of the general polyamine structure to create various polyamine homologues 115,126. The initial study of spermidine and spermine analogues provided results similar to those obtained using DFM; rather than obvious cytotoxicity, the generally observed response was a decrease in growth rate 112,127,128. The early studies demonstrated that the spermine analogues are more inhibitory of growth than spermidine analogues, a discovery that was built on considerably in designing the subsequent generations of analogues 129. The first indication that this class of compound could produce cytotoxicity came from observations that a DFM-resistant human non-small-cell lung-cancer line, CI 157, responded to treatment with the analogue 1, 8 -bis(ethyl)spermidine (BESpd; FIG. 4) in a rapidly cytotoxic manner 130. More importantly, however, was the demonstration that the response to these agents was specific to the phenotype of the cells. When a DFMsensitive human small-cell lung-cancer line, CI 82, was compared with the DFM-resistant non-small-cell lung cancer line CI 157, with respect to their response to the bis(ethyl)polyamine analogues, it was discovered that the CI 82 cells were considerably more resistant to the bis(ethyl)polyamines than the CI 157 cells MAY 2007 VLUME ature Publishing Group

7 Polyamine Polyamine transporter a e? AZ g AdoMetDC mra h Ribosome DC mra Ub SSAT SSAT f SSAT mra DC DC AZ DC AZ c DC AZ AZ b 26S d SSAT, SM i MYC, FS, JU, ERα j Anion p DAT p k Polyamines and acetylated polyamines Figure 3 Potential targets of polyamine analogues. Polyamine analogues not only compete with the natural polyamines for uptake by the polyamine transporter 129 (a), they also induce the frame shift necessary to read through the regulatory stop of the antizyme (AZ) mra 195,277 (b). AZ is known to bind to the carboxyl terminus of ornithine decarboxylase (DC) monomers (c), which leads to the breakdown of DC by the 26S proteosome 20 (d). In addition, by a currently unknown mechanism, AZ downregulates the polyamine transporter 277 (e). Although, analogues increase the rate of DC degradation they actually decrease the rate of spermidine/spermine 1 -acetyltransferase (SSAT) protein degradation by blocking the ubiquitination (Ub) of SSAT and its subsequent destruction by the 26S proteosome 278,279 (f). Excess polyamines and their analogues reduce the efficiency of DC and S-adenosylmethionine decarboxylase (AdoMetDC) mra translation 212,213 (g). owever, the analogues increase both the efficiency at which SSAT mra is translated and the stability of the mra 146,280 (h). In addition to the several levels of post-transcriptional regulation of proteins, analogue treatment and interference with polyamine metabolism also have direct effects on the transcription of several important growth regulatory genes (MYC, FS, JU, oestrogen receptor-α (ERα; also known as ESR1), as well as genes that are involved in polyamine metabolism (SSAT, spermine oxidase (SM)) 146,151,196,245,246 (i). The natural polyamines are thought to function primarily through their ionic interaction with important cellular anions (j), therefore it is highly likely that some of the antiproliferative effects of polyamine analogues and treatments that reduce intracellular polyamine concentrations are a result of either direct competition for binding to these critical sites or a displacement of natural polyamines from these critical sites. In addition to downregulating uptake and biosynthesis, while concurrently increasing catabolism, polyamine analogues also appear to lead to an increase in efflux of both the polyamines and their acetyl derivatives through the diamine transporter (DAT) 281 (k). Each of the processes can profoundly affect cellular proliferative capacity. Further study revealed that the CI 157 cells responded to exposure to the bis(ethyl)polyamine analogues with a large increase in SSAT activity that was both dose and time dependent, in addition to a significant downregulation of DC and AdoMetDC 132. Although other types of cells exhibited increases in SSAT activity in response to analogue exposure 133, this was the first example of what would later be termed SSAT super-induction. Importantly, the lack of analogue induction of SSAT in the analogue-insensitive smallcell lung-cancer lines indicated a link between SSAT induction and the cytotoxic response, which prompted the cloning of SSAT 35, Subsequent to these findings, other laboratories observed similar results in multiple tumour cell types including melanoma, pancreatic, bladder and breast cancers Several studies have also demonstrated a link between the expression of SSAT and analogue-induced apoptotic cell death 137, ne of the more effective inducers of SSAT is 1, 11 - di(ethyl)norspermine (DESpm; also known as BESpm or BE333) 118. DESpm displays many of the characteristics of the ideal analogue with respect to the criteria outlined above. It uses the polyamine transporter to gain entry into the cell effectively competing with the natural polyamines for uptake. It downregulates both DC and AdoMetDC. It upregulates polyamine catabolism, ATURE REVIEWS DRUG DISCVERY VLUME 6 MAY ature Publishing Group

8 a BESpd b BESpm c DESpm d BESpm Figure 4 Examples of symmetrically substituted polyamine analogues. a BESpd, 1, 8 bis(ethyl)sperm idine. b BESpm, 1, 12 bis(ethyl)spermine. c DESpm, 1, 11 di(ethyl)norspermine. d BEspm, 1, 14 bis(ethyl)homospermine. inducing both SSAT and SM, and it is not thought to substitute for the natural polyamines when used at pharmacological doses. There is even evidence that its activity, particularly with respect to the super-induction of SSAT, is specific to the type of tumour cell The preclinical results of DESpm in multiple models 140, were sufficiently impressive to warrant clinical trials. It is also important to emphasize that these agents are useful prototypes as multitargeting drugs in that they target uptake, biosynthesis and catabolism of polyamines. Three Phase I trials with different dosing schedules have been reported so far for DESpm Unacceptable toxicities, including a reported neurotoxicity, were observed with twice-a-day schedules with an MTD of <83 mg per m 2 per day for 5 days 160. Similar dose-limiting toxicities were observed with a threetimes-a-day scheduling for 5 days at similar total daily doses 161. owever, when administered as a single daily infusion DESpm was well tolerated in a focused Phase I trial in patients with non-small-cell lung cancer with an MTD of 185 mg per m 2 per day for 5 days, repeated every 21 days. The dose-limiting toxicity in this trial was gastrointestinal at a dose of 231 mg per m 2 per day. Although no objective responses were observed in this study, the results demonstrated that DESpm could be safely administered, and pharmacologically relevant concentrations could be obtained. It is important to note that DESpm was found to have a short serum half-life (<0.5 hours) at doses of <118 mg per m 2 per day, and one that ranged between 0.5 and 3.7 hours at higher doses 162. The only Phase II trial completed so far with DESpm was a two-stage design trial in patients with previously treated metastatic breast cancer at a dose of 100 mg per m 2 per day for 5 days, repeated every 21 days 163. Sixteen patients that were considered for evaluation, each receiving at least one full 5-day cycle of treatment, were treated on this schedule with no objective responses. Grade 1 2 toxicities consisted of abdominal pain, perioral numbness, nausea and vomiting. Two patients experienced grade 3 abdominal pain. As no objective responses were observed in the first 16 patients the trial was halted. The reasons for the differences between the preclinical studies and the clinical trials are currently not known. The pharmacokinetic data suggest that sufficient concentrations of the drug should have reached the tumour target. owever, as no biochemistry was performed on tumour tissue from treated patients, the actual amount of drug delivered to the tumour and any subsequent response with respect to induction of SSAT or polyamine-pool depletion are not known. ne lesson that can be learned from the trials with DESpm is that scheduling can be crucial with respect to toxicity. Reducing administration from three times a day to once a day allowed similar doses to be administered with completely different toxicity profiles. Consequently, it may be possible that altering the dosing schedule would result in improved efficacy. Several investigators have demonstrated an interest in combining DESpm with standard chemotherapy, as first proposed by ahm et al. 164 The recent interest is based on observations from several laboratories that exposure of many tumour cell types to various standard chemotherapeutic agents produces a significant increase in SSAT mra ector et al. 166 demonstrated that treatment of A2780 ovarian cancer cells with oxaliplatin (Eloxatin; Sanofi Aventis) increased SSAT mra 15-fold with only a doubling of SSAT activity 166. Low-dose DESpm alone produced only a fivefold increase in mra and a sevenfold increase in SSAT activity. owever, when oxaliplatin was combined with DESpm the activity of SSAT was increased more than 210-fold. This synergistic enhancement of SSAT was maintained even when low concentrations of DESpm were used. Similar results (without measuring activity) were reported by Choi et al. using 5-fluorouracil in combination with DESpm 168. The encouraging results of these combination studies are likely to stimulate new clinical trials with DESpm or with other polyamine analogues in combination with standard chemotherapeutic agents. Asymmetrically substituted polyamine analogues. Although the analogues synthesized by Bergeron and colleagues provided compounds that might still find clinical utility, the synthesis of additional congeners was constrained by the fact that, other than modifying the symmetrical end groups, the ability to modify the number of carbons between amines was limited. ne strategy used recently to extend the idea of the bis(alkyl) polyamine analogues was to make asymmetric substitutions to the basic polyamine structure. This focused primarily on the spermine and norspermine backbone, as the spermine analogues were the most active of the symmetrically substituted analogues. The initial members of this class (shown in FIG. 5) demonstrated significant antiproliferative activity The results with 1 -propargyl- 11 -ethylnorspermine (PESpm) and 1 - cyclopropyl-methyl- 11 -ethylnorspermine (CPESpm) were similar to those observed for DESpm, that is, 380 MAY 2007 VLUME ature Publishing Group

9 a PESpm b CPESpm c CESpm d IPESpm Figure 5 Examples of non-symmetrically substituted polyamine analogues. a PESpm, 1 -propargyl- 11 -ethylnorspermine. b CPESpm, 1 -cyclopropylmethyl- 11 ethylnorspermine. c CESpm, 1 -cycloheptylmethyl- 11 -ethylnorspermine. d IPESpm, (S)- 1 -(2-methyl-1-butyl)- 11 -ethyl-4,8-diaz aun decane. cell-type-specific cytotoxicity, which is associated with the high induction of SSAT 170,172. owever, results with 1 -cycloheptylmethyl- 11 -ethylnorspermine (CESpm) and (S)- 1 -(2-methyl-1-butyl)- 11 -ethyl-4,8-diazaundecane (IPESpm) demonstrated that small changes in analogue structure can result in significantly different cellular effects. CESpm does not significantly induce SSAT in comparison with the other symmetrically and asymmetrically substituted analogues. owever, CESpm rapidly induces apoptosis in non-smallcell lung cancers 173. Cell-cycle analysis revealed that CESpm treatment produces a profound G2/M block coincident with interference with normal tubulin polymerization 174. Interestingly, although IPESpm significantly induces SSAT and effects a G2/M block, the other asymmetrically substituted analogues have little effect on cell cycle with respect to G2/M transit 174,175. CESpm and IPESpm are also a potent inducers of SM, the activity of which might contribute to their antiproliferative action 176. A series of asymmetrically substituted analogues have now been successfully synthesized and examined in various types of tumour cells with encouraging results 164,172,173, In addition to demonstrating that small changes in structure can lead to significantly different biological results, these analogues demonstrate that multiple functional moieties can be linked to the same polyamine backbone, which could potentially lead to compounds with increased targeting capacity 172. Conformationally restricted analogues. Another approach to extending the boundaries of the original analogues was to introduce modifications into the methylene chains between the amines. The alkylamine structure of the natural polyamines provides them with a molecular flexibility that is thought to facilitate their interaction with multiple cellular anions. Frydman and colleagues postulated that by restricting the free rotation of the carbon-to-carbon bonds, the resulting compounds would have altered and potentially therapeutic activity (FIG. 6). Introduction of unsaturated linkages or cyclic moieties into bis(ethyl)tetramines and bis(ethyl)pentamines resulted in compounds with unique antiproliferative activities in various models. Two representatives of this class that have demonstrated encouraging preclinical results are CGC and CGC (REFS 184,186). While CGC is based on 1, 12 bis(ethyl)spermine (BESpm), with a double bond in the central 4-carbon methylene bridge, CGC is based on 1, 14 -bis(ethyl)homospermine (BESpm), with a cyclopropyl bond in the central 4-carbon methylene bridge. The introduction of the double bond into BESpm results in a compound with significantly increased antiproliferative activity compared with BESpm, but with minimal toxicity. The introduction of the cyclopropyl bond into BESpm also significantly modifies activity and toxicity. The introduction of conformational restrictions into the syn thesis of polyamine analogues has allowed for the creation of various new compounds with distinct ranges of activities and toxicities. Both CGC and CGC are in clinical trials. While CGC is still in Phase I trial, CGC is not only in Phase I trial as a single agent but also in a Phase Ib trial in combination with either bevacizumab (Avastin; Genentech), erlotinib (Tarceva; SI Pharmaceuticals), docetaxel (Taxotere; Sanofi Aventis), or gemcitabine (Gemzar; Eli Lilly). Additionally, a Phase II trial with CGC has just started to enrol patients with hormone non-responsive metastatic prostate cancer, and a Phase II trial for patients with previously treated pancreatic cancer is scheduled to start when the MTD of CGC is determined in an ongoing Phase I study. Although similar in structure, only CGC significantly induces SSAT and SM (R.A.C and L.J.M., unpublished observations). This induction seems to have a role in tumour-cell response to CGC-11047; however, such an induction is not absolutely required for activity, as CGC and other analogues have little effect on SSAT or SM expression, but are nevertheless active compounds 172. f relevance and interest are a series of studies performed with these two analogues and others in models of age-related macular degeneration (AMD). The wet form of AMD is a disease hallmarked by vascular proliferation, as is diabetic retinopathy. In various mouse models of vascular neogenesis it was shown that polyamine analogues were not only capable of preventing the progression of induced vascular lesions, but in fact could reverse vascular lesions already formed 187,188. These studies have led to the recent initiation of a Phase I clinical trial for AMD. f particular interest is the fact that the polyamine analogues are anti-angiogenic in these mouse models, an activity relevant to antineoplastic activity. Also of interest is the fact that the polyamine analogues, which are actively transported, can be administered subconjunctivally to the ATURE REVIEWS DRUG DISCVERY VLUME 6 MAY ature Publishing Group

10 Protonatable Protonatable refers here to an amine or imine nitrogen molecule that is able to accept a proton, thus existing as a positively charged molecule at a physiological p. CGC CGC Figure 6 Conformationally restricted polyamine analogues currently in clinical trials. CGC is an unsaturated cis double-bond variant of 1, 12 bis(ethyl) spermine. It is in Phase I and II trials for cancer and Phase I trials for age-related macular degeneration. CGC is a cylopropyl variant of 1,14-bis-(ethyl)-amino-5,10- diazatetradecane 1, 14 -bis(ethyl)homospermine. It is in a Phase I clinical trial for cancer. eye. This is in contrast to the recently approved drugs for AMD, which are mostly based on anti-vascularendothelial growth factor (VEGF) activity, and which must be injected directly into the eye 187,188. ligoamines. ne of the mechanisms that has been proposed for the antiproliferative effects of specific polyamine analogues is their ability to interact with nucleic acids and other intracellular polyamine-binding sites 4,5. Several studies have demonstrated the interaction of natural polyamines with DA and chromatin 6 and have implicated these interactions as being crucial to the effects of select analogues n the basis of these observations, Frydman and colleagues 193 suggested that by increasing the number of protonatable imines the resulting compounds would have increased affinity for DA, and therefore be more effective antiproliferative agents. They synthesized a series of oligoamines containing 8 14 amines in both saturated and conformationally restricted, unsaturated forms 193 (FIG. 7). As a group these compounds demonstrated potent (submicromolar) antiproliferative activity in human prostate cell lines in a rank order that correlated well with their ability to aggregate DA in vitro 193. A representative oligoamine, CGC-11144, demonstrated significant antitumour activity against human breastcancer cells in vitro and in vivo, exhibiting profound antiproliferative effects when treating established MDA- MB-231-tumour-bearing nude mice, using a twice-aweek schedule 194. CGC induces apoptotic cell death, mitochondrial cytochrome c release, caspase 3 activation, poly(adp-ribose) polymerase (PARP) cleavage, increased expression of the pro-apoptotic BCL2-associated X (BAX) protein, and decreased the anti-apoptotic B-cell CLL/lymphoma 2 (BCL-2) protein. CGC treatment also decreases DC activity with little effect on the expression of the polyamine catabolic enzymes SSAT and SM, and leads to a decrease in all three polyamines. The decrease in DC activity is probably a result of the increase in DC antizyme protein production in cells exposed to the oligoamine 195. A recent finding indicates that oligoamines can alter the expression of oestrogen receptor-α (ERα) in a highly selective manner. Specifically, the oligoamines CGC-11144, CGC and CGC downregulate the transcription of ERα through apparent disruption of Sp1-transcription factor (SP1) family member binding to the ERα promoter 196. These results suggest a novel anti-oestrogen effect by the oligoamines that might provide an additional strategy for breast-cancer therapy and/or prevention. Macrocyclic polyamines. The macrocyclic polyamines (FIG. 8) represent a unique class of analogues that possess multiple functions including DA binding, complexation of transition metals, DA cleavage and depletion of ATP As this class of interesting analogues has recently been reviewed by Liang et al. 198 they will not be discussed further here, other than to say that although they are in the early stages of development they appear to have considerable potential as antiviral and antitumour agents. Polyamine analogues as targeting vehicles The human polyamine-transport system allows molecules that are conjugated to the general polyamine backbone to be transported. Consequently, several attempts have been made to use the polyamine structure as a carrier for cytotoxic and other agents that might otherwise be poorly transported into tumour cells. These conjugates have included DA intercalating and alkylating agents such as chlorambucil, acridine and nitroimidazoles, and various other antiproliferative substituents Delcros et al. have recently reported the synthesis and testing of a series of heterocyclic amidine/polyamine homologue conjugates to examine the limits of the polyamine-transport system 208. Although putrescine and spermidine conjugates were found to be transported, their transport was limited by the size of the linked substituents. Spermine conjugates were found to be less limited by size than their putrescine or spermidine counterparts, but were only effectively transported when cells were first treated with DFM. Recently, interest has increased in drugs that alter chromatin remodelling and as such might be useful for antineoplastic therapy. Among the most interesting agents are the various class I/II histone deacetylase inhibitors (DACIs) 209. The acetylation status of various specific histones is thought to have a direct role in the regulation of gene expression and aberrant acetylation patterns might lead to inactivation of tumour-suppressor genes. Thus, one goal of treatment with DACIs is to restore tumour-suppressor expression and normal growth control. Although several classes of DACI have been synthesized and some are in clinical trials virtually all of them have been designed to target only the DAC itself (primarily the zinc cofactor), and none is designed to facilitate the targeting of the DACI to chromatin, where DACs are presumed to act. Woster and colleagues 210 used a unique approach with the goals of using the polyamine transport system to accumulate DACIs, use the polyamine structure to target the 382 MAY 2007 VLUME ature Publishing Group