Microtubule Stabilising Agents for Cancer Chemotherapy

Review Microtubule stabilising agents for cancer chemotherapy

Ying Zhao, Wei-Shuo Fang† & Klaus Pors †Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Peking 1. Introduction Union Medical College), Ministry of Education, Beijing 100050, China 2. Why target microtubules? the mechanism of MSA–microtubule Background: Microtubules are very important targets for cancer chemotherapy. binding During the past 15 years, three structurally diverse classes of microtubule 3. MSAs used as anticancer drugs stabilising agents (MSAs), namely, taxanes, epothilones and discodermolides, have been approved as anticancer agents or subjected to clinical trials, and 4. Newly discovered MSAs many novel MSAs have also been identified. Objective: This review focuses 5. Pharmacophores of MSAs on recent advances in the use of taxanes, epothilones and discodermolides, 6. Expert opinion as well as some recently described natural product based MSAs. Methods: Data were identified through the search of PubMed for research articles and reviews, the website of European patents (http://ep.espacenet.com) and US patents (www.uspto.gov) for patents up to October 2008. Only well-established MSAs are mentioned in this review. Results/conclusion: Great advances have been made in the application of MSAs in antitumour clinical practice and drug discovery. Many structure–activity relationship studies of MSAs as novel anticancer drugs have been conducted in recent years. To enhance the efficacy of MSAs as clinically useful therapeutics, numerous efforts have been made in combination chemotherapy regimens and include: i) the combination of structurally different MSAs, ii) combination of MSAs with other classes of anticancer agent with a different mechanism of action and iii) novel ways of administrating well-established MSAs.

For personal use only. Keywords: anticancer, chemotherapy, discodermolide, epothilone, microtubule stabilising agent, taxane

Expert Opin. Ther. Patents (2009) 19(5):607-622

1. Introduction

Microtubules, a fundamental component of the cytoskeleton in eukaryotic cells, are a filamentous and tube-like protein polymers composed of α- and β-tubulin heterodimers with a molecular weight of ~ 100 kD. In living cells, microtubules are characterised by their high dynamics and alternating polymerisation/ depolymerisation, which leads to net elongation/shortening of the filaments. During the cell cycle, microtubule dynamics are precisely controlled to regulate a

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science variety of important processes, including cell shape maintenance, intracellular transportation, signal transduction, mitotic spindle formation and cell division. Any disturbance of microtubule dynamics may lead to cell cycle arrest or cell death. Because of their essential roles in dividing cells, microtubules have been explored as drug targets for anticancer therapy [1]. Microtubule-targeted drugs (MTAs) have been arbitrarily classified into two large groups according to their ‘apparent’ mechanism of action: i) microtubule destabilising agents (MDAs), such as the vinca alkaloids and the colchicines, which prevent the polymerisation of tubulin and promote the depolymerisation of filamentous microtubule; and ii) microtubule stabilising agents (MSAs), such as taxanes and epothilones, which promote the polymerisation of tubulin to microtubules, further stabilising them and thereby preventing depolymerisation. Both of these microtubule-perturbing classes of agents have been successfully used in clinical cancer chemotherapies.

Since the discovery of paclitaxel (Taxol®, Bristol-Meyers growth has been inhibited by MSAs. These findings were Squibb Co., New York, NY, USA), the first defined MSA in not explained until it was observed that it is the level of sup- the 1960s, considerable progress has been made in this area. pression of microtubule dynamics, rather than the change of This review focuses on recent advances in MSAs, including polymer mass, that correlates with cell growth inhibition their structures and pharmacological activities, as well as the caused by MSAs at low concentrations [6,7]. Unlike in an in vitro achievements and problems associated with their clinical system, there are cellular factors that intrinsically control use. Owing to space limitations, the ‘putative MSAs’ (i.e., microtubule dynamics in cells. For example, a family of agents with potential microtubule stabilising activity but not proteins called microtubule-associated proteins (MAPs) bind yet confirmed) are not mentioned, and only those structure– to microtubules and stabilise them, whereas oncoprotein activity relationship (SAR) studies that have great clinical stathmin/oncoprotein 18, and some microtubule motor proteins significance are covered. For more details on the different (kinesins) tend to aid in destabilising microtubules [8]. MSAs including ‘putative’ ones and SAR studies, interested Considering the inherent factors, it is clear that the regulation readers can refer to a recent comprehensive review on natural of microtubule dynamics in cells is a much more complex product-derived MSAs [2]. procedure than that in vitro. The exact mechanism as to how MSAs induce mitotic 2. Why target microtubules? the mechanism arrest and cause subsequent apoptosis still remains unclear. of MSA–microtubule binding A universal assumption for the antimitotic action of MSAs is that they interfere with the dynamics of spindle microtubules The dynamics of microtubules comes from continuous addition in the cell, so that the daughter chromosomes cannot align and loss of tubulin heterodimers at the polymer ends (dynamic at the equatorial plate and move to the two poles of the instability). Microtubule filaments have the potential to grow mitotic spindle. Cells that fail to pass through mitotic check- and shrink; however, there is a behavioural difference at the points are arrested at the metaphase–anaphase transition, end units: at the plus end where β-tubulin is exposed, the and as a consequence are subjected to intrinsic mitochondrial addition and loss are more actively coupled with GTP apoptosis [1,9]. There is also evidence that some MSAs, such as hydrolysis than at the minus end [1-3]. In an in vitro tubulin paclitaxel, induce late-G1 arrest in mammalian fibroblast polymerisation system, the growing and shrinking of the cells without T-antigen transformation, whereas T-antigen- microtubule will continue until the system reaches equilib- transformed cells treated with paclitaxel pass through both G1 rium, which occurs when the concentration of free tubulin and G2/M checkpoints. As a consequence, the transformed reaches its ‘critical concentration’. However, as the critical cells undergo inappropriate mitosis and will rapidly die. This

For personal use only. concentration for tubulin at the plus end is lower than that at finding raises a possibility that paclitaxel may selectively kill the minus end, there exists a net addition of tubulin heterodi- cancer cells missing checkpoints [10]. Some recent studies mers at one end and balanced net loss at the other. This have also indicated that at least for some MSAs, the cell cycle phenomenon is termed “treadmilling”. Dynamic instability is arrested in the interphase after formation of multipolar together with treadmilling contributes to microtubule dynamics. spindles (mitotic slippage) at low drug concentrations, or The presence of MSAs in the system significantly perturbs terminated as tetraploid interphase cells at high drug con- the balance between polymerised microtubules and free centrations. In general, regardless of which phase of mitosis tubulin. The evidence include: i) MSAs at high concentra- is targeted by the MTAs, the cytotoxic activity results from tions (usually μM) reduce the critical concentration of tubulin, cell cycle arrest and subsequent apoptosis [11,12]. thus, leading to an increase of microtubule polymer mass; Although MSAs are named after their targeting protein, and ii) MSAs at low concentrations (nM or sub-μM) suppress more accumulating evidence has indicated that microtubules microtubule dynamics, including both dynamic instability and may not be the only target of MSAs in cells. For example,

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science treadmilling, so that the formed filaments seem to be more epothilone D, a well-recognised MSA, has been reported stable. It is interesting to note that MDAs at low concentrations to inhibit neointimal hyperplasia in vivo following rat share the same mechanism of dynamic suppression as MSAs [1]. carotid artery injury by regulating cell cycle G1-checkpoint In cells, the perturbation of microtubule formation by small proteins, such as p27 and CDKs [13]. Other intracellular molecules has been shown to affect downstream signalling protein targets of MSAs include Bcl-2 [14], heat-shock pathways, leading to cell cycle arrest and cell death, which proteins [15], CD18 [16], neuronal calcium sensor 1 [17], for many years was believed to be the primary mechanism human NFX1 [18] and MD-2 [19]. The non-microtubule of action of the MSAs. However, direct correlation between binding may contribute to the pharmaceutical activity microtubule formation/stabilisation and cytotoxic activity of MSAs, and perhaps their resistance and side effects. does not fully explain why cancer cell growth can be inhibited Additionally, it is now clear that some MSAs can directly by several MSAs at much lower concentrations (nM) than those target mitochondria and induce the disruption of the mito- needed for tubulin polymerisation promotion or stabilisation chondria membrane potential and the release of pro- (usually μM) [4,5]. In addition, no significant increase in the apoptotic factors in the cytosol, ultimately leading to mass of polymerised microtubules is observed in cells whose apoptosis induction [20,21].

608 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

3. MSAs used as anticancer drugs Cancer cells overexpressing P-gp are resistant to paclitaxel and docetaxel because they are good substrates for P-gp [36,37]. 3.1 Taxanes A number of SAR studies were carried out to develop new In 1971, the structure of a unique diterpene compound taxanes with poor affinity for P-gp to treat MDR tumours [35]. paclitaxel (1) was isolated from the Pacific yew tree Taxus Recently, a study on novel taxanes indicated that drug resis- brevifolia, possessing antileukemic and antitumour activity [22]. tance mediated by P-gp might be reversed by increasing the The discovery of paclitaxel is regarded as a landmark in the binding affinity to microtubules through synthetic manipulation history of anticancer drug development, because its potent of the C2-modified position (8) (Figure 2) [38]. antiproliferative activity is owing to its ability to stabilise With regard to microtubules and tubulin related factors, microtubules, which at the time of the discovery was an it is now universally accepted that tubulin mutations are of unknown phenomenon [23]. Subsequent preclinical and clinical little clinical significance [39]. Instead, the alternative expression studies have demonstrated its significant antitumour activity of tubulin isotypes, especially the overexpression of class III against solid tumours. In 1992, the FDA approved its use in the β-tubulin, has been reported to correlate with drug sensitivity clinic for the treatment of metastatic ovarian cancer. Paclitaxel, to taxanes in ovarian cancer [40,41], NSCLC [42,43] and breast together with its analogue docetaxel (2), also marketed, are cancer [44-46]. Some methods have been developed to deter- among the most important chemotherapeutic agents over mine the level of class III β-tubulin, which is now regarded the past 20 years. as a predictive factor for the outcome of taxane chemo- Paclitaxel binds to microtubules with high affinity, but therapy [47,48]. In 2005, the C-seco taxane IDN5390 (9) was poorly to free tubulin subunits and dimers. A 6.5 Å resolution reported to exhibit less activity in both paclitaxel-sensitive structure analysis revealed the pattern of the paclitaxel– cells and P-gp-overexpressing drug-resistant cells. In contrast, microtubule interaction [24]. The paclitaxel binding site, IDN5390 possessed higher antiproliferative activity than which is located on the β-subunit of tubulin with nearly paclitaxel in resistant cells that overexpressed class III 1:1 stoichiometry, is on the inner surface of polymerised β-tubulin [49], possibly owing to higher affinity to class III microtubule. It has been suggested that this specific binding β-tubulin than with class I β-tubulin (the major isotype in of paclitaxel induces a conformational change in tubulin, tubulins). Interestingly, although IDN5390 and paclitaxel leading to a highly stable microtubule interaction [25]. belong to the same family of taxanes, they showed synergistic Since the discoveries of paclitaxel and docetaxel, novel effects on cells overexpressing class III β-tubulin, providing new paclitaxel analogues and prodrugs thereof have been developed insights into drug combinations against tumour resistance in an attempt to improve the poor bioavailability of the drugs to taxane-based MSAs.

For personal use only. and to reduce the toxicity. Paclitaxel analogues with improved water solubility and oral bioavailability have been developed, 3.2 Epothilones thereby, reducing the need for the excipient Cremophore EL, Epothilones A (10) and B (11) are 16-membered macrolides which is used in the current paclitaxel formulation and is originally isolated from the myxobacterium Sorangium cellulosum believed to be responsible for the occurrence of severe allergic as antifungal agents. In 1995, it was reported that these side reactions [26,27]. Among the newly developed taxanes are compounds possess paclitaxel-like effects both in microtubule- IDN5109 (3) [28], MAC-321 (4) [29], DJ-927 (5) [30], BMS- based model and in cultured cells, and it was revealed that 275183 (6) [31] and MST-997 (7) [32] (Figure 1), all of which epothilones bind to microtubules at the same site as pacli- have proven to be orally effective. Several of these agents taxel [50]. However, recent studies of desoxyepothilone showed superior activity in multi-drug resistant and paclitaxel- B-resistant cells bearing tubulin mutations indicate that although resistant tumours in preclinical studies. In addition, abraxane, these drugs seem to share the same binding site, the mecha- an albuminised paclitaxel, which is water soluble in the absence nism of binding of paclitaxel and epothilones may not be

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science of excipients, was approved by the FDA in 2005 for the identical [51]. The independent binding mode of epothilones was treatment of breast cancer [33]. later confirmed by a combination of electron crystallography at In the clinic, one of the major reasons for the failure of 2.89 Å resolution and NMR-based conformational analysis [52]. chemotherapy is believed to be multi-drug resistance. Multi- In contrast to the taxanes, epothilones have two features drug resistance to MSAs arises either from the overexpres- that are ideal for drug development: i) they are easy to produce sion of P-glycoprotein (P-gp) and other ATP-binding cassette in large quantities by fermentation and ii) they possess good superfamily drug efflux pump proteins or microtubule/tubulin- water solubility, which is important for facilitating drug related factors, such as tubulin mutations, overexpression of administration. As a consequence, a number of structure tubulin isotypes with more dynamicity (β-III) and changes optimisations have been conducted [53] and several clinical in the expression of MAPs in tumour cells [34]. It should be trials of epothilones have progressed in a relatively short time. pointed out that evidence for the relevance of P-gp and Naturally occurring epothilones and synthetic derivatives similar proteins is largely based on the overwhelming data under current development in the pharmaceutical industry from in vitro studies and disappointing clinical trials with include epothilone B (EPO906, patupilone), BMS-247550 P-gp inhibitors [35]. (aza-epothilone, ixabepilone) (12), epothilone D (KOS-862,

Expert Opin. Ther. Patents (2009) 19(5) 609 Microtubule stabilising agents for cancer chemotherapy

O O OH HO O O OH O

O O NH O O NH O O O O HO O O O OH O OH O OH O O O

1 Paclitaxel (Taxol) 2 Docetaxel (Taxotere)

O OH O O O O O O HO

O O O NH O O O O NH O O O O O O O OH O O O HO O

OH O O

For personal use only. O

3 IDN-5109 4 MAC-321

N O

O O O O OH O O Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science O O NH O O NH O H O O N O O O HO O OH O O OH OH O O O F O

5 DJ-927 6 BMS-275183

Figure 1. Taxanes in clinical uses or trials (compounds 1 – 7 and 9).

610 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

HO O OH O OH O O O

O O O NH O

O O O NH O HO O O O S O HO O HO O O OH O O

7 MST-997 9 IDN5390

Figure 1. Taxanes in clinical uses or trials (compounds 1 – 7 and 9) (continued).

O affected by several factors such as P-gp overexpression, elevation in β-tubulin isotype level and β-tubulin mutations [57,58].

O In the clinic, ixabepilone was found to be effective in patients O HO with metastatic breast cancer resistant to anthracycline, taxanes and capecitabine [59]. Interestingly, the epothilones do not induce O expression of drug efflux protein genes, which is considered to be an important reason for drug resistance [60]. Several O For personal use only. NH O explanations have been proposed for the exceptional activity O of epothilones against MDR, including: i) poor substrate affinity for transporter proteins such as P-gp; ii) persistent activity in HO O O spite of altered β-tubulin; and iii) sensitive cells may exist in OH O tumours because of heterogeneity [61], but the exact mecha- O nisms are still under investigation. Another attractive feature of the epothilones is that some members, such as patupilone [62] N 3 and sagopilone [63], are able to cross the blood–brain barrier and accumulate in the brain, and show significant inhibitory 8 activity in xenograft models of either glioblastoma or CNS metastases. Thus, there is a potential use for epothilones in Figure 2. A C2-modified taxane (azido analogue) with high binding affinity to microtubules (compound 8). treating brain tumours. Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science 3.3 Discodermolide desoxyepothilone B) (13), BMS-310705 (14), KOS-1584 (15) Discodermolide (17) (Figure 4) is a polyhydroxylated lactone and ZK 219477 (ZK-EPO, sagopilone) (16) (Figure 3). isolated from the marine sponge Discodermia dissoluta in Three novel synthetic congeners, compounds 11, 13 and 16 1990 [64]. It was initially described and studied as an (ZK 219477, ZK-EPO, sagopilone), are also in Phase II clinical immunosuppressive agent [65-67], but was later found to studies. The water-soluble BMS-310705 recently finished its possess microtubule stabilising activity [68]. Similar to paclitaxel, Phase I trials (reviewed in [54,55]). Ixabepilone (12) was approved discodermolide efficiently promotes the polymerisation of by the FDA for refractory breast cancer in 2007 (Ixempra®, tubulin, even in the presence of factors favouring the depo- Bristol-Meyers Squibb) and has proved to be safe and orally lymerisation of microtubules, such as cold temperature, 2+ effective at recommended doses [56]. Ca , or the absence of GTP and MAPs. Discodermolide Although the in vitro activities of epothilones and paclitaxel competes with paclitaxel to bind to β-tubulin in microtubules are similar in drug-sensitive cancer cells, epothilones showed with 1:1 stoichiometry (apparent Ki = 0.4 μM) and its much greater effect on paclitaxel-resistant cells and xenografts binding affinity is much higher than that of paclitaxel [69,70].

Expert Opin. Ther. Patents (2009) 19(5) 611 Microtubule stabilising agents for cancer chemotherapy

R O O

S S

HO HO N N

O NH

O OH O O OH O

10 Epothilone A : R = H

11 Epothilone B : R = CH3 12 BMS-247550

S S NH 2 HO N HO N

O O

O OH O O OH O

13 Epothilone D 14 BMS-310705

S S

HO N N For personal use only. HO O O O

O OH O

O OH

15 KOS-1584 16 ZK-EPO

Figure 3. Epothilones in clinical trials (compounds 10 – 16).

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science Taken together, the results suggest that the binding sites of Discodermolide was also found to act synergistically with discodermolide and paclitaxel are similar. paclitaxel in a variety of human cancer cell lines [74,75]. The Discodermolide has been shown to inhibit the proliferation combination of paclitaxel and discodermolide produced of MDR cancer cells that either overexpress P-gp or possess positive results against a paclitaxel-resistant cell line A549-T12, point mutations in β-tubulin [68]. At very low concentrations which is ninefold resistant to paclitaxel and 20-fold resistant (IC50 < 2.5 nM), discodermolide induced apoptosis in to discodermolide, respectively [74-76]. In ovarian SKOV-3 MCF-7 and MDA-MB231 cells [71] and increased the per- carcinoma xenograft-bearing mice, the combination induced centage of cells at metaphase–anaphase by 20 – 63% as tumour regressions without notable toxicities [77]. The observed in different cell types [72], indicating that it blocks synergy of paclitaxel and discodermolide is interesting the cell cycle at metaphase–anaphase and consequently considering that these two compounds are thought to bind induces apoptosis. Additionally, a recent study indicated identically to the microtubules. The reason for the synergistic that discodermolide induced accelerated cell senescence, effect is still under investigation. It has been hypothesised which in some cases has been postulated to be a major that these two compounds bind to large numbers of mechanism of tumour growth suppression [73]. paclitaxel-binding sites on individual microtubules, which act

612 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

O O OH OO

OH NH 2

17 Discodermolide

Figure 4. Discodermolide (compound 17).

together in unique ways to alter microtubule architecture or because of its insufficient supply from nature. However, tubulin conformation [76]. chemical synthesis may help to solve this problem and The initial entry of discodermolide into clinical trials as an provide simplified analogues of dictyostatin that still possess anticancer agent in 2004 was slowed in part by its low natural biological activity [93]. abundance (0.002% w/w from frozen sponge [64]). During the past decade, numerous efforts were made towards the total 4.2 Eleutherobin and sarcodictyins synthesis of discodermolide and its derivatives [78,79] to solve the Eleutherobin (19) is a tricyclic diterpene that was isolated supply problem. Furthermore, it has been shown during SAR from the soft coral Eleutherobia sp. in 1997 [94]. It was studies that modifications of the lactone moiety [80,81] and the shown to display activity in vitro similar to paclitaxel [95]. It carbamate moiety [82] are tolerated, whereas the middle part binds competitively to the paclitaxel-binding site on of the molecule is essential for its cytotoxic activity (Figure 5) [83,84]. β-tubulin, suggesting that eleutherobin and paclitaxel share

For personal use only. Discovery and biological investigations of several natural ana- a common binding domain. The cytotoxicity of eleuther- logues of discodermolide [85] support the above-described SAR obin was comparable to that of paclitaxel in the NCI 60 cell investigations. Unfortunately, administration of discodermolide line drug panel screen with a correlation coefficient of as a single agent (30 – 40 mg once per 3 weeks [86]) in Phase I 0.84 [95]. However, eleutherobin is ineffective in P-gp over- clinical trials resulted in several cases of severe pulmonary expressing tumour cells and is also cross-resistant with pacli- toxicity, which hampers its clinical development [87,88]. taxel against a cell line that has a mutation in β-tubulin [95]. These characteristics make eleutherobin less attractive for 4. Newly discovered MSAs anticancer drug development. Sarcodictyin A (20) and B (21) are two close structural The clinical success of the taxanes encouraged the exploration relatives of eleutherobin lacking a carbohydrate moiety. They of new agents with the same cellular target. During the past were isolated from the Mediterranean stolonifer Sarcodictyon decade, several other MSAs have been discovered with great roseum in 1987, and their ability to induce tubulin polymeri-

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science potential as anticancer drug candidates and these are discussed sation was discovered in 1997 [96]. Although their ability to in the following sections (Figure 6). promote tubulin polymerisation and their antiproliferative -7 activity in vitro (IC50 10 M level) are both significantly less 4.1 Dictyostatin than that of paclitaxel, sarcodictyin A and B retain their Dictyostatin (18) is a macrocyclic lactone isolated from cytotoxicity against both P-gp overexpressing MDR cells and a marine sponge in 1994 [89]. It has been shown to effec- paclitaxel-resistant cell lines harbouring tubulin mutations [97]. tively inhibit the growth of selected NCI cancer cell lines There have been several series of analogues of eleutherobin -3 -5 with a GI50 range of 10 – 10 μg/ml [90]. However, its and sarcodictyins isolated with similar biological activity, mechanism of action was not revealed until 2003, when and synthetic derivatives have also been identified with tubulin polymerisation activity was reported by Isbrucker improved antitumour profiles [98-101]. et al. [91]. Further studies showed that the microtubule stabilising properties and potency of dictyostatin were quite similar 4.3 Laulimalide to its close structural analogue discodermolide [92]. In 1999, Mooberry et al. reported the paclitaxel-like Dictyostatin has not yet been thoroughly studied partially microtubule-stabilising activity of two 18-membered macrocyclic

Expert Opin. Ther. Patents (2009) 19(5) 613 Microtubule stabilising agents for cancer chemotherapy

Middle part

O O OH O O

OH NH Lactone 2

Carbamate OH

Figure 5. Chemical moieties of discodermolide.

O N N H

OH O

HO H O O O

For personal use only. O

O O

OH OH OH OH O 18 Dictyostatin 19 Eleutherobin

O H O HO OH O H N H N O

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science H

O O O

H H O H OH

O OR

20 Sarcodictyin A : R = CH3 21 Sarcodictyin B : R = C2H5 22 Laulimalide

Figure 6. New discovered MSAs (compounds 18 – 25). MSA: Microtubule stabilising agent.

614 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

OH H HO O O O H O O HO OH O O OH O O H H O O OH

23 Isolaulimalide 24 Peloruside A

OH HO O O H H H H

H H 25 Cyclostreptin (FR182877)

Figure 6. New discovered MSAs (compounds 18 – 25) (continued). MSA: Microtubule stabilising agent. For personal use only.

lactones, laulimalide (22) and isolaulimalide (23) [102]. Both Laulimalide or its derivatives were found to be synergistic were isolated from the marine sponge Cacospongia mycofijiensis with taxanes both in vitro and in cells [107-109]. Large scale in 1988 [103]. Although the EC50 value of laulimalide for synthetic methodologies were explored to solve the problem in vitro tubulin assembly is a little higher than that of of its supply and chemical instability (for details, see [110]). paclitaxel, longer filaments of microtubules were formed Unfortunately, a recent study revealed that the in vivo when treated with laulimalide. This phenomenon was con- tumour inhibitory activity of laulimalide is disappointing, tradictory to the observation in cells, where longer fila- despite its potent activity in cultured cells, and is accompanied ments were promoted by paclitaxel. Taken together, these by severe toxicity and mortality. These findings limit the observations suggested that the mechanisms of these two development of laulimalide as a clinically useful anticancer MSAs may be distinct [102,104]. Indeed, Pryor et al. reported drug [111].

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science later that laulimalide could not compete with taxanes when binding to microtubules, suggesting that these two 4.4 Peloruside A compounds have different binding sites [105]. Furthermore, Peloruside A (24), a polyketide-based macrolide, was first the growth inhibitory assays of laulimalide against a series isolated in 2000 from the New Zealand sponge Mycale of drug-resistant ovarian carcinoma cells showed that lauli- species [112]. In 2002, its paclitaxel-like activity was reported [113]. malide was active at nM levels both in cells overexpressing A competitive microtubule-binding assay studying peloruside A, P-gp and in cells bearing tubulin mutations. The results paclitaxel and laulimalide revealed that peloruside A did not indicated that laulimalide is a poor substrate for P-gp, and bind to the paclitaxel-site, but to the same site as laulimalide [114]. confirmed a distinct binding site to that of paclitaxel [105]. This observation was confirmed by tubulin polymerisation Further studies by computational approaches suggest that promoting assays, in which either peloruside A or laulimalide there are in fact two laulimalide-binding sites in tubulin. acted synergistically with a number of taxoid-site MSAs, but One is located at β-tubulin and the other at α-tubulin [106]. no synergism was observed between these two compounds [115]. Laulimalide is now the only MSA that seems to be able to Similar synergistic results were also obtained from cultured bind to α-tubulin. cells [116]. In a similar manner to laulimalide, peloruside A

Expert Opin. Ther. Patents (2009) 19(5) 615 Microtubule stabilising agents for cancer chemotherapy

retains antiproliferative activity in P-gp-mediated MDR cells and presence of heteroatom(s) in the lower surface of the and paclitaxel-resistant cells with altered β-tubulin [114]. macrocycle [124]. Shortly after this publication, a common However, a recent report suggested that peloruside A may pharmacophore model between taxanes and epothilones was bind to β-tubulin at a previously unknown site, reducing established by Giannakakou et al. [125]. Using this model, lateral contacts between microtubule protofilaments, which the interaction between sarcodictyins and mutant tubulins were is considered to be a novel mechanism of action [117]. proposed. Since then, a series of taxanes with a macrocycle in the southern half have been designed, including C-4 OAc 4.5 Cyclostreptin (FR182877) methyl and C-3′phenyl tethered macrocyclic taxoids mimicking Cyclostreptin (25), also known as FR182877, is a polycyclic the ‘T-shape’ paclitaxel conformation: for example, C-3′ and natural product isolated from the fermentation broth of C-4 tethered macrocyclic analogue 26 [126], C-14 and C-3′N Streptomyces sp. No. 9885 in 2000 [118]. It showed only weak linked REDOR-taxol, and SB-T-2053 (27) (Figure 7) [127]. paclitaxel-like activity to induce tubulin polymerisation Some of these compounds, especially taxoids mimicking the in vitro [119,120], although it displayed moderate in vivo anti- ‘T-shape’ conformation, exhibited higher activities than tumour activity [118]. In addition, cyclostreptin has been paclitaxel itself. Significantly, the pharmacophore studies on shown to be active in paclitaxel- or epothilone-resistant cells MSAs indicated that at least for compounds binding to the harbouring mutations in β-tubulin or overexpressing P-gp [120]. paclitaxel site, the critical residuals on β-tubulin are essential. Recent studies indicated that cyclostreptin binds irreversibly We foresee that the construction of the pharmacophore and to the taxol-binding site in β-tubulin [121], although the conformational analysis of MSAs bound to microtubules will mechanistic details of the cyclostreptin-microtubule interaction play further critical roles in the discovery of novel MSAs. remain to be fully explained. Although novel MSAs that have similar conformations have been designed to fit the binding pocket, it should be 5. Pharmacophores of MSAs noted that structural similarity does not guarantee the same mode of action. For example, laulimalide has considerable In the past 2 decades, many novel MSAs with different structural resemblance to the epothilones and had initially structural configurations have been discovered. In spite of been hypothesised to share the same binding site with such different skeletal pharmacophores, the MSAs seem to epothilones, but has since been shown not to be the case in later have affinity for the same binding sites (Table 1), indicating that studies [128]. Although structurally distinct from laulimalide, diverse structures may still act through similar mechanisms. peloruside A was found to compete with it on binding to The structural diversity of MSAs, as well as their similar the microtubules.

For personal use only. actions on tubulin/microtubules, encourages the exploration of common MSA pharmacophores to comprehensively 6. Expert opinion understand the ligand–microtubule interactions, and facilitate the design of new antimitotic drugs with simplified structures Despite substantial success in the clinic, MSAs still suffer from based on known MSAs. serious side effects, partially due to a lack of clear differential Despite the structural diversity of known MSAs, it is between microtubule compositions in cancerous and normal apparent that, with the exception of discodermolide, macrocyclic cells. It has been shown from numerous SAR studies that or polycyclic structures dominate in all the other MSAs. the ability of MSAs to promote tubulin polymerisation with However, it has been reported that discodermolide occupies subsequent cytotoxicity varies depending on the skeletal a helical conformation in solution [122], and recent NMR construction of their pharmacophores, suggesting that effective revealed a common pharmacophore model for discodermolide anticancer MSAs with fewer side effects may be achieved by and epothilone [123]. A common pharmacophore for the five appropriate structural modifications.

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science MSAs, paclitaxel, epothilone B, eleutherobin, discodermolide and In addition to tubulin and microtubules as cellular targets, nonataxel, has been proposed by Ojima et al. [124]. The 3D other factors associated with the tubulin polymerisation/ overlays of these agents succeeded in identifying the essential depolymerisation process, such as MAPs, need to be explored, structural elements of paclitaxel: that is, the C-2 benzoate and potential new drug targets may be uncovered in this and the two C-13 side-chain elements C-3′-N-benzoyl and process [8]. Small molecules targeting MAPs, in theory, can C-3′ phenyl group. It was indicated by the modelling results be combined with MSAs to modulate the interactions with that the linkage between the C-3′ phenyl group and the microtubules, the primary anticancer drug target for MSAs. C-2α benzoyl might create a conformational constraint. As we have discussed in Section 2, combined use of Based on this hypothesis, a ‘hybrid compound’ SB-TE-1120 MSAs with many other chemotherapeutic agents with was designed and synthesised, but only exhibited 37% activity different mechanisms of action can potentiate the effects of as compared to paclitaxel in the in vitro assays. In conclu- MSAs, and reduce side effects through decreasing the dosage sion, it was noted that the biological activities of these com- of both agents in combination. A recent review critically pounds show high sensitivity to steric bulk of the substituent summarised the use of MSAs in combined cancer at the C-2α position, size and flexibility of the macrocycle, chemotherapy [129].

616 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

Table 1. Summary of defined MSAs.

Compounds Binding site Effect on MDR tumour cells Status

P-gp overexpression β-tubulin related factors Taxanes Taxol-binding site Paclitaxel and docetaxel are ineffective on most Two taxanes have been clinically MDR cells. Novel taxanes that can reverse MDR approved, and several are in are in development clinical trials Epothilones Taxol-binding site + + Ixabepilone has been clinically approved, and several are in clinical trials Discodermolide Taxol-binding site or overlapped + + Phase I clinical trials are hampered by severe pulmonary toxicity Dictyostatin Same as discodermolide + + Preclinical (probably) Eleutherobin Taxol-binding site (probably) _ _ Preclinical Sarcodictyins Taxol-binding site (probably) + + Preclinical Cyclostreptin Taxol-binding site (irreversibly) + + Preclinical Laulimalide Laulimalide-binding site + + Preclinical Peloruside A The same or overlapped + + Preclinical laulimalide-binding site, or a new site

MDR: Multi-drug resistance; MSA: Microtubule stabilising agent; P-gp: P-glycoprotein.

O O For personal use only.

O O O O OH OH

O O

HO O O O O HN O O O OH O O OH O O O OH O N O H O O Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science

26 27 SB-T-2053

Figure 7. Some synthesised taxanes with macrocycles (compounds 26 and 27).

Expert Opin. Ther. Patents (2009) 19(5) 617 Microtubule stabilising agents for cancer chemotherapy

It can be appreciated why anticancer agents with different which P-gp inhibitors have been given to cancer patients mechanisms of action are synergistic, but it is somewhat along with anticancer drugs have revealed poor systemic clear- surprising that the combination of MSAs with similar binding ance of the administered anticancer drug. Consequently, this sites on the microtubules, in some cases, are also synergistic [74]. has necessitated a reduction of the maximum tolerated doses To a certain extent, it can be explained by the possible of anticancer drugs, thereby, diminishing the therapeutic subtle differences in the mode of binding. Further studies as opportunity as well as increasing the risk of the patient to the rationale behind the synergy between MSAs are developing tumours resistant to MTAs among other classes required, and such studies may also aid in understanding of drug [132]. why MSAs with similar mechanisms of action on tubulin Considering accumulating evidences for the involvement of assembly behave very differently in cells. It should also be β-III tubulin overexpression in drug resistance to MSAs [40-46] taken into consideration whether differences in biological and the effectiveness of taxanes [49] and epothilones [60] against activity are actually due to mechanisms of action or perhaps drug-resistant tumours, we believe that β-III has potential as a are also a reflection of pharmacokinetic and pharmacodynamic novel attractive target for cancer chemotherapeutics against parameters including absorption, distribution, metabolism drug resistance. and poor blood supply of such drugs. Although several classes of MSAs have been reported, A potentially serious problem encountered in clinical there are still many other structurally diverse MSAs yet to chemotherapy with MSAs is MDR, in which overexpressed be identified owing to the complex modulation of assembly/ ATP-binding cassette transporters and tubulin-related drug disassembly of microtubules. Some plant-derived natural resistance (e.g., mutations around MSA-binding sites in products, including terpenoids and steroids [2], have been tubulin/microtubule and high expression of insensitive tubulin deduced to be microtubule-pertubing agents, but further isotypes such as β-III) are present. Whereas P-gp mediated direct evidence is necessary to confirm their interactions with MDR has been well studied in anticancer drug design, other microtubules. The growing family of MSAs will continue to clinically relevant factors contributing to the resistance to expand the range of antitumour agents and deepen our MSAs, for example, overexpression of β-III tubulin, have understanding in ligand–microtubule interactions and biology not been considered seriously. It should be noted that the of microtubule-related processes. efforts to overcome P-gp-mediated drug resistance in clinical trials have proven unsuccessful as observed either by combina- Acknowledgments tion of taxanes and P-gp inhibitors [130] or by the use of potent taxanes such as ortataxel effective against P-gp overexpressing We thank JF Díaz of CIB, CSIC for critical reading of

For personal use only. tumours in animal models [35]. This may be owing to two our manuscript, and J-z Shi for assistance in drawing conflicting issues. First, cancer cells need to lose their chemo- chemical structures. protection mediated by efflux pump proteins including, MDR1 and MRP-1; and second, sensitive non-cancerous Declaration of interest cells, such as rapidly proliferating bone marrow cells, need to retain this intrinsic resistance. Approaches that target Our research works are financially supported by NSFC MDR1 and MRP-1 must be achieved in a way that is (Grant Nos. 20572135, 30630069) and MOST of China cancer specific, so as not to affect the normal function of (Grant No. 2006DFA31490) to W-S.F., and Yorkshire Cancer these proteins in healthy cells [131]. Clinical investigations in Research to K.P. Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science

618 Expert Opin. Ther. Patents (2009) 19(5) Zhao, Fang & Pors

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Expert Opin. Ther. Patents (2009) 19(5) 621 Microtubule stabilising agents for cancer chemotherapy

106. Pineda O, Farràs J, Maccari L, et al. with each other, on tubulin assembly. 125. Giannakakou P, Gussio R, Nogales E, Computational comparison of Mol Pharmacol 2006;70:1555-64 et al. A common pharmacophore for microtubule-stabilising agents laulimalide 116. Wilmes A, Bargh K, Kelly C, et al. epothilone and taxanes: molecular basis and peloruside with taxol and colchicine. Peloruside A synergizes with other for drug resistance conferred by tubulin Bioorg Med Chem Lett 2004;14:4825-9 microtubule stabilizing agents in cultured mutations in human cancer cells. 107. Gapud EJ, Bai R, Ghosh AK, Hamel E. cancer cell lines. Mol Pharm Proc Natl Acad Sci USA 2000;97:2904-9 Laulimalide and paclitaxel: a comparison 2007;4:269-80 126. Ganesh T, Guza RC, Bane S, et al. The of their effects on tubulin assembly and 117. Huzil JT, Chik JK, Slysz GW, et al. A bioactive taxol conformation on β-tubulin: their synergistic action when present unique mode of microtubule stabilization Experimental evidence from highly simultaneously. Mol Pharmacol induced by peloruside A. J Mol Biol active constrained analogs. 2004;66:113-21 2008;378:1016-30 Proc Natl Acad Sci USA 2004;101:10006-11 108. Clark EA, Hills PM, Davidson BS, et al. 118. Sato B, Muramatsu H, Miyauchi M, et al. Laulimalide and synthetic laulimalide A new antimitotic substance, FR182877: I. 127. Geney R, Sun L, Pera P, et al. Use of the analogues are synergistic with paclitaxel Taxonomy, fermentation, isolation, tubulin bound paclitaxel conformation for and 2-methoxyestradiol. Mol Pharm physico-chemical properties and biological structure-based rational drug design. 2006;3:457-67 activities. J Antibiot (Tokyo) Chem Biol 2005;12:339-48 109. Lu H, Murtagh J, Schwartz EL. 2000;53:123-30 128. Ghosh AK. Microtubule stabilizing The microtubule binding drug laulimalide 119. Sato B, Nakajima H, Hori Y, compounds. US20030203929; 2003 inhibits vascular endothelial growth et al. A new antimitotic substance, 129. Bergstralh DT, Ting JP-Y. Microtubule factor-induced human endothelial cell FR182877: II. The mechanism of action. stabilizing agents: their molecular signaling migration and is synergistic when J Antibiot 2000;53:204-6 consequences and the potential for combined with docetaxel (Taxotere). 120. Edler MC, Buey RM, Gussio R, et al. enhancement by drug combination. Mol Pharmacol 2006;69:1207-15 Cyclostreptin (FR182877), an antitumor Cancer Treat Rev 2006;32:166-79 110. Mulzer J, Öhler E. Microtubule-stabilizing tubulin-polymerizing agent deficient in 130. Kaye SB. Multidrug resistance: clinical marine metabolite laulimalide and its enhancing tubulin assembly despite its high relevance in solid tumours and strategies derivatives: synthetic approaches and affinity for the taxoid site. Biochemistry for circumvention. Curr Opin Oncol antitumor activity. 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Sánchez-Pedregal VM, Kubicek K, Meiler J, †Author for correspondence 2000;65:445-9 et al. The tubulin-bound conformation 1Key Laboratory of Bioactive Substances and 113. Hood KA, West LM, Rouwé B, et al. of discodermolide derived by NMR Resources Utilization of Chinese Herbal Medicine Peloruside A, a novel antimitotic agent with studies in solution supports a common (Peking Union Medical College), paclitaxel-like microtubule-stabilizing pharmacophore model for epothilone and Ministry of Education, activity. Cancer Res 2002;62:3356-60 discodermolide. Angew Chem Int Ed Engl Beijing 100050, China 114. Gaitanos TN, Buey RM, Díaz JF, et al. 2006;45:7388-94 Tel: +86 10 6316 5229; Fax: +86 10 6301 7757; Expert Opin. Ther. Patents Downloaded from informahealthcare.com by Chinese Academy Of Medical Science Peloruside A does not bind to the taxoid 124. Ojima I, Chakravarty S, Inoue T, E-mail: [email protected] 2 site on β-tubulin and retains its activity in et al. A common pharmacophore for Institute of Materia Medica, multidrug-resistant cell lines. Cancer Res cytotoxic natural products that stabilize Chinese Academy of Medical Sciences, 2004;64:5063-7 microtubules. Proc Natl Acad Sci USA 1 Xian Nong Tan Street, 115. Hamel E, Day BW, Miller JH, et al. 1999;96:4256-61 Beijing 100050, China 3 Synergistic effects of peloruside A and • Describes the common pharmacophore of University of Bradford, laulimalide with taxoid site drugs, but not five MSAs. Institute of Cancer Therapuetics, Richmond Road, Bradford, West Yorkshire, BD7 1DP, UK

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