Estradiol dimer inhibits tubulin polymerization and microtubule dynamics
Graphical abstract
Introduction
Steroids and their synthetic analogs belong to a class of compounds exhibiting diverse biological activities. In present days, steroids are used as anti-inflammatory, anti-cancer, anabolic, progestational, contraceptive and neuroactive agents [1,2]. Dimeric steroids are unique entities which offer special features applicable in various fields [3]. From the pool of steroid dimers isolated from natural sources, mainly from marine organisms, cephalostatins [4], crellastatins [3] and ritterazins [5] are notable due to their high cytotoxic activity against cancer cells. To date, a number of steroidal dimers, including dimers of estradiol [6], testosterone [[7], [8], [9], [10]] and pregnenolone [11,12] have been synthesized and studied. From those analyzed to date, it is evident that biologically important properties of dimerized steroids largely depend upon the particular connecting bridge and structural features of the steroid ring system.
Microtubules are cytoskeletal filaments composed of tubulin subunits. They are a key component of the cytoskeleton and are essential in all eukaryotic cells. Microtubules are highly dynamic protein polymers composed of α-tubulin and β-tubulin heterodimers and their polymerization dynamics is tightly regulated both spatially and temporally. Microtubule-active drugs mostly bind to one of three main sites on tubulin, the paclitaxel site, the Vinca domain or the colchicine domain. As microtubules remain one of the most effective cancer chemotherapeutic targets, new drugs targeting microtubules are in different stages of clinical trials and a large number of microtubule-active compounds are being developed [13]. For a long time, it was understood that these compounds exerted their biological activities by either stabilizing or destabilizing microtubules and thereby increasing or decreasing microtubule-polymer mass, and by suppressing microtubule dynamics [14]. Extensive new data in vitro and in vivo demonstrate that the effects of microtubule targeting drugs are not due only to their ability to suppress microtubule dynamics of the mitotic spindle leading to antimitotic effects. Disruption of tubulin assembly and microtubule dynamics has extensive effects on multiple cellular processes including transport of proteins, organelles and RNA [15].
Within the long list of synthetic or naturally occurring compounds interacting with microtubules, small molecules with steroid structure represent an important class. Estramustine is an estradiol synthetic conjugate with nitrogen mustard, exhibiting antitubulin activity [16], and which has long been used in the treatment of advanced prostate cancer [17]. 2-Methoxyestradiol (2-ME) is by far the most widely studied steroid molecule with antiproliferative and antiangiogenic properties [18], interfering with microtubules by competitively binding to the colchicine site in tubulin [19]. 2-ME is a naturally occurring metabolite of estradiol with very low toxicity and good oral availability. It was investigated under various clinical trials under the name Panzem (EntreMed), alone or in combination therapy. Promising results were collected in clinical trials for the treatment of hormone-refractory prostate cancer [20], multiple myeloma [21], and recurrent and platinum resistant epithelial ovarian cancer [22]. Nevertheless, these studies also revealed that the bioavailability of 2-ME might be a limiting factor [20,21]. 2-ME inspired synthesis and biological evaluation of a large number of analogs and prodrugs [23,24]. From these, ENMD-1198 exhibited significantly better antiproliferative and antitumor activity compared to 2-ME [25]. Although the outcome from phase I clinical trial was encouraging, there is no evidence that ENMD-1198 progressed into Phase II clinical trials [26].
In contrast to a variety of estrogens reported to interact with microtubules, we found no evidence of other steroid hormone-based microtubule destabilizers in the literature. This finding together with our previous work on dimerized steroids [27,28] led us to design four dimeric molecules with different steroid moieties connected with short linker and investigated their biological properties with respect to steroid hormones receptors, antimitotic and anticancer activities and their ability to interact specifically with microtubules.
Section snippets
Materials
17α-Ethynylestradiol, mestranol and ethisterone were purchased from Steraloids Inc. Paclitaxel was a gift from the National Cancer Institute, (USA, Bethesda). Nocodazole, E2, DHT and 2-ME were purchased from Sigma-Aldrich.
Chemistry: general techniques
All chemicals, reagents and solvents were used without further purification as purchased from commercial sources. Coupling unit 2,6-bis(azidomethyl)pyridine [29] and 24-norchol-5-en-22-yn-3β-ol [30] were synthesized according to protocols found in literature. Plates coated by
Design and synthesis of steroid dimers
Four different steroid molecules including estradiol, 3-O-methyl estradiol, testosterone and pregnenolone were linked via bis(azidomethyl)pyridine connected to steroid D rings. These steroids structurally differ in the shape of A-ring, presence of functional groups and position of double bonds. To keep the A-ring untouched, we used corresponding ethynylated steroids at the C-17 position, in the case of estradiol, 3-O-methyl estradiol and testosterone, and at the C-20 position in the case of
Discussion
Wide biological characterization of series of steroidal dimers derived from estradiol, testosterone and pregnenolone revealed that the dimerization introduced a surprising array of biological properties. First, the dimers showed markedly reduced ability to modulate the activity of the respective steroid receptors, ERα and AR. At the same time, both estradiol derivatives 1 and 2 and testosterone dimer 3 exerted general cytotoxicity in the micromolar range, a property that was not observed with
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
We are grateful to E. Zborníková for HPLC analyses of tested compounds and T. Epp for language correction. This work was supported by grants 20-SVV/2017, LO1220, LO1304, LO1601 and LM2015063(MEYS), IGA-LF-2017-030(Palacky University), 15-22194S and 16-25159S(GAČR), GAUK 888713(Grant Agency of Charles University), RVO 68378050(Institutional Research Support) and OPPCCZ.2.16/3.1.00/21537. Computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085.
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These authors contributed equally to this work.