Targeting an Old Foe for Cancer: A Molecular Dynamics Perspective to Unravel the Specific Binding Nature of 2-Methoxy Estradiol to Human ß-Tubulin Isotypes.

Microtubules, composed of α- and ß-tubulin subunits are crucial for cell division with their dynamic tissue-specificity which is dictated by expression of isotypes. These isotypes differ in carboxy-terminal tails (CTTs), rich in negatively charged acidic residues in addition to the differences in the composition of active site residues. 2-Methoxy estradiol (2-ME) is the first antimicrotubule agent that showed less affinity toward hemopoietic-specific ß1 isotype consequently preventing myelosuppression toxicity. The present study focuses on the MD-directed conformational analysis of 2-ME and estimation of its binding affinity in the colchicine binding pocket of various ß-tubulin isotypes combined with the α-tubulin isotype, α1B. AlphaFold 2.0 was used to predict the 3D structure of phylogenetically divergent human ß-tubulin isotypes in dimer form with α1B. The dimeric complexes were subjected to induced-fit docking with 2-ME. The statistical analysis of docking showed differences in the binding characteristics of 2-ME with different isotypes. The replicas of atom-based molecular dynamic simulations of the best conformation of 2-ME provided insights into the molecular-level details of its binding pattern across the isotypes. Furthermore, the MM/GBSA analyses revealed the specific binding energy profile of 2-ME in ß-tubulin isotypes. It also highlighed, 2-ME exhibits the lowest binding affinity toward the ß1 isotype as supported by experimental study. The present study may offer useful information for designing next-generation antimicrotubule agents that are more specific and less toxic.

The multifaceted NF-kB: are there still prospects of its inhibition for clinical intervention in pediatric central nervous system tumors?

Despite advances in the understanding of the molecular mechanisms underlying the basic biology and pathogenesis of pediatric central nervous system (CNS) malignancies, patients still have an extremely unfavorable prognosis. Over the years, a plethora of natural and synthetic compounds has emerged for the pharmacologic intervention of the NF-kB pathway, one of the most frequently dysregulated signaling cascades in human cancer with key roles in cell growth, survival, and therapy resistance. Here, we provide a review about the state-of-the-art concerning the dysregulation of this hub transcription factor in the most prevalent pediatric CNS tumors: glioma, medulloblastoma, and ependymoma. Moreover, we compile the available literature on the anti-proliferative effects of varied NF-kB inhibitors acting alone or in combination with other therapies in vitro, in vivo, and clinical trials. As the wealth of basic research data continues to accumulate, recognizing NF-kB as a therapeutic target may provide important insights to treat these diseases, hopefully contributing to increase cure rates and lower side effects related to therapy.

Synthesis and biological evaluation of new 2-methoxyestradiol derivatives: Potent inhibitors of angiogenesis and tubulin polymerization.

Here, we report the structural optimization of a hit natural compound, 2-ME2 (2-methoxyestradiol), which exhibited inhibitory activity but low potency on tubulin polymerization, anti- angiogenesis, MCF-7 proliferation and metastasis in vitro and in vivo. A novel series of 3,17-modified and 17-modified analogs of 2-ME2 were synthesized and investigated for their antiproliferative activity against MCF-7 and another five different human cancer cell lines leading to the discovery of 9i. 9i bind to tubulin colchicine site tightly, inhibited tubulin polymerization and disrupted cellular microtubule networks. Cellular mechanism studies revealed that 9i could induce G2/M phase arrest by down-regulated expression of p-Cdc2, P21 and cell apoptosis by regulating apoptosis-related proteins (Parp, Caspase families) in a dose-dependent manner. Importantly, 9i significantly inhibited HUVEC tube formation, proliferation, migration and invasion. The inhibitory effect against angiogenesis in vivo was confirmed by zebrafish xenograft. Furthermore, 9i could effectively inhibit the proliferation and metastasis of MCF-7 cells in vitro and in zebrafish xenograft. The satisfactory physicochemical property and metabolic stability of 9i further indicated that it can act as a promising and potent anti-angiogenesis, inhibiting proliferation and metastasis of breast cancer agent via targeting tubulin colchicine binding site.

PEG-PCL modification and intestinal sustained-release of solid lipid nanoparticles for improving oral bioavailability of 2-methoxyestradiol.

The primary purpose of the present study was to design and optimize a solid lipid nanoparticle (SLN) formulation of the poorly water-soluble drug 2-methoxyestradiol (2-ME) to improve its oral bioavailability and prolong the duration of therapeutic drug level. SLN was modified by amphipathic PEG-PCL (PLN) and then encapsulated in pH-sensitive microparticles (MP) by spray drying technology. Several properties of 2-ME PLN-MP were characterized including particle size, drug loading, and drug or PLN release. After oral administration of 2-ME PLN-MP, retention time in mice was evaluated by in vivo imaging technology and the pharmacokinetic parameters in rats were determined by HPLC. The results demonstrated that PEG-PCL modification of 2-ME SLN significantly decreased particle size and delayed drug release without influencing IC50 in 4T1 cells. 2-ME PLN in the microparticles showed significant pH-sensitive release in the simulated gastrointestinal fluid and controlled release in the intestine. The PLN (labelled with IR-780 iodide) prolonged significantly fluorescence duration time compared to the SLN and the prolongation was further enhanced by the PLN-MP formulation. Furthermore, compared with the suspension, the PLN-MP formulation showed a 56.66-fold delay in Tmax, a 10.36-fold extension in MRT and a 140.86-fold increase in the relative bioavailability in the rat. The research work in the paper suggests that the PLN-MP could serve as a practical oral preparation for 2-ME in future cancer therapy.

Liposomal 2-Methoxyestradiol Nanoparticles for Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model.

Uterine leiomyomas represent a challenging problem with limited medical treatment options. The anti-tumor agent 2-methoxyestradiol (2-ME) shows promising results but its efficacy is limited by inadequate pharmacokinetics. We previously demonstrated that 2-ME nanoparticles can be successfully formulated and that they show improved in vitro anti-leiomyoma cell activity. Here, we examined the effects of the in vivo delivery of 2-ME nanoparticles in a patient-derived xenograft (PDX) leiomyoma mouse model. Patient-derived leiomyoma tumor tissues were xenografted subcutaneously in estrogen/progesterone pretreated immunodeficient NOG mice. Animals (n = 12) were treated with liposomal 2-ME nanoparticles by intra-peritoneal (IP) injection (50 mg/kg/dose, three times weekly) or control for 28 days. Tumor volume was measured weekly by calipers and prior to sacrifice by ultrasound. In addition, the expression of the cell proliferation marker Ki67 and the apoptosis marker cleaved caspase-3 in tumor tissues after treatment were measured by immunohistochemistry. Liposomal 2-ME treatment was associated with a significant tumor growth inhibition (30.5% less than controls as early as 2 weeks, p = 0.025). In addition, injections of liposomal 2-ME inhibited the expression of the proliferation marker Ki67 (55.8% reduction, p < 0.001). Furthermore, liposomal 2-ME treatment was associated with a 67.5% increase of cleaved caspase-3 expression of increase (p = 0.048). Our findings suggest that liposomal nanoparticle formulation can successfully deliver 2-ME and can be a promising therapeutic strategy for uterine leiomyoma. Further characterization of the liposomal-2ME, including pharmacokinetics, maximal tolerated dose, and safety, is needed in preclinical models prior to clinical trials.

Concise synthesis of 2-methoxyestradiol from 17ß-estradiol through the C(sp2)-H hydroxylation.

A four-step route for the synthesis of 2-methoxyestradiol (5) starting from 17ß-estradiol (1) has been achieved with a 51% overall yield. The key step was the ruthenium-catalyzed ortho-C(sp2)-H bond hydroxylation of aryl carbamates. Using dimethyl carbamate as the directing group, [RuCl2(p-cymene)]2 as the catalyst, PhI(OAc)2 as the oxidant and trifluoroacetate/trifluoroacetic anhydride (1:1) as the co-solvent, the hydroxyl group could be singly installed at the 2-position of 3-dimethylcarbamoyloxyestradiol (2) with 65% yield. Subsequent methylation of hydroxy and removal of dimethyl carbamate afforded 2-methoxyestradiol (5).

MgO nanoparticles coated with polyethylene glycol as carrier for 2-Methoxyestradiol anticancer drug.

Novel Magnesium Oxide (MgO) nanoparticles (NPs) modified with the polymer polyethylene glycol (PEG) were synthesized as carrier for the anticancer drug 2-Methoxyestradiol (2ME) to improve its clinical application. The functionalized NPs were characterized by Infrared spectroscopy with Fourier transform to elucidate the vibration modes of this conjugate, indicating the formation of the MgO-PEG-2ME nanocomposite. The studies of absorption and liberation determined that MgO-PEG-2ME NPs incorporated 98.51 % of 2ME while liberation of 2ME was constant during 7 days at pH 2, 5 and 7.35. Finally, the MgO-PEG-2ME NPs decreased the viability of the prostate cancer cell line LNCap suggesting that this nanocomposite is suitable as a drug delivery system for anticancer prostate therapy.

Angiopep-2 modified PEGylated 2-methoxyestradiol micelles to treat the PC12 cells with oxygen-glucose deprivation/reoxygenation.

2-Methoxyestradiol (2ME2), as a microtubule and hypoxia-inducible factor-1 (HIF-1) inhibitor, can be used to treat cerebral ischemia-reperfusion (I/R) injury. However, its poor water solubility compromises its efficacy as a neuroprotectant. Herein, we synthesized PEGylated 2ME2 and angiopep-2 capped PEGylated 2ME2 and fabricated angiopep-2 modified PEGylated 2ME2 micelles containing free 2ME2 (ANG-PEG-2ME2/2ME2) via emulsion-solvent evaporation method. The effect of the micelles on ischemia-reoxygenation injury was evaluated by oxygen-glucose deprivation/reoxygenation (OGD/R) models with different degrees of PC12 cell damage. In comparison with free 2ME2, the micelles significantly increased the cell viability, inhibited reactive oxygen species (ROS) generation and apoptosis for PC12 cells with 0.5 and 4 h OGD followed by 24 h reoxygenation. Taken together, the angiopep-2 modified 2ME2-loaded micelles could effectively reduce the injury of PC12 cells induced by OGD/R.

Synthesis, molecular modeling and biological evaluation of potent analogs of 2-methoxyestradiol.

The endogenous steroid 2-methoxyestradiol (1) has attracted a great interest as a lead compound towards the development of new anti-cancer drugs. Herein, the synthesis, molecular modeling, anti-proliferative and anti-angiogenic effects of ten 2-ethyl and four 2-methoxy analogs of estradiol are reported. The ethyl group was introduced to the steroid A-ring using a novel Friedel-Crafts alkylation protocol. Several analogs displayed potent anti-proliferative activity with IC50-values in the submicromolar range towards the CEM human leukemia cancer cell line. As such, all of these compounds proved to be more active than the lead compound 2-methoxyestradiol (1) in these cells. The six most cytostatic analogs were also tested as anti-angiogenic agents using an in vitro tube formation assay. The IC50-values were determined to be in the range of 0.1 µM ±â€¯0.03 and 1.1 µM ±â€¯0.2. These six compounds were also modest inhibitors against tubulin polymerization with the most potent inhibitor was 14b (IC50 = 2.1 ±â€¯0.1 µM). Binding studies using N,N'-ethylene-bis(iodoacetamide) revealed that neither14a or 14b binds to the colchicine binding site in the tubulin protein, in contrast to 2-methoxyestradiol (1). These observations were supported by molecular modeling studies. Results from a MDA-MB-231 cell cycle assay showed that both 10e and 14b gave accumulation in the G2/M phase resulting in induction of apoptosis. The results presented herein shows that the novel analogs reported exhibit their anticancer effects via several modes of action.