Potent taccalonolides, AF and AJ, inform significant structure-activity relationships and tubulin as the binding site of these microtubule stabilizers

The taccalonolides are a class of microtubule stabilizing agents isolated from plants of the genus Tacca. In efforts to define their structure-activity relationships, we isolated five new taccalonolides, AC-AF and H2, from one fraction of an ethanol extract of Tacca plantaginea. The structures were elucidated using a combination of spectroscopic methods, including 1D and 2D NMR and HR-ESI-MS. Taccalonolide AJ, an epoxidation product of taccalonolide B, was generated by semisynthesis. Five of these taccalonolides demonstrated cellular microtubule-stabilizing activities and antiproliferative actions against cancer cells, with taccalonolide AJ exhibiting the highest potency with an IC(50) value of 4.2 nM. The range of potencies of these compounds, from 4.2 nM to >50 μM, for the first time provides the opportunity to identify specific structural moieties crucial for potent biological activities as well as those that impede optimal cellular effects. In mechanistic assays, taccalonolides AF and AJ stimulated the polymerization of purified tubulin, an activity that had not previously been observed for taccalonolides A and B, providing the first evidence that this class of microtubule stabilizers can interact directly with tubulin/microtubules. Taccalonolides AF and AJ were able to enhance tubulin polymerization to the same extent as paclitaxel but exhibited a distinct kinetic profile, suggesting a distinct binding mode or the possibility of a new binding site. The potencies of taccalonolides AF and AJ and their direct interaction with tubulin, together with the previous excellent in vivo antitumor activity of this class, reveal the potential of the taccalonolides as new anticancer agents.     

α-Trifluoromethyl Chalcones as Potent Anticancer Agents for Androgen Receptor-Independent Prostate Cancer

α-Trifluoromethyl chalcones were prepared and evaluated for their antiproliferative activities against androgen-independent prostate cancer cell lines as well as five additional types of human tumor cell lines. The most potent chalcone 5 showed superior antitumor activity in vivo with both oral and intraperitoneal administration at 3 mg/kg. Cell-based mechanism of action studies demonstrated that 5 induced cell accumulation at sub-G1 and G2/M phases without interfering with microtubule polymerization. Furthermore, several cancer cell growth-related proteins were identified by using chalcone 5 as a bait for the affinity purification of binding proteins.     

In Vivo Evaluation of (−)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (−)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (−)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (−)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.     

Synthesis of Novel Thiol Taxoid Based on the 7,10-Di-(2,2,2-trichloroethyl-oxycarbonyl)-10-deacetylbaccatin Ⅲ and (4R,5S)-2,4-Diphenyl-2-oxazoline-5-carboxylic acid:(2''R,3''R)-10-Deacetyl- 2''-deoxy-mercaptopaclitaxel

The complex natural product paclitaxel (TaxolR) 1, first isolated from Taxus brevifolia, and its semisynthetic analog,docetaxel 2, represent a large family of taxane diterpenoids, which revealed excellent clinical activity against ovarian and breast cancers, and showed promising results in the treatment of other cancers.[1] Extensive studies on the structure activity relationship (SAR) have been explored. It is already well known that a free hydroxyl group at the C-2' position on the C-13 side chain is crucial for microtubule binding[2] and may act as a hydrogen bond donor.     

Enhancement of Anti-Tumoral Properties of Paclitaxel Nano-Crystals by Conjugation of Folic Acid to Pluronic F127: Formulation Optimization,In Vitro and In Vivo Study

A brand-new nano-crystal (NC) version of the hydrophobic drug Paclitaxel (PT) were formulated for cancer treatment. A stable NC formulation for the administration of PT was created using the triblock co-polymer Pluronic F127. To achieve maximum entrapment effectiveness and minimal particle size, the formulation was improved using the central composite design by considering agitation speed and vacuum pressure at five levels (coded as +1.414, +1, 0, −1, and −1.414). According to the Design Expert software’s predictions, 13 runs were created and evaluated for the chosen responses. The formulation prepared with an agitation speed of 1260 RPM and a vacuum pressure of 77.53 mbar can meet the requirements of the ideal formulation in order to achieve 142.56 nm of PS and 75.18% EE, according to the level of desirability (D = 0.959). Folic acid was conjugated to Pluronic F127 to create folate receptor-targeted NC. The drug release profile of the nano-crystals in vitro demonstrated sustained release over an extended period. Folate receptor (FR)-targeted NC (O-PT-NC-Folate) has also been prepared by conjugating folic acid to Pluronic F127. MTT test is used to validate the targeting efficacy on the FR-positive human oral cancer cell line (KB). At pharmacologically relevant concentrations, the PT nano-crystal formulation did not cause hemolysis. Compared to non-targeted NC of PT, the O-PT-NC-Folate showed a comparable but more sustained anti-cancer effect, according to an in vivo anti-tumor investigation in NCI/ADR-RES cell lines. The remarkable anti-tumor effectiveness, minimal toxicity, and simplicity of scale-up manufacturing of the NC formulations indicate their potential for clinical development. Other hydrophobic medications that are formulated into nano-systems for improved therapy may benefit from the formulation approach.     

Evaluation of In Vitro Cytotoxic Potential of Avarol towards Human Cancer Cell Lines and In Vivo Antitumor Activity in Solid Tumor Models

The goal of this study was to determine the activity in vitro and in vivo of avarol, a sesquiterpene hydroquinone originating from the Dysidea avara sponge from the south Adriatic Sea, against different cancer cell lines and two types of mouse carcinoma. To investigate the in vitro cytotoxicity, a human cervix adenocarcinoma cell line (HeLa), human colon adenocarcinoma (LS174), human non-small-cell lung carcinoma (A549), and a normal human fetal lung fibroblast cell line (MRC-5) were used. The in vivo antitumor activity was investigated against two transplantable mouse tumors, the Ehrlich carcinoma (EC) and cervical cancer (CC-5). The effect of avarol on cancer cell survival, which was determined by the microculture tetrazolium test, confirmed a significant in vitro potency of avarol against the investigated cell lines, without selectivity towards MRC-5. The highest cytotoxicity was exhibited against HeLa cancer cells (10.22 ± 0.28 μg/mL). Moreover, potent antitumor activity against two tumor models was determined, as the intraperitoneal administration of avarol at a dose of 50 mg/kg resulted in a significant inhibition of tumor growth in mice. After three administrations of avarol, a 29% inhibition of the EC growth was achieved, while in the case of CC-5, a 36% inhibition of the tumor growth was achieved after the second administration of avarol. Therefore, the results indicate that this marine sesquiterpenoid hydroquinone could be a promising bioactive compound in the development of new anticancer medicine.     

Magnetic nanoparticle-peptide conjugates for in vitro and in vivo targeting and extraction of cancer cells

Magnetic cobalt spinel ferrite nanoparticles coated with biocompatible polygalacturonic acid were functionalized with ligands specific for targeting expressed EphA2 receptors on ovarian cancer cells. By using such magnetic nanoparticle-peptide conjugates, targeting and extraction of malignant cells were achieved with a magnetic field. Targeting ovarian cancer cells with receptor specific peptide-modified magnetic nanoparticles resulted in cell capture from a flow stream in vitro and from the peritoneal cavity of mice in vivo. Successful removal of metastatic cancer cells from the abdominal cavity and circulation using magnetic nanoparticle conjugates indicate the feasibility of a dialysis-like treatment and may improve long-term survival rates of ovarian cancer patients. This approach can be applied for fighting other cancers, such as leukemia, once the receptors on malignant cells are identified and the efficacy of targeting ligands is established.     

Boosting the photodynamic therapy efficiency with a mitochondria-targeted nanophotosensitizer

A nanophotosensitizer with outstanding mitochondrion-targeting ability was developed and the enhanced photodynamic therapy efficiency both in cancer cells and xenograft tumor models was successfully realized.     

BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.     

Chemo-enzymatic synthesis of ester-linked taxol-oligosaccharide conjugates as potential prodrugs

7-Glycolylpaclitaxel 2″-O-α-glucooligosaccharides, novel taxol (paclitaxel) prodrugs of ester-linked oligosaccharide series compounds, were synthesized by chemo-enzymatic procedures, including enzymatic transglycosylations with α-glucosidase and cyclodextrin glucanotransferase. The water-solubility of 7-glycolylpaclitaxel 2″-O-α-glucopentaoside was 2.7 mM, which was 6.8 thousand-fold higher than that of paclitaxel. C-7 modification of paclitaxel with a longer oligosaccharide chain decreased the in vitro cytotoxicity of paclitaxel against KF human ovarian cancer cells.     

Synthesis and antitumor activity of heterocyclic acid ester derivatives of 20S-camptothecins

A series of heterocyclic acid esters of camptothecins as new compounds had been synthesized by acylation method,their in vitro and in vivo antitumor activities were evaluated.The cytotoxic results showed that 6 possessed the best efficacy on six human cancer cell lines in the six classes of CPTs' derivatives.In vivo testing results indicated that 9 had better antitumor activity against mouse liver carcinoma H_(22) than topotecan.     

Carnosine Potentiates Doxorubicin-Induced Cytotoxicity in Resistant NCI/ADR-RES Cells by Inhibiting P-Glycoprotein—In Silico and In Vitro Evidence

The activity of the P-glycoprotein (P-gp) transporter encoded by the ABCB1 gene confers resistance to anticancer drugs and contributes to cancer-related mortality and morbidity. Recent studies revealed the cytotoxic effects of the endogenous dipeptide carnosine. The current study aimed to investigate the role of carnosine as a potential inhibitor of P-gp activity. We used molecular docking and molecular dynamic simulations to study the possible binding and stability of carnosine-P-gp interactions compared with verapamil. In vitro assays using doxorubicin-resistant NCI/ADR-RES cells were established to test the effects of carnosine (10–300 µM) on P-gp activity by the rhodamine-123 efflux assay and its effect on cell viability and doxorubicin-induced cytotoxicity. Verapamil (10 µM) was used as a positive control. The results showed that carnosine binding depends mainly on hydrogen bonding with GLU875, GLN946, and ALA871, with a higher average Hbond than verapamil. Carnosine showed significant but weaker than verapamil-induced rhodamine-123 accumulation. Carnosine and verapamil similarly inhibited cell viability. However, verapamil showed a more significant potentiating effect on doxorubicin-induced cytotoxicity than a weaker effect of carnosine at 300 µM. These results suggest that carnosine inhibits P-gp activity and potentiates doxorubicin-induced cytotoxicity at higher concentrations. Carnosine might be a helpful lead compound in the fight against multidrug-resistant cancers.     

Use of the tubulin bound paclitaxel conformation for structure-based rational drug design

A new computational docking protocol has been developed and used in combination with conformational information inferred from REDOR-NMR experiments on microtubule bound 2-(p-fluorobenzoyl)paclitaxel to delineate a unique tubulin binding structure of paclitaxel. A conformationally constrained macrocyclic taxoid bearing a linker between the C-14 and C-3'N positions has been designed and synthesized to enforce this "REDOR-taxol" conformation. The novel taxoid SB-T-2053 inhibits the growth of MCF-7 and LCC-6 human breast cancer cells (wild-type and drug resistant) on the same order of magnitude as paclitaxel. Moreover, SB-T-2053 induces in vitro tubulin polymerization at least as well as paclitaxel, which directly validates our drug design process. These results open a new avenue for drug design of next generation taxoids and other microtubule-stabilizing agents based on the refined structural information of drug-tubulin complexes, in accordance with typical enzyme-inhibitor medicinal chemistry precepts.     

The Design,Synthesis, and Biological Activities of Pyrrole-Based Carboxamides: The Novel Tubulin Inhibitors Targeting the Colchicine-Binding Site

Microtubule targeting agents (MTAs) that interfere with the dynamic state of the mitotic spindle are well-known and effective chemotherapeutic agents. These agents interrupt the microtubule network via polymerization or depolymerization, halting the cell cycle progression and leading to apoptosis. We report two novel pyrrole-based carboxamides (CAs) (CA-61 and -84) as the compounds exhibiting potent anti-cancer properties against a broad spectrum of epithelial cancer cell lines, including breast, lung, and prostate cancer. The anti-cancer activity of CAs is due to their ability to interfere with the microtubules network and inhibit tubulin polymerization. Molecular docking demonstrated an efficient binding between these ligands and the colchicine-binding site on the tubulin. CA-61 formed two hydrogen bond interactions with THR 179 (B) and THR 353 (B), whereas two hydrogen bonds with LYS 254 (B) and 1 with ASN 101 (A) were identified for CA-84. The binding energy for CA-84 and CA-61 was −9.910 kcal/mol and −9.390 kcal/mol. A tubulin polymerization assay revealed a strong inhibition of tubulin polymerization induced by CA-61 and -84. The immunofluorescence data revealed the disruption of the tubulin assembly in CA-treated cancer cells. As an outcome of the tubulin inhibition, these compounds halted the cell cycle progression in the G2/M phase, leading to the accumulation of the mitotic cells, and further induced apoptosis. Lastly, the in vivo study indicated that CAs significantly inhibited the HCC1806 breast cancer xenograft tumor growth in a nude mouse model. Collectively, we identified the novel CAs as potent MTAs, inhibiting tubulin polymerization via binding to the colchicine-binding site, disrupting the microtubule network, and exhibiting potent pro-apoptotic activities against the epithelial cancer cell lines both in vitro and in vivo.     

PBK drives PARP inhibitor resistance through the TRIM37/NFκB axis in ovarian cancer

Resistance to PARP inhibitors (PARPi) remains a therapeutic challenge in ovarian cancer patients. PDZ-binding kinase (PBK) participates in the chemoresistance of many malignancies. However, the role of PBK in PARPi resistance of ovarian cancer is obscure. In the current study, we demonstrated that overexpression of PBK contributed to olaparib resistance in ovarian cancer cells. Knockdown of PBK sensitized olaparib-resistant SKOV3 cells to olaparib. Inhibition of PBK using a specific inhibitor enhanced the therapeutic efficiency of olaparib. Mechanically, PBK directly interacted with TRIM37 to promote its phosphorylation and nuclear translocation. which subsequently activates the NFκB pathway. Additionally, PBK enhanced olaparib resistance of ovarian cancer by regulating the NFκB/TRIM37 axis in vitro and in vivo. In conclusion, PBK confers ovarian cancer resistance to PARPi through activating the TRIM37-mediated NFκB pathway, and targeted inhibition of PBK provided the new therapy to improve PARPi treatment outcomes for ovarian cancer patients.Subject terms: Ovarian cancer, Cancer therapeutic resistance, Mechanisms of disease, Translational research     

Benzofuran-isatin conjugates as potent VEGFR-2 and cancer cell growth inhibitors

A series of benzofuran-isatin conjugates 6a-l and 7a,b tethered by various alkyl linkers were synthesized and evaluated for their VEGFR-2 inhibitory activity and in vitro activity against a panel of cancer cell lines. Seven of them were comparable with or better than Sunitinib against all tested cancer cells, demonstrating benzofuran-isatin conjugates were potential anticancer candidates. The mechanism study revealed that VEGFR-2 was at least one of the targets for this kind of conjugates. The structure-activity relationship demonstrated that the carbon spacer between benzofuran and isatin moieties, substituents on the C-2 position of benzofuran moiety, and substituents on C-3 as well as C-5 position of isatin motif influenced the anticancer activity significantly, and the enriched structure-activity relationship may provide an insight for rational design of more effective conjugates.     

Effects of C-17 heterocyclic substituents on the anticancer activity of 2-ethylestra-1,3,5(10)-triene-3-O-sulfamates: synthesis, in vitro evaluation and computational modelling

The potent activity of 2-substituted estra-1,3,5(10)-triene-3-O-sulfamates against the proliferation of cancer cells in vitro and tumours in vivo highlights the therapeutic potential of such compounds. Optimal activity is derived from a combination of a 2-XMe group (where X = CH(2), O or S), a 3-O-sulfamate group in the steroidal A-ring and a H-bond acceptor around C-17 of the D-ring. Herein, we describe the synthesis and anti-proliferative activities of a series of novel 2-substituted estra-1,3,5(10)-triene-3-O-sulfamates bearing heterocyclic substituents (oxazole, tetrazole, triazole) tethered to C-17. In vitro evaluation of these molecules revealed that high anti-proliferative activity in breast and prostate cancer cells lines (GI(50) of 340-850 nM) could be retained when the heterocyclic substituent possesses H-bond acceptor properties. A good correlation between the calculated electron density of the heterocyclic ring and anti-proliferative activity was observed. Docking of the most active compounds into their putative site of action, the colchicine binding site of tubulin, suggests that they bind through a different mode to the previously described bis-sulfamate derivatives and 1 and 2, which possess similar in vitro activity.     

Design,synthesis and cytotoxicity of nitrogen-containing tanshinone derivatives

Tanshinones were used as starting material to synthesize a small library of nitrogen heterocyclic derivatives featured with oxazole, imidazole, and pyrazine ring between C-11/C-12 by simple methods. Except for salviamine A and isosalviamine A, 22 new derivatives were synthesized. Their structures were confirmed by spectroscopic analysis. Moreover, 11 derivatives exhibited moderate cytotoxic activities against five human cancer lines in vitro.     

MPEG-hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics

Ovarian cancer is the most common gynecological cancer diagnosed in Western countries. Detection of micrometastases at an early stage of the disease could lead to a cure rate of 90% by limiting the spread of the disease outside the ovaries. In this article, hypericin (Hy), a hydrophobic photosensitizer used for the photodynamic diagnosis (PD) of ovarian cancer, was efficiently incorporated into a core of micelles made from methoxy-poly(ethylene glycol) and hexyl-substituted poly(lactides) copolymers. The fate of these micelles following intravenous injection was studied in vivo in two ovarian tumor-bearing animal models. In the chick embryo chorioallantoic membrane model, 17 times more Hy accumulated in tumor nodules when Hy was delivered with micelles than when Hy was delivered as an ethanol solution. Studies of the biodistribution of Hy in Fisher rats revealed escape of these nanosized micelles (<32 nm) from the mononuclear phagocyte system. Hy-loaded micelles showed maximal accumulation in tumors and demonstrated the best tumor/muscle contrast visible 3 h after injection in the rat model. The rapid and highly selective accumulation of Hy in tumors that we demonstrated in this study suggests that these micelle formulations could be used for the PD of ovarian cancer in the future.     

Synthesis and evaluation of new β-carboline-3-(4-benzylidene)-4H-oxazol-5-one derivatives as antitumor agents

In the present work, we report the synthesis and in vitro anticancer and antimicrobial activity evaluation of a new series of 1-substituted-β-carboline derivatives bearing a 4-benzylidene-4H-oxazol-5-one unity at C-3. The compound 2-[1-(4-methoxyphenyl)-9H-β-carbolin-3-yl]-4-(benzylidene)-4H-oxazol-5-one (11) was the most active derivative, exhibiting a potent cytotoxic activity against glioma (U251), prostate (PC-3) and ovarian (OVCAR-03) cancer cell lines with IC50 values of 0.48, 1.50 and 1.07 μM, respectively. An in silico study of the ADME properties of the novel synthesized β-carboline derivatives was also performed.