On the Road to Fight Cancer: The Potential of G-quadruplex Ligands as Novel Therapeutic Agents.

Nucleic acid sequences able to adopt a G-quadruplex conformation are overrepresented within the human genome. This evidence strongly suggests that these genomic regions have been evolutionary selected to play a pivotal role in several aspects of cell biology. In the present review article, we provide an overview on the biological impact of targeting G-quadruplexes in cancer. A variety of small molecules showing good G-quadruplex stabilizing properties has been reported to exert an antitumor activity in several preclinical models of human cancers. Moreover, promiscuous binders and multiple targeting G-quadruplex ligands, cancer cell defense responses and synthetic lethal interactions of G-quadruplex targeting have been also highlighted. Overall, evidence gathered thus far indicates that targeting G-quadruplex may represent an innovative and fascinating therapeutic approach for cancer. The continued methodological improvements, the development of specific tools and a careful consideration of the experimental settings in living systems will be useful to deepen our knowledge of G-quadruplex biology in cancer, to better define their role as therapeutic targets and to help design and develop novel and reliable G-quadruplex-based anticancer strategies.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Redox-Sensitive PEG-Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells.

We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.

Naphthalene diimides as red fluorescent pH sensors for functional cell imaging.

A small library of hydrosoluble naphthalene diimides (NDIs) was designed and synthesized, as cell permeable pH "turned-on" fluorescent sensors, for cellular applications. The NDIs exhibit a non-emitting twisted intramolecular charge transfer (TICT) state, which has been described by a DFT computational investigation. These NDIs do not emit as a free base, but they become strong emitters when protonated. Switching of the red emission was achieved in the pH window 2.5-6, tuning steric and electronic features of the amine moiety. The least acidic protonated NDI (pKa 5.1), was investigated in normal and cancer cells. Its selective redistribution in cancer cells from acidic vesicular organelles to the cytoplasm and the nucleus describes an effective application of these NDIs as a valuable functional tool.

Targeting loop adenines in G-quadruplex by a selective oxirane.

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

Nestling telomere length does not predict longevity, but covaries with adult body size in wild barn swallows.

Telomere length and dynamics are increasingly scrutinized as ultimate determinants of performance, including age-dependent mortality and fecundity. Few studies have investigated longevity in relation to telomere length (TL) in the wild and none has analysed longevity in relation to TL soon after hatching, despite the fact that telomere shortening may mostly occur early in life. We show that TL in nestling barn swallows (Hirundo rustica) in the wild does not predict longevity. However, TL positively covaries with body size, suggesting that individuals with large TL can afford to grow larger without paying the cost of reduced TL, and/or that benign rearing conditions ensure both large body size and low rates of telomere shortening. Overall, our study hints at a role of TL in developmental processes, but also indicates a need for further analyses to assess the expectation that TL in young individuals predicts longevity in the wild.

G-quadruplex structures in the human genome as novel therapeutic targets.

G-quadruplexes are secondary structures that may form within guanine-rich nucleic acid sequences. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular G-quadruplexes, leading to the rational design and development of G-quadruplex­stabilizing molecules. These ligands were shown to selectively exert an antiproliferative and chemosensitizing activity in in vitro and in vivo tumor models, without appreciably affecting normal cells. Such findings point to them as possible drug candidates for clinical applications. Other than in telomeres, G-quadruplexes may form at additional locations in the human genome, including gene promoters and untranslated regions. For instance, stabilization of G-quadruplex structures within the promoter of MYC, KIT, or KRAS resulted in the down-regulation of the corresponding oncogene either in gene reporter assays or in selected experimental models. In addition, the alternative splicing of a number of genes may be affected for a therapeutic benefit through the stabilization of G-quadruplexes located within pre-mRNAs. It is now emerging that G-quadruplex structures may act as key regulators of several biological processes. Consequently, they are considered as attractive targets for broad-spectrum anticancer therapies, and much effort is being made to develop a variety of ligands with improved G-quadruplex recognition properties. Quarfloxin, a fluoroquinolone derivative designed to target a G-quadruplex within ribosomal DNA and disrupt protein-DNA interactions, has entered clinical trials for different malignancies. This review will provide some hints on the role of G-quadruplex structures in biological processes and will evaluate their implications as novel therapeutic targets.

Hybrid ligand-alkylating agents targeting telomeric G-quadruplex structures.

The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI-QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand-alkylating NDIs as possible tools for the development of novel targeted anticancer therapies.

Teloxantron inhibits the processivity of telomerase with preferential DNA damage on telomeres.

Telomerase reactivation is one of the hallmarks of cancer, which plays an important role in cellular immortalization and the development and progression of the tumor. Chemical telomerase inhibitors have been shown to trigger replicative senescence and apoptotic cell death both in vitro and in vivo. Due to its upregulation in various cancers, telomerase is considered a potential target in cancer therapy. In this study, we identified potent, small-molecule telomerase inhibitors using a telomerase repeat amplification protocol assay. The results of the assay are the first evidence of telomerase inhibition by anthraquinone derivatives that do not exhibit G-quadruplex-stabilizing properties. The stability of telomerase in the presence of its inhibitor was evaluated under nearly physiological conditions using a cellular thermal shift assay. Our data showed that the compound induced aggregation of the catalytic subunit (hTERT) of human telomerase, and molecular studies confirmed the binding of the hit compound with the active site of the enzyme. The ability of new derivatives to activate DNA double-strand breaks (DSBs) was determined by high-resolution microscopy and flow cytometry in tumor cell lines differing in telomere elongation mechanism. The compounds triggered DSBs in TERT-positive A549 and H460 lung cancer cell lines, but not in TERT-negative NHBE normal human bronchial epithelial and ALT-positive U2OS osteosarcoma cell lines, which indicates that the induction of DSBs was dependent on telomerase inhibition. The observed DNA damage activated DNA damage response pathways involving ATM/Chk2 and ATR/Chk1 cascades. Additionally, the compounds induced apoptotic cell death through extrinsic and intrinsic pathways in lung cancer cells. Taken together, our study demonstrated that anthraquinone derivatives can be further developed into novel telomerase-related anticancer agents.

Telomere as a Therapeutic Target in Dedifferentiated Liposarcoma.

BACKGROUND: Well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS) accounts for ~60% of retroperitoneal sarcomas. WDLPS and DDLPS divergently evolve from a common precursor and are both marked by the amplification of the 12q13-q15 region, leading to the abnormal expression of MDM2, CDK4, and HMGA2 genes. DDLPS is a non-lipogenic disease associated with aggressive clinical behavior. Patients have limited therapeutic options, especially for advanced disease, and their outcome remains largely unsatisfactory. This evidence underlines the need for identifying and validating DDLPS-specific actionable targets to design novel biology-driven therapies. METHODS: Following gene expression profiling of DDLPS clinical specimens, we observed the up-regulation of "telomere maintenance" (TMM) pathways in paired DD and WD components of DDLPS. Considering the relevance of TMM for LPS onset and progression, the activity of a telomeric G-quadruplex binder (RHPS4) was assessed in DDLPS patient-derived cell lines. RESULTS: Equitoxic concentrations of RHPS4 in DDLPS cells altered telomeric c-circle levels, induced DNA damage, and resulted in the accumulation of γ-H2AX-stained micronuclei. This evidence was paralleled by an RHPS4-mediated reduction of in vitro cell migration and induction of apoptosis/autophagy. CONCLUSIONS: Our findings support telomere as an intriguing therapeutic target in DDLPS and suggest G-quadruplex binders as innovative therapeutic agents.