Trisubstituted acridine derivatives as potent and selective telomerase inhibitors

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.     

The Truncated Human Telomeric Sequence forms a Hybrid‐Type Intramolecular Mixed Parallel/antiparallel G‐quadruplex Structure in K+ Solution

In 80–90% tumor cells, telomerase becomes active and stabilizes the length of telomeres. The formation and stabilization of G‐quadruplexes formed from human telomeric sequences have been proved able to inhibit the activity of telomerase, thus human telomeric G‐quadruplex structure has become a potential target for the development of cancer therapy. Hence, structure of G‐quadruplex formed in K⁺ solution has been an attractive hotspot for further studies. However, the exact structure of human telomeric G‐quadruplex in K⁺ is extremely controversial, this study provides information for the understanding of different G‐quadruplexes. Here, we report that 22nt and 24nt human telomeric sequences form unimolecular hybrid‐type mixed parallel/antiparallel G‐quadruplex in K⁺ solution elucidated utilizing Circular Dichroism, Differential Scanning Calorimetry, and gel electrophoresis. Moreover, individual configuration of these two sequences was speculated in this study. The detailed structure information of the G‐quadruplex formed under physiologically relevant condition is necessary for structure‐based rational drug design.     

Molecular modeling studies on G-quadruplex complexes of telomerase inhibitors: structure-activity relationships

Inhibition of the ability of the enzyme telomerase to add telomeric repeats to the end of chromosomes is a novel target for potential anticancer therapy. This paper examines the hypothesis that compounds possessing a planar aromatic chromophore inhibit telomerase via stabilization of, and binding to, a folded guanine quadruplex structure. Two series of telomerase inhibitors have been designed based on the 2,6-disubstituted amidoanthracene-9,10-dione and 3,6-disubstituted acridine chromophores in order to investigate structure-activity relationships between biological activity and substituent group size. The relative binding energies between these compounds and the folded human telomere DNA quadruplex were determined using molecular simulation methods, involving explicitly solvated structures. The results obtained are in excellent agreement with the biological activity as measured in vitro using a modified TRAP assay and in general agreement with the ranking order of binding enthalpies found in isothermal titration calorimetry studies. This broad agreement provides strong support for the hypothesis that guanine quadruplexes are the primary target for telomerase inhibitors with extended planar chromophores.     

Mechanism of acridine-based telomerase inhibition and telomere shortening

The trisubstituted acridine compound BRACO-19 has been developed as a ligand for stabilising G-quadruplex structures. It is shown here that BRACO-19 produces short- and long-term growth arrest in cancer cell lines, and is significantly less potent in a normal cell line. BRACO-19 reduces telomerase activity and long-term telomere length attrition is observed. It is also shown that BRACO-19 binds to telomeric single-stranded overhang DNA, consistent with quadruplex formation, and the single-stranded protein hPOT1 has been shown to be displaced from the overhang in vitro and in cellular experiments. It is concluded that the cellular activity of BRACO-19 can be ascribed both to the uncapping of 3' telomere ends and to telomere shortening that may preferentially affect cells with short telomeres.     

Telomerase inhibition and cancer: might platinum based drugs have a future as anti-telomerase pharmacological approach?

Telomerase is a ribonucleoprotein polymerase that maintains the length of telomeric DNA by adding hexameric units (TTAGGG) to the ends of the chromosomes. This mechanism prevents replicative senescence, thus conferring unlimited proliferative potential to cells. Telomerase reactivation has been detected in most human tumour tissue, indicating that the enzyme may be useful as a specific tumour marker. The inhibition of telomerase causes a progressive and critical reduction of telomeres, leading to a potent signal for the blockage of cell proliferation and the induction of apoptosis. Since normal somatic cells lack telomerase activity, the anti-telomerase approach is highly specific for tumour cells and metastases. Prolonged treatment is required before enzyme deactivation causes the telomeres to be shortened enough to induce senescence and apoptosis. Therefore, the drugs employed in anti-telomerase therapy should be of only moderate non-specific cytotoxicity. Certain cis-Pt(II)-complexes have recently been shown to be effective inhibitors of telomerase in both cell-free and in vitro assays, most likely by targeting the nucleobases of the RNA component of the enzyme.     

Quadruplex-forming oligonucleotides as tools in anticancer therapy and aptamers design: energetic aspects

Recent investigations on the G-quadruplex motif propose a new strategy for the making of antitumour drugs. Quadruplex-drug complexes have been suggested to inhibit telomerase activity; further, aptamers based on the quadruplex motif have been proved useful as tools aimed at binding and inhibiting particular proteins, thus serving as pharmaceutically active agents. However, the design of new aptamers is difficult because many factors affecting their activity and stability have not still been clarified. The knowledge of the energetics of quadruplex formation is a crucial point in view of their potential therapeutic utilization both as targets as well as therapeutic agents. In this review the energetic aspects of both quadruplex assembly and quadruplex-ligand interactions are discussed together with a summary of recent studies on physico-chemical properties in solution of quadruplex structures obtained from synthetic aptamers, including PNA-DNA chimeras.     

A new telomerase inhibitor and apoptosis‐inducing agent in leukemia: perylene derivative G‐quadruplex ligand Tel03

Tel03 (N,N′‐Bis‐(2‐(dimethylamino)ethyl)‐3,4,9,10‐perylentetra‐carboxylic acid diimide), a perylene derivative, was synthesized and its interaction with different forms of human telomeric DNA examined . The effect of Tel03 on leukemia cells in short‐term cultures was also used to explore its molecular actions. Electrospray ionization mass spectrometry (ESI‐MS) analysis indicated that Tel03 specifically recognizes telomeric quadruplex, not duplex DNA. After the treatment with Tel03 (0.1–10 µM) for 48 h, telomerase activity in K562 leukemia cell extracts was inhibited in a dose‐dependent manner as assessed with a telomere repeat amplification protocol (TRAP) assay. MTT assay results showed that Tel03 (0.1–10 µM) inhibited the proliferation of K562 cells after treatment for 72 h. In addition, apoptosis was observed. Using real‐time quantitative PCR and Western blot analysis, the expression of c‐myc and bcl‐2 was markedly down‐regulated at both the mRNA and protein levels. However, no changes were observed in the expression of bax. We conclude that G‐quadruplex ligand, Tel03, is a novel telomerase inhibitor and apoptosis‐inducing agent in leukemia cells acting via down‐regulation of bcl‐2 and c‐myc. Tel03 has the potential to be developed as an antileukemia drug. Drug Dev Res 69:235–241, 2008. © 2008 Wiley‐Liss, Inc.     

Remarkable interference with telomeric function by a G-quadruplex selective bisantrene regioisomer

The use of small molecules able to induce and stabilize selected G-quadruplex arrangements can cause telomerase inhibition and telomere dysfunction in cancer cells, thus providing very selective therapeutic approaches. Effective stabilizers usually comprise a planar aromatic portion to grant effective stacking onto the G-quartet and positively charged side chains to exploit the highly negative charge density on the quadruplex grooves. Since the relative position of these two pharmacophoric moieties is expected to play an important role in DNA folding stabilization, we evaluated a series of anthracene derivatives substituted with one or two 4,5-dihydro-1H-imidazol-2-yl-hydrazonic groups (the bisantrene side chain) at different positions of the aromatic system. Indeed, the various regioisomers showed distinct binding affinities for telomeric G-quadruplex, and the most effective was the 1,5 and 1,7 bis-substituted analogues. On turn, the 1,8 regioisomer was poorly effective. Interestingly, G-quadruplex binding is clearly related to telomerase inhibition in this class of compounds, thus confirming their ability to shift the nucleic acid conformational equilibrium upon binding and consequently produce interference with the telomere processing enzyme. Additionally, the 1,5 regioisomer was shown to inhibit telomerase activity at lower concentrations than those required to reduce tumor cell proliferation. Comparative analysis of drug effects in telomerase-positive and telomerase-negative cancer cells showed consistent cell growth impairment, as a consequence of activation of the senescence pathway, which was mainly attributable to anthracene-mediated telomere dysfunction.     

Telomeres and telomerase, new targets for anticancer chemotherapy

Telomeres are composed of single-strand DNA rich in guanine which can adopt particular structures such as T-loop or G-quadruples, a four-strand DAN structure formed by guanine repeats. Telomeric single-strand DNA is the substrate of telomerase, an enzyme necessary for telomeric replication which is suppressed in most cancer cells and which participates in tumor genesis. The formation of a telomeric G-quadruplex blocks telomerase activity and offers an original strategy for new anti-cancer agents. Using an original approach combining rational screening and synthesis, several series of compounds have been identified which specifically bind to the telomeric quadruplex. These derivatives, called "G-quadruplex DNA ligands", are able to block telomeric replication in cancer cells and provoke replicative senescence and/or apoptosis after a few cell cycles. Our team is working on characterizing the cellular and molecular mechanisms of action of these ligands. Using mutant cell models resistant to these ligands or expressing a protein cuff covering the telomere in tumor lines, we have demonstrated that the telomere is the principal intracellular target of action of these compounds and the implicit existence of the G-quadruplex structure. In collaboration with academic and industrial partners, optimization of these ligands to develop pharmacologically active products should enable in vivo validation of a new therapeutic concept.     

荷包牡丹碱对人肺癌A549细胞生长及端粒酶活性的抑制作用

目的探讨荷包牡丹碱对人肺癌A549细胞生长的抑制作用及其作用机制。方法 A549细胞加入荷包牡丹碱0～200μmol.L-1分别作用24,48和72 h,MTT法测定A549细胞的生长抑制作用。荷包牡丹碱0～20μmol.L-1作用72 h,端粒重复序列扩增(TRAP)法测定端粒酶活性。变温紫外法检测荷包牡丹碱9μmol.L-1对端粒酶G-四链体的稳定作用。结果荷包牡丹碱25,50,100和200μmol.L-1作用细胞72 h后的抑制率分别为33.4%,88.2%,88.6%和89.4%,明显高于正常对照组细胞(P<0.05),并具有量效(r=0.906,P<0.05)和时效(r=0.949,P<0.05)性。与正常对照组相比,荷包牡丹碱5,10,15和25μmol.L-1可有效抑制A549细胞端粒酶的活性(P<0.05),相对TRAP端粒酶活性从正常对照组的1.471±0.102分别降低为1.093±0.054,1.013±0.016,0.554±0.034,0.365±0.081(P<0.05)。荷包牡丹碱9μmol.L-1使G-四链体的熔点值从正常对照组的48℃提高到54℃。结论荷包牡丹碱可以通过稳定G-四链体结构,抑制端粒酶活性,有效抑制人肺腺癌细胞A549的生长。     

Antitumor polycyclic acridines. 8.(1) Synthesis and telomerase-inhibitory activity of methylated pentacyclic acridinium salts.

Two short routes to novel methylated pentacyclic quinoacridinium salts have been devised. New compounds display telomerase-inhibitory potency (<1 microM) in the TRAP assay. 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (12d, RHPS4, NSC 714187) has a higher selectivity for triplex and quadruplex DNA structures than the 3,6,8,11,13-pentamethyl analogue (12c, RHPS3, NSC 714186) and a low overall growth-inhibitory activity in the NCI 60 cell panel (mean GI(50) 13.18 microM); in addition, the activity profile of 12d does not COMPARE with agents of the topoisomerase II class. Compound 12d is soluble in water, stable in the pH range of 5-9, efficiently transported into tumor cells, and is currently the lead structure for further elaboration in this new class of telomerase inhibitor.     

Molecular basis of structure-activity relationships between salphen metal complexes and human telomeric DNA quadruplexes

The first X-ray crystal structures of nickel(II) and copper(II) salphen metal complexes bound to a quadruplex DNA are presented. Two structures have been determined and show that these salphen-metal complexes bind to human telomeric quadruplexes by end-stacking, with the metal in each case almost in line with the potassium ion channel. Quadruplex and duplex DNA binding is presented for these two and other related salphen complexes, all with side-chains terminating in pyrrolidino end-groups and differing patterns of substitution on the salphen core. The crystal structures are able to provide rationalizations for the structure-activity data, and in particular for the superior quadruplex-binding of the nickel complexes compared to that of the copper-containing ones. The complexes show significant antiproliferative activity for the compounds in a panel of cancer cell lines. They also show telomerase inhibitory activity in the telomerase TRAP-LIG assay.     

On the significance of telomerase activity in human malignant glioma cells

Telomerase is critical for tumor cell immortalization and is a novel target for cancer chemotherapy. Here, we examined whether telomerase is expressed in glioma cell lines, whether telomerase activity is regulated by bcl-2 or p53, and whether telomerase activity predicts response to chemotherapy. Further, we characterized the effects of a candidate telomerase inhibitor, penclomedine, in glioma cells. All 12 human malignant glioma cell lines examined were telomerase positive. Telomerase activity was not modulated during cell cycle progression, did not correlate with p53 status or bcl-2 family protein expression, and did not predict drug sensitivity, except for an association with resistance to carmustine. Ectopic bcl-2 expression did not enhance telomerase activity. Wild-type p53 reduced telomerase activity in cell lines retaining p53 activity but not in p53-mutant cell lines. Penclomedine killed glioma cells via an apoptotic, but death receptor-, bcl-2- and caspase-independent pathway, but did not inhibit telomerase and did not act synergistically with cytotoxic drugs. We conclude that telomerase activity does not account for the differential chemosensitivity of human glioma cells and that penclomedine kills glioma cells via a telomerase-independent pathway.