Downregulation of androgen receptor transcription by promoter g-quadruplex stabilization as a potential alternative treatment for castrate-resistant prostate cancer.

Androgen receptor (AR) signaling remains an important regulatory pathway in castrate-resistant prostate cancer, and its transcriptional downregulation could provide a new line of therapy. A number of small-molecule ligands have previously demonstrated the ability to stabilize G-quadruplex structures and affect gene transcription for those genes whose promoters contain a quadruplex-forming sequence. Herein, we report the probable formation of new G-quadruplex structure present in the AR promoter in a transcriptionally important location. NMR spectroscopy, circular dichroism, UV spectroscopy, and UV thermal melting experiments for this sequence are consistent with G-quadruplex formation. Fluorescence resonance energy transfer (FRET) melting studies have identified a novel compound, MM45, which appears to stabilize this G-quadruplex at submicromolar concentrations. The effects of MM45 have been investigated in prostate cancer cell lines where it has been shown to inhibit cell growth. A reporter assay intended to isolate the effect of MM45 on the G-quadruplex sequence showed dose-dependent transcriptional repression only when the AR promoter G-quadruplex sequence is present. Dose-dependent transcriptional repression of the AR by MM45 has been demonstrated at both a protein and mRNA level. This proof of concept study paves the route toward a potential alternative treatment pathway in castrate-resistant prostate cancer.

Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands.

The folding of the single-stranded 3' end of the human telomere into G-quadruplex arrangements inhibits the overhang from hybridizing with the RNA template of telomerase and halts telomere maintenance in cancer cells. The ability to thermally stabilize human telomeric DNA as a four-stranded G-quadruplex structure by developing selective small molecule compounds is a therapeutic path to regulating telomerase activity and thereby selectively inhibit cancer cell growth. The development of compounds with the necessary selectivity and affinity to target parallel-stranded G-quadruplex structures has proved particularly challenging to date, relying heavily upon limited structural data. We report here on a structure-based approach to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA. Crystal structures have been determined of complexes between a 22-mer intramolecular human telomeric quadruplex and two potent tetra-substituted naphthalene diimide compounds, functionalized with positively charged N-methyl-piperazine side-chains. These compounds promote parallel-stranded quadruplex topology, binding exclusively to the 3' surface of each quadruplex. There are significant differences between the complexes in terms of ligand mobility and in the interactions with quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups. The data presented here provides a structural rationale for the biophysical (effects on quadruplex thermal stabilization) and biological data (inhibition of proliferation in cancer cell lines and evidence of in vivo antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules.

Sequences in the HSP90 promoter form G-quadruplex structures with selectivity for disubstituted phenyl bis-oxazole derivatives.

The HSP90 protein is an important target in cancer. We report here that stable quadruplex DNAs can be formed from a promoter sequence in the HSP90 gene, on the basis of melting, circular and NMR studies, and show that these can be selectively targeted by non-macrocyclic quadruplex-stabilizing phenyl bis-oxazole derivatives. These do not bind significantly to duplex DNA and show low stabilization of the human telomeric quadruplex. These results suggest an approach to targeting HSP90 at the DNA level.

Structure-based design and evaluation of naphthalene diimide G-quadruplex ligands as telomere targeting agents in pancreatic cancer cells.

Tetra-substituted naphthalene diimide (ND) derivatives with positively charged termini are potent stabilizers of human telomeric and gene promoter DNA quadruplexes and inhibit the growth of human cancer cells in vitro and in vivo. The present study reports the enhancement of the pharmacological properties of earlier ND compounds using structure-based design. Crystal structures of three complexes with human telomeric intramolecular quadruplexes demonstrate that two of the four strongly basic N-methyl-piperazine groups can be replaced by less basic morpholine groups with no loss of intermolecular interactions in the grooves of the quadruplex. The new compounds retain high affinity to human telomeric quadruplex DNA but are 10-fold more potent against the MIA PaCa-2 pancreatic cancer cell line, with IC50 values of ~10 nM. The lead compound induces cellular senescence but does not inhibit telomerase activity at the nanomolar dosage levels required for inhibition of cellular proliferation. Gene array qPCR analysis of MIA PaCa-2 cells treated with the lead compound revealed significant dose-dependent modulation of a distinct subset of genes, including strong induction of DNA damage responsive genes CDKN1A, DDIT3, GADD45A/G, and PPM1D, and repression of genes involved in telomere maintenance, including hPOT1 and PARP1.

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents.

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.

Design, synthesis, and biological evaluation of substituted naphthalene imides and diimides as anticancer agent.

Naphthalimmide (NI) and 1,4,5,8-naphthalentetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity. NDI derivatives 1-9 were more cytotoxic than the corresponding NI derivatives 10-18. The molecular mechanisms of 1 and 2 were investigated in comparison to mitonafide. They interacted with DNA, were not topoisomerase IIalpha poisons, triggered caspase activation, caused p53 protein accumulation, and down-regulated AKT survival. Furthermore, 1 and 2 caused a decrease of ERK1/2 and, unlike mitonafide, inhibited ERKs phosphorylation.

Structure-activity relationships of acetylcholinesterase noncovalent inhibitors based on a polyamine backbone. 4. Further investigation on the inner spacer.

Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimer's disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted beta-amyloid aggregation in the micromolar range.