Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

The self-assembly and stability of DNA G-quadruplexes (GQs) are affected by the intrinsic stability of different GpG base steps embedded in their G-quartet stems. We have carried out MD simulations followed by MM-PBSA (molecular mechanics Poisson–Boltzmann surface area) free energy calculations on all the experimentally observed three-quartet intramolecular human telomeric GQ topologies. We also studied antiparallel GQ models with alternative <i>syn</i>-<i>anti</i> patterns of the G-quartets. We tested different ions, dihedral variants of the DNA force field, water models, and simulation lengths. In total, ∼35 μs of simulations have been carried out. The systems studied here are considerably more complete than the previously analyzed two-quartet stems. Among other effects, our computations included the stem–loop coupling and ion–ion interactions inside the stem. The calculations showed a broad agreement with the earlier predictions. However, the increase in the completeness of the system was associated with increased noise of the free energy data which could be related, for example, to the presence of long-lived loop substates and rather complex dynamics for the two bound ions inside the G-stem. As a result, the MM-PBSA data were noisy and we could not improve their quantitative convergence even by expanding the simulations to 2.5 μs long trajectories. We also suggest that the quality of MM-based free energy computations based on MD simulations of complete GQs is more sensitive to the neglect of explicit polarization effects, which, in real systems, are associated with the presence of multiple closely spaced ions inside the GQs. Thus, although the MM-PBSA procedure provides very useful insights that complement the structural-dynamics data from MD trajectories of GQs, the method is far from reaching quantitative accuracy. Our conclusions are in agreement with critical assessments of the MM-PBSA methodology available in contemporary literature for other types of problems

Symmetric Bis-benzimidazoles Are Potent Anti-Staphylococcal Agents with Dual Inhibitory Mechanisms against DNA Gyrase

Various bis-benzimidazole derivatives have been reported to possess activity against Gram-positive pathogens. No mechanism of action has been elucidated to fully account for the antibacterial activity of this class of compounds. A group of symmetric bis-benzimidazoles (BBZ) designed as anticancer agents have previously been shown to possess moderate antiproliferative activity. We sought to assess the antibacterial activity and mechanism of action of BBZ compounds against <i>Staphylococcus aureus</i>. Antibacterial activities were assessed by determination of minimal inhibitory concentrations (MICs), time-kill curves, and scanning electron microscopy. Transcriptional responses to BBZ treatment were determined using whole genome microarrays. Activities against bacterial type II topoisomerases were investigated using in vitro supercoiling, decatenation, DNA binding, and DNA cleavage inhibition assays. MICs for EMRSA-16 were between 0.03 and 0.5 μg/mL. The compounds showed concentration-dependent bactericidal activity and induced cell swelling and lysis. Transcriptional responses to BBZ were consistent with topoisomerase inhibition and DNA damage. A subset of BBZ compounds inhibited <i>S. aureus</i> DNA gyrase supercoiling activity with IC₅₀ values in the range of 5–10 μM. This inhibition was subsequently shown to operate through both inhibition of binding of DNA gyrase to DNA and accumulation of single-stranded DNA breaks. We conclude that BBZ compounds are potent anti-staphylococcal agents and operate at least in part through DNA gyrase inhibition, leading to the accumulation of single-stranded DNA breaks, and by preventing the binding of gyrase to DNA

Molecular Dynamics Studies of the STAT3 Homodimer:DNA Complex: Relationships between STAT3 Mutations and Protein–DNA Recognition

Signal Transducers and Activators of Transcription (STAT) proteins are a group of latent cytoplasmic transcription factors involved in cytokine signaling. STAT3 is a member of the STAT family and is expressed at elevated levels in a large number of diverse human cancers and is now a validated target for anticancer drug discovery.. Understanding the dynamics of the STAT3 dimer interface, accounting for both protein–DNA and protein–protein interactions, with respect to the dynamics of the latent unphosphorylated STAT3 monomer, is important for designing potential small-molecule inhibitors of the activated dimer. Molecular dynamics (MD) simulations have been used to study the activated STAT3 homodimer:DNA complex and the latent unphosphorylated STAT3 monomer in an explicit water environment. Analysis of the data obtained from MD simulations over a 50 ns time frame has suggested how the transcription factor interacts with DNA, the nature of the conformational changes, and ways in which function may be affected. Examination of the dimer interface, focusing on the protein–DNA interactions, including involvement of water molecules, has revealed the key residues contributing to the recognition events involved in STAT3 protein–DNA interactions. This has shown that the majority of mutations in the DNA-binding domain are found at the protein–DNA interface. These mutations have been mapped in detail and related to specific protein–DNA contacts. Their structural stability is described, together with an analysis of the model as a starting-point for the discovery of novel small-molecule STAT3 inhibitors

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 <i>in vivo</i> antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules

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 <i>N</i>-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

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

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

Exposure to IQ3A compounds induces cell death, mainly by apoptosis in HCT116 cells.

<p>Cell populations were obtained by Guava ViaCount flow cytometry following 72 h incubation of HCT116 colon cancer, SW620 metastatic colon cancer, CCD18co human colon fibroblasts and HEK293T embryonic kidney cell lines with 5-FU, TMPyP4 and IQ3A at equitoxic (IC₅₀ and IC₆₅) concentrations, or DMSO (vehicle control). Results are expressed as the mean percentage (%) of viable, mid-apoptotic and dead cells ± SEM, of at least three different experiments, for <b>A.</b> IC₅₀, and for <b>B.</b> IC₆₅ compound concentrations. a, p < 0.05; b, p < 0.01 from DMSO (vehicle control); c, p < 0.05; and d, p < 0.01 from 5-FU.</p

Exposure to IQ3A compounds increases apoptosis in HCT116 cells.

<p><b>A.</b> Nuclear morphology and representative images of HCT116 human colon cancer cells and CCD18co human colon fibroblasts after Hoechst staining, evaluated by fluorescence microscopy after 72 h exposure to equitoxic (IC₅₀) concentrations of 5-FU, TMPyP4 and IQ3A or DMSO (vehicle control) at 400x magnification. White Arrows indicate nuclear fragmentation and chromatin condensation, and <b>B.</b> Apoptotic cell populations obtained by Guava Nexin flow cytometry following 72 h incubation of HCT116 and CCD18co cells with 5-FU, TMPyP4 and IQ3A at equitoxic (IC₅₀) concentrations, or DMSO (vehicle control). Results are expressed as the mean percentage (%) of early (LR quadrant) and late (UR quadrant) apoptotic cells. Results are expressed as mean ± SEM of at least three independent experiments; *p < 0.05 and §p < 0.01 from DMSO (vehicle control); and †p <0.05 and ‡p < 0.01 from 5-FU.</p

Exposure to IQ3A compounds decreases <i>HSP90</i> and <i>KRAS</i> mRNA, protein expression and <i>KRAS</i> transcription.

<p><b>A.</b> KRAS and HSP90 protein steady-state expression evaluated by immunoblot relative to DMSO (vehicle control), after 72 h exposure to equitoxic (IC₅₀) concentrations of 5-FU, TMPyP4 and IQ3A treatment; <b>B.</b><i>KRAS</i> mRNA steady-state expression was evaluated by Taqman Real-time RT-PCR using specific Taqman Assays for KRAS and β-Actin for normalization. <i>KRAS</i> mRNA steady-state expression levels were calculated by the ΔΔCt method, using DMSO (vehicle control) for calibration; and <b>C.</b> HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with <i>KRAS</i> promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5.000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC₅₀ equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO); <b>D.</b> HCT116, SW620 and HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5,000 cells per well and exposed to IC₅₀ equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO). <i>KRAS</i> promoter activity levels were evaluated by Dual-Luciferase assay 72 h (<b>C.</b>) or 24 h (<b>D.</b>) after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. Results are expressed as mean ± SEM of at least three independent experiments; *p < 0.05 and §p < 0.01 from DMSO (vehicle control); and †p <0.05 and ‡p < 0.01 from 5-FU.</p