RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

Impact of cationic surfactant-induced DNA compaction on the characteristics of a minor groove bound flavonol

3-Hydroxyflavone (3-HF), which binds to the minor groove of DNA, is a strong antioxidant and hence a potent therapeutic and diagnostic agent. A special photo-property, called excited state intramolecular proton...

Depicting the DNA Binding and Cytotoxicity Studies against Human Colorectal Cancer of Aquabis (1-Formyl-2-Naphtholato-k2O,O′) Copper(II): A Biophysical and Molecular Docking Perspective

In this study, we attempted to examine the biological activity of the copper(II)–based small molecule aquabis (1-formyl-2-naphtholato-k2O,O′)copper(II) (1) against colon cancer. The characterization of complex 1 was established by analytical and spectral methods in accordance with the single-crystal X-ray results. A monomeric unit of complex 1 exists in an O4 (H2O) coordination environment with slightly distorted square pyramidal geometry (τ = ~0.1). The interaction of complex 1 with calf thymus DNA (ctDNA) was determined by employing various biophysical techniques, which revealed that complex 1 binds to ctDNA at the minor groove with a binding constant of 2.38 × 105 M–1. The cytotoxicity of complex 1 towards human colorectal cell line (HCT116) was evaluated by the MTT assay, which showed an IC50 value of 11.6 μM after treatment with complex 1 for 24 h. Furthermore, the apoptotic effect induced by complex 1 was validated by DNA fragmentation pattern, which clarified that apoptosis might be regulated through the mitochondrial-mediated production of reactive oxygen species (ROS) causing DNA damage pathway. Additionally, molecular docking was also carried out to confirm the recognition of complex 1 at the minor groove.

Dissociation pathways of the p53 DNA binding domain from DNA and critical roles of key residues elucidated by dPaCS-MD/MSM

The dissociation process of the DNA binding domain of p53 (p53-DBD) from a DNA duplex that contains the consensus sequence, which is the specific target of p53-DBD, was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). Based on an all-atom model including explicit solvent, we first simulated the p53-DBD dissociation processes by 75 trials of dPaCS-MD, which required an average simulation time of 11.2 ± 2.2 ns per trial. By setting the axis of the DNA duplex as the Z-axis and the binding side of p53-DBD on DNA as the + side of the X-axis, we found that dissociations took place along the +X and −Y directions (−Y directions) in 93% of the cases, while 7% of the cases moved along +X and +Y directions (+Y directions). Toward the −Y directions, p53-DBD dissociated first from the major groove and then detached from the minor groove, while unbinding from the minor groove occurred first in dissociations along the +Y directions. Analysis of the free energy landscape by MSM showed that loss of the minor groove interaction with p53-DBD toward the +Y directions incurred a relatively high energy cost (1.1 kcal/mol) upon a critical transition, whereas major groove detachment more frequently occurred with lower free energy costs. The standard binding free energy calculated from the free energy landscape was −10.9 ± 0.4 kcal/mol, which agrees with an experimental value of –11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.

Amidine- and Amidoxime-Substituted Heterocycles: Synthesis, Antiproliferative Evaluations and DNA Binding

The novel 1,2,3-triazolyl-appended N- and O-heterocycles containing amidine 4–11 and amidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activities in vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (SW620), with IC50 values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adenocarcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.

1052. Characterisation of the DNA binding properties of ridinilazole, a selective antibiotic currently in phase III trials for the treatment of Clostridioides difficile

Background Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile. Methods High resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. Results Confocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (Kd) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved. Conclusion Ridinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole. Disclosures Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

BINDING PECULIARITIES OF POLY(rA)-POLY(rU) WITH MINOR GROOVE LIGAND HOECHST 33258

Study on the interaction of DNA-specific ligands – classical intercalator acridine orange (AO) and groove binding compound Hoechst 33258 (H33258) with poly(rA)-poly(rU), being a model for double-stranded (ds-) RNA, has been carried out. The absorption and fluorescence spectra of the complexes of these ligands with ds-polynucleotide were obtained. It was revealed that the optic and fluorescent characteristics of the complexes of both ligands with ds-RNA are similar with those at the complex-formation with DNA.