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

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S617-S617
Author(s):  
Clive Mason ◽  
Tim Avis ◽  
Chris Coward ◽  
David Powell ◽  
Esther Duperchy ◽  
...  
Keyword(s):  
Dna Binding ◽  
Confocal Microscopy ◽  
Mechanism Of Action ◽  
Tight Binding ◽  
Minor Groove ◽  
Phase Iii ◽  
Sequence Specificity ◽  
Binding Properties ◽  
Clostridioides Difficile ◽  
Dna Binding Properties

Abstract 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)

scholarly journals Evidence that sequence homologous region in LRAT-like proteins possesses anti-proliferative activity and DNA binding properties: translational implications and mechanism of action

2005 ◽  
Vol 27 (4) ◽  
pp. 693-707 ◽  
Author(s):  
Denise Perry Simmons ◽  
Megan L. Peach ◽  
Jonathan R. Friedman ◽  
Michael M.B. Green ◽  
Marc C. Nicklaus ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mechanism Of Action ◽  
Proliferative Activity ◽  
Homologous Region ◽  
Binding Properties ◽  
Dna Binding Properties

scholarly journals Identification and Characterization of Saccharomyces cerevisiae Cdc6 DNA-binding Properties

2000 ◽  
Vol 11 (5) ◽  
pp. 1673-1685 ◽  
Author(s):  
Luo Feng ◽  
Bin Wang ◽  
Barbara Driscoll ◽  
Ambrose Jong
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Activity ◽  
Temperature Sensitive ◽  
Sequence Specificity ◽  
Binding Properties ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Dna Binding Properties ◽  
Mutant Cells

Recent studies have shown that Cdc6 is an essential regulator in the formation of DNA replication complexes. However, the biochemical nature of the Cdc6 molecule is still largely unknown. In this report, we present evidence that the Saccharomyces cerevisiaeCdc6 protein is a double-stranded DNA-binding protein. First, we have demonstrated that the purified yeast Cdc6 can bind to double-stranded DNA (dissociation constant ∼ 1 × 10−7 M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in its native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-Cdc6 binds to various DNA probes without apparent sequence specificity. Third, the minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the GST-NP6 protein competes for DNA binding with distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the29KRKK region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstrated that a binding mutant of Cdc6 fails to complement either cdc6-1 temperature-sensitive mutant cells orΔcdc6 null mutant cells at the nonpermissive temperature. The mutant gene also conferred growth impairments and increased the plasmid loss in its host, indicative of defects in DNA synthesis. Because the mutant defective in DNA binding also fails to stimulate Abf1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding activity may play a pivotal role in the initiation of DNA replication.

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