Novel antibiotics from DNA adenine methyltransferase inhibitors

Abstract

The re-emergence of plague as a world-wide health concern and the potential risk posed by bioterrorism has led to an increased interest in available treatments for the disease. The bacterial DNA adenine-N6 methyltransferase, Dam, is involved in the regulation of a range of pathogenic bacteria and has been validated as a target for the development of antimicrobial agents with activity against Yersinia pestis, the causative agent of plague. The lack of a functionally similar enzyme in mammals suggests that highly selective Dam inhibitors could be developed. A coupled, real-time break light Dam activity assay has been optimised for HTS, and assays for the validation and characterisation of screening hits have also been developed. Screening of random and in silico enriched compound libraries, and the subsequent application of counter-screening and hit confirmation assays, resulted in the identification of a single viable lead, (4-(N-(2-hydroxyethyl)sulfamoyl)phenyl) stibonic acid (13776). Screening of compounds analogous to 13776 identified a series of arylstibonic acids with activity against Dam. Kinetic characterisation of the most potent arylstibonic acid, 4-stibonobenzenesulfonic acid (13746), revealed a DNA-competitive mode of action, and a Ki of 6.46 ± 0.07 nM. However, selectivity assays have revealed a potentially non-specific mode of action for the stibonic acids, which have shown activity against a range of DNA and protein binding enzymes. Yersinia cell culture experiments have shown a single compound, (3-((2-hydroxyethyl)carbamoyl)phenyl)stibonic acid (13782), to be capable of penetrating Yersinia cells and partially inhibiting methylation, and mRNA profiling experiments have shown 13782 to induce a statistically significant change in several genes involved in the pathogenicity of Y. pestis. Attempts at resynthesising 13782 have proved challenging, with only a fraction of the activity of the original sample reproduced. HPLC analysis of the original and resynthesised samples has shown the former to comprise two components, with only one present in both samples. The in vitro evaluation of a series of bisubstrate analogues designed to mimic both the methyl donor S-adenosylmethionine (AdoMet), and the methylation target (adenine) has shown that substitution of the AdoMet sulfur for nitrogen results in a significant but not total loss of activity. Furthermore, the addition of a bicyclic heteroaromatic adenine analogue mimic to this scaffold led to an increase in potency and selectivity for Dam over the human cytosine methyltransferase DNMT1 but a reduction in selectivity for Dam over the restriction enzyme DpnI. These results suggest that a selective and potent Dam inhibitor can be obtained by carefully modifying both components of the bisubstrate analogue inhibitor.

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ORCID for Peter L. Roach: orcid.org/0000-0001-9880-2877

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