Antibiotic discovery and development using structural analysis of DNA adenine methyltransferase inhibitors

Abstract

The requirement for developing antibiotics is continually increasing due to the rise in antibiotic resistance and lack of novel compounds that bypass the known resistance mechanisms. A well-characterised target for such development is DNA adenine methyltransferase (Dam), a suitable candidate because it is connected with virulence in a number of highly pathogenic bacteria as well as the lack of a human equivalent enabling selectivity. A fluorescence-based activity assay has been developed to identify potential Dam inhibitors, which then require structural analysis to confirm mode of binding and allow structure-activity relationships to be identified. The bi-substrate nature of the enzyme, requiring DNA and S-adenosylmethionine (SAM), provides the opportunity for it to be targeted in two pockets. This has been exploited by the development of a library of SAM analogues, including the attachment of an adenine mimic using different linker lengths to the core scaffold. The kinetic characterisation of these compounds has revealed selective inhibition of Dam over the human equivalent cytosine methyltransferase Dnmt1. To understand the reasons for such inhibitory properties, a number of compounds were co-crystallised with E. coli Dam so that a detailed 3D representation of the mechanism of inhibition could be probed. The active site of the enzyme revealed the addition of the ethylindole or ethylbenzothiophene moiety displaces a key residue (W10) in the SAM binding domain, rather than occupying the pocket typically filled by the adenine base as expected. This alternative mode of binding has resulted in a selective inhibitor over Dnmt1, a feature which must be retained during further rounds of compound modification. The screening of a fragment library yielded sixteen hits for structural analysis with E. coli Dam, but relatively high IC 50 values restricted the crystallisation of the enzyme:inhibitor complexes. The crystal structure for one fragment bound in the active site was solved, revealing the adenine-like fragment binding the pocket typically occupied by the adenine moiety of SAM. The crystal structures of more enzyme:fragment complexes are needed to enable the growing, merging or linking of the compounds, resulting in an inhibitor that fully occupies the active site whilst lowering the IC 50 value for further inhibitory potency.

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

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