New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase
Graphical abstract
Introduction
Antibacterial resistance continues to be a growing threat to human health worldwide. The rate of discovery of new antibacterials is far outstripped by the rate at which resistance is spreading, and there remains an urgent need for the development of new antibacterial drugs.1 Of particular concern is the emergence of bacterial strains that appear to be totally resistant to all current antibacterials. This was recently underlined by the CDC, who reported a case in which a patient in the US was infected with a strain of Klebsiella pneumoniae that was resistant to all available antibiotics.2
DNA topoisomerases are an essential class of enzymes responsible for maintaining the topological state of DNA (e.g. supercoiling)3. The ATP-dependent bacterial type II topoisomerases, DNA gyrase and DNA topoisomerase IV, contribute to the maintenance of the correct level of supercoiling in bacterial DNA. These enzymes are hetero-tetramers, e.g. gyrase consists of two subunits, GyrA and GyrB, which form an A2B2 complex4. These enzymes have been well-validated as antibacterial targets as the fluoroquinolone antibiotics act at the DNA-cleavage site of these enzymes. The aminocoumarin antibiotics, including novobiocin, which was used clinically in the 1960′s, target the ATP-binding site. Efforts to discover novel inhibitors of the ATP-binding site of these enzymes have been ongoing for more than four decades. However, only two compounds have progressed into the clinic.5
We have previously reported a series of pyridine-3-carboxamide inhibitors of DNA gyrase and topoisomerase IV which displayed antibacterial activity6. These compounds, designed to bind to the ATP-binding site within the gyrase using the de novo molecular design program SPROUT,7 were developed into a series possessing promising antibacterial activity. However, the computationally predicted binding mode of these molecules has not been previously validated experimentally and little assessment was made of the potential “drug-likeness” of compounds in this series. We now report the detailed structural characterisation of the binding mode of these inhibitors within bacterial GyrB as well as delineation of a number of key physicochemical parameters of importance in considering their potential for progression into antibacterial drugs.
Section snippets
Biological activity of novel pyridine-2-urea-3-carboxamide (PUC) inhibitors
As part of a program designed to expand upon our previously reported studies,6 a small library of novel PUC inhibitors was synthesised with a view to establishing the best candidates for subsequent co-crystallisation studies with the target enzymes. As with previous examples,6 the synthetic strategy was informed by a number of molecular modelling techniques. Putative compounds were initially developed via de novo design utilising SPROUT. The most likely inhibitors of E. coli GyrB were then
Conclusions
In this study, protein X-ray crystallography has been used to explore the detailed mode of binding of the pyridine carboxamide class of antibacterial agents, to their bacterial DNA gyrase target. These studies reveal that the binding mode of these inhibitors closely corresponds to that predicted via the structure-based design approach used in their conception, in particular, confirming the importance of an exquisite network of H-bonds involving all three nitrogen atoms shared between the urea
Experimental section
The Supporting Information contains a complete general Experimental Section, including all procedures and equipment used. Chemicals were from commonly used suppliers (Aldrich, Acros, and Alfa Aesar) and used without purification. The purities of compounds submitted for screening were ≥95% as determined by UV analysis of liquid chromatography (HPLC) chromatograms at 254 nM.
Notes and acknowledgements
The authors declare no competing financial interest.
Crystal data for compounds 2, 4, 5, and 10 have been deposited in the Protein Data Bank with accession codes 6F86, 6F8J, 6F94 and 6F96.
The authors thank Alison Howells (Inspiralis Ltd.) for carrying out supercoiling and relaxation assays.
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