Abstract
The bifunctional enzyme methylenetetrahydrofolate dehydrogenase – cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.
Highlights
The Gram-negative Pseudomonas aeruginosa is a serious nosocomial pathogen, accounting for a significant level of hospitalacquired infections and is troublesome for burn victims, immunocompromised and cystic fibrosis patients [1,2]
Structural analysis An efficient supply of recombinant material, yielding over 30 mg of enzyme per litre of bacterial culture, and an efficient purification protocol were established. This provided a source of enzyme for structural studies and a highthroughput screening (HTS) campaign
The active site is assessed as possessing the right combination of properties in terms of size, and juxtaposition of hydrophilic and hydrophobic components to warrant being described as druggable
Summary
The Gram-negative Pseudomonas aeruginosa is a serious nosocomial pathogen, accounting for a significant level of hospitalacquired infections and is troublesome for burn victims, immunocompromised and cystic fibrosis patients [1,2]. Numerous drug resistant strains of P. aeruginosa, employing the common mechanisms of resistance such as modification of the target, active efflux and/or decreased uptake of drugs, have emerged [3,4,5,6]. The need for novel antibiotics to tackle, in particular Gram-negative bacteria such as P. aeruginosa, and drug resistant bacteria in general, has been well publicized along with the practical difficulties associated with antibacterial drug development [7,8]. There are genome sequences available for important pathogens and increasing knowledge of the mechanism of action of existing drugs. Data are available on which genes encode essential activities and we have an improved understanding of what types of molecules are likely to provide either the drug targets or appropriate lead compounds [9,10]. It is appropriate and timely to identify and carefully assess potential targets that might provide a foundation for the future of antimicrobial research
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