Abstract
Divalent metal ions play a critical role in the removal of N-terminal methionine from nascent proteins by methionine aminopeptidase (MetAP). Being an essential enzyme for bacteria, MetAP is an appealing target for the development of novel antibacterial drugs. Although purified enzyme can be activated by several divalent metal ions, the exact metal ion used by MetAP in cells is unknown. Many MetAP inhibitors are highly potent on purified enzyme, but they fail to show significant inhibition of bacterial growth. One possibility for the failure is a disparity of the metal used in activation of purified MetAP and the metal actually used by MetAP inside bacterial cells. Therefore, the challenge is to elucidate the physiologically relevant metal for MetAP and discover MetAP inhibitors that can effectively inhibit cellular MetAP. We have recently discovered MetAP inhibitors with selectivity toward different metalloforms of Escherichia coli MetAP, and with these unique inhibitors, we characterized their inhibition of MetAP enzyme activity in a cellular environment. We observed that only inhibitors that are selective for the Fe(II)-form of MetAP were potent in this assay. Further, we found that only these Fe(II)-form selective inhibitors showed significant inhibition of growth of five E. coli strains and two Bacillus strains. We confirmed their cellular target as MetAP by analysis of N-terminal processed and unprocessed recombinant glutathione S-transferase proteins. Therefore, we conclude that Fe(II) is the likely metal used by MetAP in E. coli and other bacterial cells.
Highlights
methionine aminopeptidase (MetAP) is an appealing target for the development of antibacterial and antifungal drugs with novel mechanisms of action [6]
We demonstrated that the Fe(II)-form selective inhibitors inhibit the cellular MetAP activity and growth of bacterial cells
Development of a Cell-based MetAP Enzyme Activity Assay— To evaluate the ability of a compound to inhibit MetAP inside cells, we employed a strategy to make live E. coli cells permeable to small molecules, including substrates and inhibitors, and to assess inhibition by the compound when MetAP carries out hydrolysis of its substrate in a cellular environment
Summary
Materials—Fluorogenic substrate, Met-AMC, is a methionine derivatized with 7-amino-4-methylcoumarin (AMC), which was purchased from Bachem Bioscience (King of Prussia, PA). MetAP Activity and Inhibition Assay on Purified Enzyme— Enzyme activity was monitored by fluorescence on a SpectraMax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA), following hydrolysis of the fluorogenic substrate MetAMC (ex 360 nm, em 460) at room temperature [12, 24, 29]. A suspension of bacterial cells grown to exponential phase in Mueller Hinton medium was adjusted to 0.5 McFarland optical density [30] and further diluted by 1000-fold in the same medium containing 100 mM Tris, pH 7.5. The conversion from resazurin to resofurin was monitored kinetically by fluorescence (ex 530 nm and em 590 nm) using a SpectraMax Gemini XPS plate reader Both absorbance and fluorescence kinetic experiments were carried out for 10 h at 37 °C, with readings taken every 5 min. Physicochemical parameters describing Lipinski’s rule of five (such as molecular weight, hydrogen bond donors, and acceptors) were evaluated by Molinspiration Property Calculation Service
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