The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis/trans isomerase (PPIase) enzymes. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms such as Burkholderia pseudomallei and Neisseria meningiditis. It has previously been demonstrated that small molecule compounds designed to target Mip from the Gram-negative bacterium B. pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip.
This study utilised co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. Two of the three lead small-molecule compounds were effective in inhibiting the PPIase activity of Mip proteins from Niesseria spp. and Klebsiella pneumoniae and Coxiella burnettii. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. This study demonstrates that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds and show potential as broad-spectrum drug candidates.