A Structural Biology Approach Enables the Development of Antimicrobials Targeting Bacterial Immunophilins

Abstract

f Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila

“…However, the compounds inhibited the prototypic human FKBP12 30-fold more efficiently than Mip, indicating low specificity for bacterial FKBPs (Juli et al, 2011 ). In a follow-up study, the inhibitory activities of these derivatives were also confirmed for BpML1 and Mip-like proteins of the pathogens Yersinia pestis and Francisella tularensis (Begley et al, 2014 ).…”

FKBPs in bacterial infections

“…All but one of the tested substances were also able to prevent B. mallei-induced cytotoxicity in J744A.1 macrophage cells. However, here again no strict correlation to the PPIase inhibitory activity could be drawn (Begley et al, 2014).…”

“…Recently, small molecule inhibitors based on this core inhibitory structure were tested on Mip and BpML1 (Begley et al, 2014;Juli et al, 2011). In order to find novel inhibitors of Mip, two previously reported pipecoline ring containing inhibitors A and B with K i (app) of 10 nM and 0.23 µM, respectively, were used as lead structures.…”

Recent contributions of structure-based drug design to the development of antibacterial compounds

“…Numerous NMR experiments, based on differences in the NMR properties between big (target) and small (fragment) molecules, have been designed to identify and validate lead compounds [24]. These experiments, some of which can be used in high-throughput mode, include WaterLOGSY [26], SLAPSTIC [27], TINS [28], transferred NOEs [29] DOSY-NMR [30] and saturation transfer difference (STD)-NMR [31, 32] For example, STD-NMR was used to identify a series of compounds with low-micromolar affinity for the macrophage infectivity potentiator BpML1 from Burkholderia pseudomallei , the etiological agent for melioidosis [33]. In terms of lead optimization, the use of NMR to determine structures for protein less than ~25 kDa in size is well established[21, 34].…”