Rational design of TFDs as novel oligonucleotide antimicrobials to treat Gram-negative infections

Abstract

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.