Tolfenpyrad Derivatives Exhibit Potent <i>Francisella</i>-Specific Antibacterial Activity without Toxicity to Mammalian Cells <i>In Vitro</i>

Tularemia is a deadly disease caused by <i>Francisella tularensis</i>, an emerging intracellular bacterial pathogen that can be disseminated rapidly through aerosols and vector-borne transmission. Recent surveillance data demonstrate an increasing incidence in several countries. Although clinical isolates of <i>Francisella</i> strains are sensitive to currently used antibiotics, engineered or horizontal acquisition of antibiotic resistance is a constant threat to public health. Therefore, the identification of antibiotics that target previously undrugged pathways is required to safeguard human health. An environmental pesticide that is registered for use in multiple countries, tolfenpyrad, shows promising activity to block <i>Francisella</i> growth; however, it is not a suitable antimicrobial candidate for use <i>in vivo</i> due to potential toxicity in humans and other animals. In this study, we applied a structure-activity relationship approach to tolfenpyrad to generate compounds with improved antibacterial activity and reduced toxicity. Through screening of a library of derivatives, we identified analogs with improved therapeutic windows compared with tolfenpyrad. Although structural diversity exists among these analogs, they inhibit the growth of <i>Francisella</i> species but not other Gram-negative or Gram-positive species. These compounds block intramacrophage growth of <i>F. novicida</i> and pathogenesis in an <i>in vivo</i> arthropod model of infection. Although the biochemical activity of these drugs is unknown, they appear to target the same pathway as the parent molecule because <i>F. novicida</i> mutants that are resistant to tolfenpyrad are also resistant to its analogs. Taken together, these findings suggest that these tolfenpyrad-derived compounds comprise a new class of <i>Francisella</i>-targeted antimicrobials and merit further evaluation and development.