Abstract The increased incidence of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis, especially for the treatment of infections caused by Gram-negative bacteria. Here, we identify and characterize a molecule, NM102, that displays antimicrobial activity exclusively in the context of infection. NM102 inhibits the activity of the non-essential Mutation Frequency Decline (Mfd) protein by competing with ATP binding to its active site. Inhibition of Mfd by NM102 sensitizes pathogenic bacteria to the host immune response and blocks infections with clinically- relevant Klebsiella pneumoniae and Pseudomonas aeruginosa , without inducing host toxicity. Finally, NM102 inhibits the function of Mfd as a mutation and evolvability factor, thus reducing the bacterial capacity to develop antimicrobial resistance. These data provide a potential roadmap to expand the arsenal of drugs to combat antimicrobial resistance. Highlight NM102 is a “first in class” molecule specifically targeting the active site of the bacterial Mfd protein NM102 has a new mode of action: it inhibits Mfd function during immune stress response NM102 also inhibits Mfd evolvability function and thereby decreases bacterial resistance to known antibiotics NM102 effectively treats Gram-negative infections in animal models NM102 is efficient against clinically relevant resistant bacteria and provides an increased efficacy in combination with the β-lactam meropenem

An anti-virulence drug targeting the evolvability protein Mfd protects against infections with antimicrobial resistant ESKAPE pathogens