Cooperative Membrane Damage as a Mechanism for Pentamidine–Antibiotic Mutual Sensitization

Most
Gram-positive-selective antibiotics have low activity against
Gram-negative bacteria due to the presence of an outer membrane barrier.
There is, therefore, interest in developing combination therapies
that can penetrate the outer membrane (OM) with known antibiotics
coupled with membrane-active sensitizing adjuvants. However, two unanswered
questions hinder the development of such combination therapies: the
sensitization spectrum of the sensitizer and the mechanism of antibiotic–sensitizer
mutual potentiation. Here, with pentamidine as an example, we screened
a library of 170 FDA-approved antibiotics in combination with pentamidine,
a compound known to disturb the OM of Gram-negative bacteria. We found
that four antibiotics, minocycline, linezolid, valnemulin, and nadifloxacin,
displaced enhanced activity in combination with pentamidine against
several multidrug-resistant Gram-negative bacteria. Through a descriptor-based
structural-activity analysis and multiple cell-based biochemical assays,
we found that hydrophobicity, partial charge, rigidity, and surface
rugosity were key factors that affected sensitization via a cooperative
membrane damage mechanism in which lipopolysaccharides and phospholipids
were identified as sites of synergy. Finally, in vitro experiments showed that the linezolid–pentamidine combination
slowed the generation of drug resistance, and there was also potent
activity in in vivo experiments. Overall, our results
highlight the importance of the physicochemical properties of antibiotics
and cooperative membrane damage for synergistic pentamidine–antibiotic
drug combinations.