title: Characterization of a novel small molecule that potentiates β-lactam activity against gram-positive and gram-negative pathogens.
abstract: In a loss-of-viability screen using small molecules against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 with a sub-MIC of a β-lactam, we found a small molecule, designated DNAC-1, which potentiated the effect of oxacillin (i.e., the MIC of oxacillin decreased from 64 to 0.25 μg/ml). Fluorescence microscopy indicated a disruption in the membrane structures within 15 min of exposure to DNAC-1 at 2× MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by use of the DiOC2 (3,3'-diethyloxacarbocyanine iodide) dye. Macromolecular analysis showed the inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall, together with mesosome-like projections into the cytosol. The exposure of USA300 cells to DNAC-1 was associated with the mislocalization of FtsZ accompanied by the localization of penicillin-binding protein 2 (PBP2) and PBP4 away from the septum, as well as mild activation of the vraRS-mediated cell wall stress response. However, DNAC-1 does not have any generalized toxicity toward mammalian host cells. DNAC-1 in combination with ceftriaxone is also effective against an assortment of Gram-negative pathogens. Using a murine subcutaneous coinjection model with 10(8) CFU of USA300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with a sub-MIC of oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and cell wall, is a viable candidate in the development of combination therapy against many common bacterial pathogens.
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Dhanalakshmi R
authors.initials: DR
authors.lastname: Nair
authors.affiliation: Bacterial Cell Biology Laboratory, Instituto de Technologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: João M
authors.initials: JM
authors.lastname: Monteiro
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Guido
authors.initials: G
authors.lastname: Memmi
authors.affiliation: Achillion Pharmaceuticals, New Haven, Connecticut, USA.
authors.forename: Jane
authors.initials: J
authors.lastname: Thanassi
authors.affiliation: Achillion Pharmaceuticals, New Haven, Connecticut, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Pucci
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Schwartzman
authors.affiliation: Bacterial Cell Biology Laboratory, Instituto de Technologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Pinho
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA ambrose.cheung@dartmouth.edu.
authors.forename: Ambrose L
authors.initials: AL
authors.lastname: Cheung
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001424:Bacterial Infections / Q000382:microbiology*; D002460:Cell Line; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D015169:Colony Count, Microbial; D003600:Cytosol / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D006090:Gram-Negative Bacteria / Q000187:drug effects*; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D008807:Mice, Inbred BALB C; D046915:Penicillin-Binding Proteins / Q000378:metabolism; D011809:Quinones / Q000737:chemistry / Q000494:pharmacology; D054852:Small Molecule Libraries; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D046915:Penicillin-Binding Proteins; D011809:Quinones; D054852:Small Molecule Libraries; D047090:beta-Lactams; C030786:dnacins