Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD.
The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin-permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria.
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Ramkumar
R
Iyer
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Mark A
MA
Sylvester
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Camilo
C
Velez-Vega
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Ruben
R
Tommasi
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Thomas F
TF
Durand-Reville
Entasis Therapeutics, Inc. , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
Alita A
0000-0002-7110-0493
AA
Miller
D015780:Carbapenems / Q000737:chemistry* / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism; D013329:Structure-Activity Relationship
D015780:Carbapenems; D018272:Porins; C077157:OprD protein, Pseudomonas aeruginosa
OprD; Pseudomonas aeruginosa; antibiotic; carbapenem; permeation; porin