Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach.

Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE

Original Publication: Washington, DC : National Academy of Sciences

Anti-Bacterial Agents/*pharmacology ; Carbapenem-Resistant Enterobacteriaceae/*drug effects ; DNA Replication/*drug effects ; Drug Resistance, Multiple, Bacterial/*drug effects ; Enterobacteriaceae Infections/*drug therapy ; High-Throughput Screening Assays/*methods; Amines/pharmacology ; Amines/therapeutic use ; Aminoglycosides/pharmacology ; Aminoglycosides/therapeutic use ; Anti-Bacterial Agents/therapeutic use ; Bacterial Proteins/antagonists & inhibitors ; Bacterial Proteins/genetics ; Benzimidazoles/pharmacology ; Benzimidazoles/therapeutic use ; Carbapenem-Resistant Enterobacteriaceae/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Drug Synergism ; Drug Therapy, Combination ; Enterobacteriaceae Infections/microbiology ; Escherichia coli/genetics ; Meropenem/pharmacology ; Meropenem/therapeutic use ; Microbial Sensitivity Tests ; Mutagenesis, Site-Directed ; Plasmids/genetics ; Quinazolines/pharmacology ; Quinazolines/therapeutic use ; Repressor Proteins/antagonists & inhibitors ; Repressor Proteins/genetics ; Repressor Proteins/metabolism ; Triazoles/pharmacology ; Triazoles/therapeutic use ; beta-Lactamases/genetics

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representative Escherichia coli CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into an E. coli K12 tolC host. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.
Competing Interests: Competing interest statement: The authors declare a competing interest (as defined by PNAS policy). This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R33AI119114 to J.E.K. K.E.Z. was supported in part by a National Institute of Allergy and Infectious Diseases training grant (T32AI007061). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The HP D300 digital dispenser and TECAN M1000 were provided for our use by TECAN (Morrisville, NC). Tecan had no role in study design, data collection/interpretation, manuscript preparation, or decision to publish.

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R33 AI119114 United States AI NIAID NIH HHS; T32 AI007061 United States AI NIAID NIH HHS

Keywords: antimicrobial; carbapenem-resistant Enterobacteriaceae; high-throughput screening; multidrug resistance; plasmid

0 ((2'-(4-aminophenyl)-(2,5'-bi-1H-benzimidazol)-5-amine))
0 (Amines)
0 (Aminoglycosides)
0 (Anti-Bacterial Agents)
0 (Bacterial Proteins)
0 (Benzimidazoles)
0 (Quinazolines)
0 (Repressor Proteins)
0 (Triazoles)
EC 3.5.2.6 (beta-Lactamases)
EC 3.5.2.6 (carbapenemase)
FV9J3JU8B1 (Meropenem)
O957UYB9DY (kasugamycin)
Y5A5D5E2AQ (9-chloro-2-(2-furyl)-(1,2,4)triazolo(1,5-c)quinazolin-5-imine)

Date Created: 20201110 Date Completed: 20210119 Latest Revision: 20210902

20240105

PMC7703651

10.1073/pnas.2005948117

33168749