Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach Katelyn E. Zulaufa,b and James E. Kirbya,b,1 aDepartment of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; and bHarvard Medical School, Boston, MA 02215 Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved October 12, 2020 (received for review March 30, 2020) Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug- DNA, cause DNA strand breakage, affect supercoiling, inhibit resistant pathogens for which new treatments are desperately conjugation, or cause plasmid-dependent fitness defects (5). needed. Carbapenemases and other types of antibiotic resistance Almost all, where investigated, acted nonspecifically and were genes are carried almost exclusively on large, low-copy-number highly toxic to the host bacterial cell, for example chemothera- plasmids (pCRE). Accordingly, small molecules that efficiently evict peutic intercalators. Of interest, however, DeNap et al. (6) and pCRE plasmids should restore much-needed treatment options. We Thomas et al. (7) identified several positively charged amino- therefore designed a high-throughput screen to identify such com- glycosides that affected the RNA-regulated plasmid replication pounds. A synthetic plasmid was constructed containing the plas- machinery of the IncB incompatibility group plasmid, pMU2403. mid replication machinery from a representative Escherichia coli These findings supported the possibility of identifying anti- CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid agents with greater mechanistic specificity. plasmid maintenance. The synthetic plasmid was then introduced As pCRE plasmids encode carbapenemase genes, we hypoth- into an E. coli K12 tolC host. We used this screening strain to test a esized that interference with plasmid maintenance would restore library of over 12,000 known bioactive agents for molecules that carbapenem susceptibility and offer new therapeutic options. We selectively reduce plasmid levels relative to effects on bacterial therefore designed a high-throughput screening approach to iden- growth. From 366 screen hits we further validated the antiplasmid tify antiplasmid agents that did not presuppose a particular mech- activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleo- anism of action. For proof of principle, we screened known MICROBIOLOGY side analog; and Ro 90-7501, a bibenzimidazole. All three com- bioactive compounds that might more quickly advance our under- pounds exhibited significant antiplasmid activity including up to standing of antiplasmid mechanisms and would be available in complete suppression of plasmid replication and/or plasmid evic- sufficient quantities for follow-up studies. To do so, we constructed tion in multiple orthogonal readouts and potentiated activity of a synthetic screening plasmid (pScreening) containing the IncFIA the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening Significance 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 Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug- validated our approach to identify compounds that alter plasmid resistant bacterial pathogens designated as urgent threats by maintenance, confer resensitization to antimicrobials, and have spe- the US Centers for Disease Control and Prevention. Resistance to cific mechanisms of action. carbapenems and other antibiotics is usually carried on diverse large, low-copy-number plasmids. Interfering with the replica- plasmid | multidrug resistance | carbapenem-resistant Enterobacteriaceae | tion of such resistance plasmids could potentially restore anti- antimicrobial | high-throughput screening biotic susceptibility. Therefore, we designed a high-throughput screen to identify compounds with anti-CRE plasmid activity. We identified several compounds which by blocking plasmid repli- he US Centers for Disease Control and Prevention lists cation and/or evicting a representative IncFIA CRE plasmid po- Tcarbapenem-resistant Enterobacteriaceae (CRE) in its most tentiated carbapenem activity, thereby providing proof of urgent antimicrobial resistant threat category (1). CRE strains β principle for our approach. Our findings underscore the potential are resistant to carbapenems, a class of last-resort -lactam an- of antiplasmid therapeutics to restore treatment options for tibiotics. CRE strains are also largely resistant to multiple other highly drug-resistant pathogens and validate a high-throughput classes of antibiotics as well. As a result, CRE infections are screening approach to identify these agents. often incredibly difficult, if not impossible, to treat. Carbapenemase and other antibiotic resistance genes are Author contributions: K.E.Z. and J.E.K. designed research; K.E.Z. performed research; typically carried on diverse, large, low-copy-number plasmids (2, K.E.Z. and J.E.K. analyzed data; and K.E.Z. and J.E.K. wrote the paper. 3). CRE resistance plasmids (pCRE) are maintained at only 1 to This article is a PNAS Direct Submission. 10 copies per cell and can be as large as 300 kilobases (kb) in Competing interest statement: The authors declare a competing interest (as defined by size. These large, low-copy-number plasmids utilize a combination 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. of precise replication machinery and maintenance strategies to K.E.Z. was supported in part by a National Institute of Allergy and Infectious Diseases ensure their persistence, including specific plasmid replication training grant (T32AI007061). The content is solely the responsibility of the authors and genes, partitioning mechanisms that segregate plasmids to progeny 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 (Morris- bacteria, resolvases to disentangle plasmid multimers, conjugation ville, NC). Tecan had no role in study design, data collection/interpretation, manuscript systems, and plasmid addiction systems (4). Plasmid addiction preparation, or decision to publish. systems encode a stable toxin and unstable antidote. Bacterial host Published under the PNAS license. cells that lose their plasmids can no longer make antidote and are 1To whom correspondence may be addressed. Email: [email protected]. thus killed by the persisting toxin. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Previously, a limited number of compounds with antiplasmid doi:10.1073/pnas.2005948117/-/DCSupplemental. activity were identified. They were found to intercalate into First published November 9, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2005948117 PNAS | November 24, 2020 | vol. 117 | no. 47 | 29839–29850 Downloaded by guest on September 26, 2021 replication and maintenance machinery from a representative pCRE drugs, and a variety of compounds with at least partially known plasmid as well as a fluorescent reporter that would allow us to easily biological effects including active biolipids, kinase inhibitors, ion monitor plasmid loss upon exposure to small-molecule inhibitors. channel inhibitors, and G protein-coupled receptor inhibitors. Here, we describe the results of a screen of over 12,000 known- To distinguish between compounds that affect plasmid main- bioactive compounds for antiplasmid activity in which we identify tenance directly rather than indirectly through inhibiting growth several potent antiplasmid small molecules. We further characterize of the host cell, we imposed two constraints in selecting positive their activity and find support for the predicted ability to restore screening hits: 1) the RFU must be reduced greater than two antibiotic susceptibility to CRE strains. times more than any reduction in the A600 in both replicate plates and 2) screening hits must not be overtly antibacterial, that Results and Discussion is, the A600 was not reduced by more than 80% compared to the High-Throughput Screening Assay to Identify Antiplasmid Agents. To median of all treatment wells in both replicate plates. Using identify prospective anti-pCRE agents, we designed a high- these criteria, we identified 366 compounds with antiplasmid throughput screening approach to identify small molecules that activity (Dataset S1) and 1,205 compounds with antibacterial disrupt bacterial plasmid maintenance. Our screening strategy activity (Dataset S2). The latter was not unexpected as the made use of a synthetic screening plasmid (pScreening) that known bioactive collection contains over 250 known antimicro- contains the maintenance, replication, and resolvase machinery of a bials, which are often represented several times in the libraries carbapenemase-encoding plasmid from a representative Escherichia (23, 24). coli CRE isolate, BIDMC20a (8). The CRE plasmid in BIDMC20a belongs to the IncF incompatibility group, a leading incompatibility Validation of Antiplasmid Activity. We chose to characterize three group associated with multidrug resistance (9–12). Specifically, we antiplasmid compounds based on potency and