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

2020 ◽  
Vol 117 (47) ◽  
pp. 29839-29850
Author(s):  
Katelyn E. Zulauf ◽  
James E. Kirby
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Replication Initiation ◽  
Plasmid Replication ◽  
Complete Suppression ◽  
Fluorescent Reporter ◽  
Plasmid Maintenance

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 representativeEscherichia coliCRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into anE. coliK12tolChost. 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.

scholarly journals Identification of Antifungal Compounds against Multidrug Resistant Candida auris Utilizing a High Throughput Drug Repurposing Screen

2021 ◽  
Author(s):  
Yu-Shan Cheng ◽  
Jose Santinni Roma ◽  
Min Shen ◽  
Caroline Mota Fernandes ◽  
Patricia S. Tsang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Drug Repurposing ◽  
Multidrug Resistant ◽  
Current Treatment ◽  
Drug Combinations ◽  
Serine Palmitoyltransferase ◽  
Candida Auris ◽  
Pharmacologically Active

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identifying new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug resistant Candida auris. The assay is robust and suitable for assessing large compound collections by high throughput screening. Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds which yielded 25 compounds including 6 novel anti-Candida auris compounds and 13 sets of potential two drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

scholarly journals CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Valerie J. Price ◽  
Wenwen Huo ◽  
Ardalan Sharifi ◽  
Kelli L. Palmer
Keyword(s):  
Antibiotic Resistance ◽  
High Risk ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Treatment Options ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Content Type ◽  
New Genes ◽  
Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

scholarly journals Methods for High-Throughput Drug Combination Screening and Synergy Scoring

2016 ◽  
Author(s):  
Liye He ◽  
Evgeny Kulesskiy ◽  
Jani Saarela ◽  
Laura Turunen ◽  
Krister Wennerberg ◽  
...  
Keyword(s):  
Cancer Cells ◽  
High Throughput ◽  
Drug Combination ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Drug Combinations ◽  
Molecular Medicine ◽  
Targeted Drugs ◽  
Functional Profiling ◽  
Automated Screening

AbstractGene products or pathways that are aberrantly activated in cancer but not in normal tissue hold great promises for being effective and safe anticancer therapeutic targets. Many targeted drugs have entered clinical trials but so far showed limited efficacy mostly due to variability in treatment responses and often rapidly emerging resistance. Towards more effective treatment options, we will critically need multi-targeted drugs or drug combinations, which selectively inhibit the cancer cells and block distinct escape mechanisms for the cells to become resistant. Functional profiling of drug combinations requires careful experimental design and robust data analysis approaches. At the Institute for Molecular Medicine Finland (FIMM), we have developed an experimental-computational pipeline for high-throughput screening of drug combination effects in cancer cells. The integration of automated screening techniques with advanced synergy scoring tools allows for efficient and reliable detection of synergistic drug interactions within a specific window of concentrations, hence accelerating the identification of potential drug combinations for further confirmatory studies.

DDRE-45. HIGH-THROUGHPUT SCREENING OF EPIGENETIC COMPOUNDS FOR THE TREATMENT OF CHORDOMA IDENTIFIES POTENTIAL NOVEL THERAPEUTICS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi84-vi84
Author(s):  
Tadeusz Wroblewski ◽  
Philip Tatman ◽  
Anthony Fringuello ◽  
Sam Scherer ◽  
William Foreman ◽  
...  
Keyword(s):  
Survival Rate ◽  
Cell Viability ◽  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Spinal Tumors ◽  
Mobile Spine ◽  
Hc Toxin ◽  
Sacral Tumors ◽  
Inhibitor Screen

Abstract BACKGROUND Chordoma is a rare malignant tumor with few treatment options. While surgical resection is deemed the most effective treatment, the 5-year overall survival rate is 61% and 5-year recurrence free survival rate is approximately 50%. To date, no FDA approved pharmacotherapies exist for the treatment of chordoma, and adjuvant therapy remains highly debated. This necessitates the need for further research to provide clinicians with more options to treat this patient population. METHODS In this study, we conducted a high-throughput 139-compound epigenetic inhibitor screen against 12 chordoma patient-derived cell lines; 4 were resected at our institution and 8 were graciously donated by the Chordoma Foundation. RESULTS 8 tumors were located in the sacrum, 3 were located in the mobile spine, and 1 tumor was located in the clivus. 5 tumors were primary, 5 were recurrent, and 2 were metastatic. 6 tumors came from female patients and 6 tumors came from male patients. The top three most effect compounds across the cohort were the G9a inhibitor UNC0631 (cell viability = 64.5% +/-25.1SD; p=1.53x10-9), the KDM inhibitor JIB-04 (cell viability = 68.4% +/-27.2SD; p=9.81x10-8), and the G9a inhibitor BIX01294 (cell viability = 68.6% +/-27.9SD; p=1.27x10-7). No single compound significantly reduced viability in every tumor in the cohort, although the HDAC inhibitor HC Toxin significantly reduced viability in 9 tumors (cell viability = 69.7% +/-16.6SD; p=2.6x10-12). The most effective compound for sacral tumors was UNC0631 (viability = 68.6% +/-22.1SD; p=4x10-7), UNC0631 was also the most effective for spinal tumors (viability = 54.4% +/-32.1SD; p=2.72x10-3), and notably, no significant compounds were identified for the single clival tumor. CONCLUSIONS Based on our drug screen results, epigenetic inhibition, particularly methyltransferase inhibition, may be a promising therapeutic avenue for the treatment of chordomas.

scholarly journals High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

2021 ◽  
Author(s):  
Brandon M. Murareanu ◽  
Jessica Knox ◽  
Peter Roy ◽  
Aaron W. Reinke
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Screening Method ◽  
C Elegans ◽  
Automated Method ◽  
Intracellular Parasites ◽  
Chemical Inhibitors ◽  
Animal Phyla ◽  
Small Molecule Screen

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.

scholarly journals Structure of Mpro from COVID-19 virus and discovery of its inhibitors

2020 ◽  
Author(s):  
Zhenming Jin ◽  
Xiaoyu Du ◽  
Yechun Xu ◽  
Yongqiang Deng ◽  
Meiqin Liu ◽  
...  
Keyword(s):  
Drug Design ◽  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Screening Strategy ◽  
Drug Candidates ◽  
Approved Drugs ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Drug Leads

SUMMARYA new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

scholarly journals Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents

2002 ◽  
Vol 46 (10) ◽  
pp. 3118-3124 ◽  
Author(s):  
David J. Payne ◽  
William H. Miller ◽  
Valerie Berry ◽  
John Brosky ◽  
Walter J. Burgess ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Enoyl Acp Reductase

ABSTRACT Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.

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