Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo

In this study, we sought to determine if an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC 50 in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. While it’s thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. But perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that BCP can identify multiple pathways that are inhibited by an individual molecule. Importance: We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

Technologies for High-Throughput Identification of Antibiotic Mechanism of Action

There are two main strategies for antibiotic discovery: target-based and phenotypic screening. The latter has been much more successful in delivering first-in-class antibiotics, despite the major bottleneck of delayed Mechanism-of-Action (MOA) identification. Although finding new antimicrobial compounds is a very challenging task, identifying their MOA has proven equally challenging. MOA identification is important because it is a great facilitator of lead optimization and improves the chances of commercialization. Moreover, the ability to rapidly detect MOA could enable a shift from an activity-based discovery paradigm towards a mechanism-based approach. This would allow to probe the grey chemical matter, an underexplored source of structural novelty. In this study we review techniques with throughput suitable to screen large libraries and sufficient sensitivity to distinguish MOA. In particular, the techniques used in chemical genetics (e.g., based on overexpression and knockout/knockdown collections), promoter-reporter libraries, transcriptomics (e.g., using microarrays and RNA sequencing), proteomics (e.g., either gel-based or gel-free techniques), metabolomics (e.g., resourcing to nuclear magnetic resonance or mass spectrometry techniques), bacterial cytological profiling, and vibrational spectroscopy (e.g., Fourier-transform infrared or Raman scattering spectroscopy) were discussed. Ultimately, new and reinvigorated phenotypic assays bring renewed hope in the discovery of a new generation of antibiotics.

Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo

In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While it’s generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase.ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic’s in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

Identifying the effect of vancomycin on health care–associated methicillin-resistant Staphylococcus aureus strains using bacteriological and physiological media

Background The evolving antibiotic-resistant behavior of health care–associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern. They are resistant to a broad class of antibiotics such as macrolides, aminoglycosides, fluoroquinolones, and many more. Findings The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (cation-adjusted Mueller-Hinton broth) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behavior of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. Conclusions The study reveals the vancomycin resistance behavior of HA-MRSA USA100 strains in dual media conditions using wide-ranging experiments.

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals: Streptomyces strains and phages for antibiotic discovery

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare’s most potent antibiotics are becoming obsolete. Approximately two-thirds of the world’s antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, in order to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 101 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

Identifying the effect of vancomycin on HA-MRSA strains using bacteriological and physiological media

Healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern due to their evolving antibiotic resistant. They are resistant to a broad class of antibiotics like macrolides, aminoglycosides, fluoroquinolones, and many more. The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (CA-MHB) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behaviour of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. The study reveals the vancomycin resistance behavior of USA100 strains in dual media conditions using wide-ranging experiments.

Profiling the effect of nafcillin on HA-MRSA D592 using bacteriological and physiological media

Staphylococcus aureus is a leading human pathogen associated with both hospital-acquired and community-acquired infections. The bacterium has steadily gained resistance to β-lactams and other important first-line antibiotics culminating in its categorization as an urgent threat by the U.S. Centers for Disease Control and Prevention. Observations of a varying response to antimicrobial exposure as a function of media type has revealed that clinical susceptibility testing performed in standard bacteriological media might not adequately represent pharmacological responses in the patient. Such observations have encouraged research designed to identify media types that more closely mimic the in vivo environment. In this study, we examine the response of a hospital-acquired USA100 lineage methicillin-resistant, vancomycin-intermediate S. aureus (MRSA/VISA) strain (D592) to nafcillin in a bacteriological compared to a more physiological tissue culture-based medium. We performed multi-dimensional analysis including growth and bacterial cytological profiling, RNA sequencing, and exo-metabolomics measurements (both HPLC and LC/MS) to shed light on the media-dependent activity of the commonly prescribed β-lactam antibiotic nafcillin.

Profiling the effect of nafcillin on HA-MRSA D712 using bacteriological and physiological media

Staphylococcus aureus strains have been continuously evolving resistance to numerous classes of antibiotics including methicillin, vancomycin, daptomycin and linezolid, compounding the enormous healthcare and economic burden of the pathogen. Cation-adjusted Mueller-Hinton broth (CA-MHB) is the standard bacteriological media for measuring antibiotic susceptibility in the clinical lab, but the use of media that more closely mimic the physiological state of the patient, e.g. mammalian tissue culture media, can in certain circumstances reveal antibiotic activities that may be more predictive of effectiveness in vivo. In the current study, we use both types of media to explore antibiotic resistance phenomena in hospital-acquired USA100 lineage methicillin-resistant, vancomycin-intermediate Staphylococcus aureus (MRSA/VISA) strain D712 via multidimensional high throughput analysis of growth rates, bacterial cytological profiling, RNA sequencing, and exo-metabolomics (HPLC and LC-MS). Here, we share data generated from these assays to shed light on the antibiotic resistance behavior of MRSA/VISA D712 in both bacteriological and physiological media.

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active againstAcinetobacter baumannii

ABSTRACTAn increasing number of multidrug-resistantAcinetobacter baumannii(MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) ofA. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB. We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds inEscherichia coliandBacillus subtilis. Given how distantly relatedA. baumanniiis to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform forA. baumannii. We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth ofA. baumanniivia targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in therpoBgene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active againstA. baumannii.