HALO384: A Halo-Based Potency Prediction Algorithm for High-Throughput Detection of Antimicrobial Agents

Woehrmann, Marcos H.; Gassner, N.C.; Bray, Walter M.; Stuart, Joshua M.; Lokey, Scott

doi:10.1177/1087057109355060

Cited by 5 publications

(12 citation statements)

References 7 publications

(11 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using an automated yeast halo assay that we had developed previously[33], we screened the benomyl- and rapamycin-resistant mutants for multi-drug resistance in the presence of 14 antifungal drugs representing a variety of MOA classes (Figure 2). Each mutant was seeded in agar and poured into a 384-well-format “omni-tray”, and DMSO stock solutions of benomyl, rapamycin, and the 14 drugs in the cross-resistance panel were pin-transferred to each mutant tray.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

et al. 2014

Self Cite

View full text Add to dashboard Cite

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug’s molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach “Mutagenesis to Uncover Targets by deep Sequencing, or “MUTseq”, and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.a

show abstract

Section: Resultsmentioning

confidence: 99%

“…Mutants that yielded colonies within 3 days were considered resistant and evaluated further in a multi-drug resistance cross-screen. This screen was performed using the 384 halo assay as previously reported [26]. Overnight cultures of resistant mutants were seeded in YPD top-agar at an OD600 of 0.06 and poured into OmniTrays.…”

Section: Methodsmentioning

confidence: 99%

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…These assays have the advantage of requiring small volumes of compound, and compound hits can easily be confirmed through visual inspection. 5–7 Unpredictable compound diffusion rates and overlapping halos were once considered a limitation of this assay type. However, it has been demonstrated that these effects occur only when a compound is extremely toxic.…”

Section: Introductionmentioning

confidence: 99%

“…However, it has been demonstrated that these effects occur only when a compound is extremely toxic. 8 Halo assays to date have been used to screen only single strains against compound libraries 5,7,8 ; therefore, screening for differential sensitivities via this method remains labor and material intensive.…”

Section: Introductionmentioning

confidence: 99%

A Chemogenomic Screening Platform Used to Identify Chemotypes Perturbing HSP90 Pathways

et al. 2017

View full text Add to dashboard Cite

Compounds that modulate the heat shock protein (HSP) network have potential in a broad range of research applications and diseases. A yeast-based liquid culture assay that measured time-dependent turbidity enabled the high-throughput screening of different Saccharomyces cerevisae strains to identify HSP modulators with unique molecular mechanisms. A focused set of four strains, with differing sensitivities to Hsp90 inhibitors, was used to screen a compound library of 3680 compounds. Computed turbidity curve functions were used to classify strain responses and sensitivity to chemical effects across the compound library. Filtering based on single-strain selectivity identified nine compounds as potential heat shock modulators, including the known Hsp90 inhibitor macbecin. Haploid yeast deletion strains (360), mined from previous Hsp90 inhibitor yeast screens and heat shock protein interaction data, were screened for differential sensitivities to known N-terminal ATP site-directed Hsp90 inhibitors to reveal functional distinctions. Strains demonstrating differential sensitivity (13) to Hsp90 inhibitors were used to prioritize primary screen hit compounds, with NSC145366 emerging as the lead hit. Our follow-up biochemical and functional studies show that NSC145366 directly interacts and inhibits the C-terminus of Hsp90, validating the platform as a powerful approach for early-stage identification of bioactive modulators of heat shock-dependent pathways.

show abstract

“…From the pathway’s perspective, we refer to such a compound as a hit compound. Current high-throughput screening (HTS) technology allows cells harboring a deletion in an indicator gene to be screened against a large library of compounds[12]. Compounds that show differential lethality toward the mutant relative to wild type are selected as hits and then moved into secondary screens to test their effect on the pathway of interest.…”

Section: Introductionmentioning

confidence: 99%

The synthetic genetic interaction network reveals small molecules that target specific pathways in Sacchromyces cerevisiae

et al. 2011

Self Cite

View full text Add to dashboard Cite

High-throughput elucidation of synthetic genetic interactions (SGIs) has contributed to a systems-level understanding of genetic robustness and fault-tolerance encoded in the genome. Pathway targets of various compounds have been predicted by comparing chemical-genetic synthetic interactions to a network of SGIs. We demonstrate that the SGI network can also be used in a powerful reverse pathway-to-drug approach for identifying compounds that target specific pathways of interest. Using the SGI network, the method identifies an indicator gene that may serve as a good candidate for screening a library of compounds. The indicator gene is selected so that compounds found to produce sensitivity in mutants deleted for the indicator gene are likely to abrogate the target pathway. We tested the utility of the SGI network for pathway-to-drug discovery using the DNA damage checkpoint as the target pathway. An analysis of the compendium of synthetic lethal interactions in yeast showed that superoxide dismutase 1 (SOD1) has significant SGI connectivity with a large subset of DNA damage checkpoint and repair (DDCR) genes in Saccharomyces cerevisiae, and minimal SGIs with non-DDCR genes. We screened a sod1Δ strain against three National Cancer Institute (NCI) compound libraries using a soft agar high-throughput halo assay. Fifteen compounds out of ~3100 screened showed selective toxicity toward sod1Δ relative to the isogenic wild type (wt) strain. One of these, 1A08, caused a transient increase in growth in the presence of sublethal doses of DNA damaging agents, suggesting that 1A08 inhibits DDCR signaling in yeast. Genome-wide screening of 1A08 against the library of viable homozygous deletion mutants further supported DDCR as the relevant targeted pathway of 1A08. When assayed in human HCT-116 colorectal cancer cells, 1A08 caused DNA-damage resistant DNA synthesis and blocked the DNA-damage checkpoint selectively in S-phase.

show abstract

HALO384: A Halo-Based Potency Prediction Algorithm for High-Throughput Detection of Antimicrobial Agents

Cited by 5 publications

References 7 publications

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

A Chemogenomic Screening Platform Used to Identify Chemotypes Perturbing HSP90 Pathways

The synthetic genetic interaction network reveals small molecules that target specific pathways in Sacchromyces cerevisiae

Contact Info

Product

Resources

About