In Vitro High-Throughput RNAi Screening to Accelerate the Process of Target Identification and Drug Development

Abstract Background Nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM. Methods We are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria. Results We are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well. Conclusion To enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology. Disclosures All authors: No reported disclosures.

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A Multi-Omics Human Liver Organoid Screening Platform for DILI Risk Prediction

10.1101/2021.08.26.457824 ◽

2021 ◽

Author(s):

Charles J. Zhang ◽

Matthew J. O’Meara ◽

Sophia R. Meyer ◽

Sha Huang ◽

Meghan M. Capeling ◽

...

Keyword(s):

Drug Development ◽

Risk Prediction ◽

High Throughput ◽

High Throughput Screening ◽

Human Liver ◽

Drug Induced ◽

Predictive Capacity ◽

On Chip

AbstractBackground and AimsDrug-induced liver injury (DILI) is a prominent failure mode in drug development resulting in clinical trial failures and post-approval withdrawal. Improved in vitro models for DILI risk prediction that can model diverse genetics are needed to improve safety and reduce high attrition rates in drug development. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. The recent clinical failure of inarigivir soproxil due to DILI underscores the need for improved models.MethodsHLOs were adapted for high-throughput drug screening in dispersed-cell 384-well format and a collection of DILI-associated drugs were screened. HLOs were also adapted to a liver-chip system to investigate enhanced in vivo-like function. Both platforms were benchmarked for their ability to predict DILI using combined biochemical assays, microscopy-based morphological profiling, and transcriptomics.ResultsDispersed HLOs retained DILI predictive capacity of intact HLOs and are amenable to high-throughput screening allowing for measurable IC50 values for cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of action. HLOs on chips were shown to increase albumin production, CYP450 expression and also release ALT/AST when treated with known DILI drugs. Importantly, HLO liver chips were able to predict hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis.ConclusionsThe high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the utility of the platforms in early and late-stage drug development. Tenofovir-inarigivr associated hepatotoxicity was observed and highly correlates with the clinical manifestation of DILI.

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Target Discovery for New Antitubercular Drugs Using a Large Dataset of Growth Inhibitors from PubChem

Infectious Disorders - Drug Targets ◽

10.2174/1871526519666181205163810 ◽

2020 ◽

Vol 20 (3) ◽

pp. 352-366

Author(s):

Robert C. Goldman

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Target Identification ◽

Growth Inhibitors ◽

Antitubercular Drugs ◽

Whole Cells ◽

Culture Cells ◽

Target Binding ◽

Multiple Drugs

The number of drugs available for treatment of active tuberculosis is diminishing due to increased multidrug resistance selection in Mycobacterium tuberculosis leading to multiple (MDR) and extensively (XDR) resistant strains. Also, TB is treated with multiple drugs to minimize further resistance development, mandating a sustained effort to identify new lead compounds for treating drug-resistant TB and shortening time to cure for all TB infections. High throughput screening, a well-known approach to discovery of new leads, is conducted in two basic modes 1) using whole cells and screening for inhibition of growth, or whole cell reporter cells that signal when a specific pathway is perturbed, and 2) in vitro non-cell based enzyme or other functional assays for direct ligand-target binding. Combining high throughput screening for inhibitors of growth (to identify and chemically assess inhibitors active on whole cells), followed by target identification abrogates the problem of discovering new leads in non-cell based systems that are inactive on whole cells due to issues with target access (e.g., uptake). High throughput screening of 341,778 compounds by the National Institutes of Health identified 8,950 primary hit, growth inhibitors of M. tuberculosis. Final evaluation based on reproducibility, potency, medicinal chemistry inspection, and cytotoxicity on tissue culture cells identified 1,113 priority compounds. These data were deposited in PubChem, making data available to TB research labs for follow up studies on target identification. This effort led to the identification of compounds targeting Pks13, MmpL3, DprE1, AspS, EthA, GuaB2, nonreplicating cells, and VKOR (Vitamin K epoxide reductase).

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A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111412183 ◽

2011 ◽

Vol 16 (8) ◽

pp. 852-861 ◽

Cited By ~ 16

Author(s):

Brian J. Geiss ◽

Hillary J. Stahla-Beek ◽

Amanda M. Hannah ◽

Hamid H. Gari ◽

Brittney R. Henderson ◽

...

Keyword(s):

Drug Development ◽

High Throughput ◽

High Throughput Screening ◽

Antiviral Drug ◽

Functional Assay ◽

Screening Assay ◽

Gtp Binding ◽

High Throughput Screening Assay ◽

Capping Enzyme

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5′ end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

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