High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

Abstract Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of ATP13A2-associated disorders is not yet well understood and currently, these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for ATP13A2 deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. In this study, we show that the deletion of YPK9 (the yeast orthologue of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish—N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new in vivo model for investigating ATP13A2 deficiency.

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High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

Journal of Biological Chemistry ◽

10.1074/jbc.m501100200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 21847-21853 ◽

Cited By ~ 128

Author(s):

Jeffrey Baldwin ◽

Carolyn H. Michnoff ◽

Nicholas A. Malmquist ◽

John White ◽

Michael G. Roth ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Binding Site ◽

High Throughput ◽

High Throughput Screening ◽

De Novo ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Dihydroorotate Dehydrogenase ◽

Selective Inhibitors ◽

Diverse Range

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC50 values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC50 against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01305-20 ◽

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.

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High-Throughput Identification of Antibacterials Against Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2020.591426 ◽

2020 ◽

Vol 11 ◽

Author(s):

Shijia Li ◽

Pengfei She ◽

Linying Zhou ◽

Xianghai Zeng ◽

Lanlan Xu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Throughput ◽

High Throughput Screening ◽

Combined Therapy ◽

Drug Repurposing ◽

Antimicrobial Activities ◽

Health Concern ◽

Phenotypic Analysis ◽

Screening Assay ◽

High Throughput Screening Assay

Antibiotic resistance is a growing public health concern, though the constant development of new antibiotics. The combination of high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses of drugs. In this study, we screened a drug library consisting of 1,573 drugs already approved by the Food and Drug Administration and 903 drugs from the natural product library, to identify antimicrobials against Pseudomonas aeruginosa. A high-throughput screening assay based on microtiter plate was used to screen 39 drugs that inhibit the planktonic or biofilm formation of P. aeruginosa while most of them are antibiotics. The antimicrobial activities of these drugs were evaluated by phenotypic analysis. Further studies showed the combined therapy of tetracycline antibiotics demeclocycline hydrochloride (DMCT) and the novel antimicrobial peptide SAAP-148 has an effective synergistic antibacterial effect on P. aeruginosa PAO1 and P. aeruginosa ATCC27853. Moreover, the time-kill curve assay and murine model of cutaneous abscesses further confirmed the synergistic effect. In addition, the combination of DMCT and SAAP-148 has the potential to combat clinically isolated multidrug-resistant (MDR) P. aeruginosa strains. Our results clearly indicate that DMCT and SAAP-148 combined therapy could be an effective method to combat MDR P. aeruginosa-related infections.

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Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220914532 ◽

2020 ◽

Vol 25 (5) ◽

pp. 434-446

Author(s):

Hongkang Zhang ◽

Bryan D. Moyer ◽

Violeta Yu ◽

Joseph G. McGivern ◽

Michael Jarosh ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

High Reliability ◽

Selective Inhibitors ◽

New Class ◽

Random Library ◽

Automated Patch Clamp ◽

Patch Clamp Electrophysiology ◽

Screening Platform ◽

Subtype Selective

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

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