Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

2020 ◽  
Vol 25 (9) ◽  
pp. 1064-1071
Author(s):  
Maikel Izquierdo ◽  
De Lin ◽  
Sandra O’Neill ◽  
Martin Zoltner ◽  
Lauren Webster ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Signal To Noise Ratio ◽  
Screening Method ◽  
Cellular Functions ◽  
Screening Assay ◽  
High Throughput Screening Assay

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin–based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)–based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

scholarly journals Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

2017 ◽  
Vol 13 (2) ◽  
pp. e1006168 ◽  
Author(s):  
Mart Toots ◽  
Mart Ustav ◽  
Andres Männik ◽  
Karl Mumm ◽  
Kaido Tämm ◽  
...  
Keyword(s):  
High Risk ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
High Risk Hpv

A Generic High-Throughput Screening Assay for Kinases: Protein Kinase A as an Example

2003 ◽  
Vol 8 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Rommel Mallari ◽  
Elissa Swearingen ◽  
Wei Liu ◽  
Arnold Ow ◽  
Stephen W. Young ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Signal To Noise Ratio ◽  
Product Formation ◽  
Screening Assay ◽  
Chemical Libraries ◽  
Coulombic Interactions ◽  
High Throughput Screening Assay ◽  
Peptide Product

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated 33P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5-to 9-fold and a Z′ factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors. ( Journal of Biomolecular Screening 2003:198-204)

scholarly journals High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using RapidFire® Mass Spectrometry

2011 ◽  
Vol 16 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Maureen K. Highkin ◽  
Matthew P. Yates ◽  
Olga V. Nemirovskiy ◽  
William A. Lamarr ◽  
Grace E. Munie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Mass Spectrometry Technology ◽  
Sphingosine 1 Phosphate ◽  
Mass Spectrometry System

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system.

scholarly journals Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Filip Zmuda ◽  
Lalitha Sastry ◽  
Sharon M. Shepherd ◽  
Deuan Jones ◽  
Alison Scott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Space ◽  
Protozoan Parasite ◽  
Current Treatment ◽  
Screening Assay ◽  
Content Type ◽  
High Throughput Screening Assay

ABSTRACT Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.

scholarly journals A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

2019 ◽  
Author(s):  
André Alcântara ◽  
Denise Seitner ◽  
Fernando Navarrete ◽  
Armin Djamei
Keyword(s):  
Unfolded Protein Response ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Method ◽  
Screening Assay ◽  
Unfolded Protein ◽  
High Throughput Screening Assay ◽  
Protein Response

AbstractBackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signalingin planta. A set of 35 genes from a library of secreted proteins from the maize pathogenUstilago maydiswere transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress inNicotiana benthamianaplants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

Sign in / Sign up

Export Citation Format

Share Document