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

Filip Zmuda, Lalitha Sastry, Sharon M. Shepherd, Deuan Jones, Alison Scott, Peter D. Craggs, Alvaro Cortes, David W. Gray (Lead / Corresponding author), Leah S. Torrie, Manu De Rycker (Lead / Corresponding author)

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Article numbere00309-19
Pages (from-to)1-15
Number of pages15
JournalAntimicrobial Agents and Chemotherapy
Volume63
Issue number9
Early online date15 Jul 2019
DOIs
Publication statusPublished - Sep 2019

Fingerprint

High-Throughput Screening Assays
Proteasome Inhibitors
Trypanosoma cruzi
Proteasome Endopeptidase Complex
Luminescence
Chymotrypsin
Drug Discovery
Chagas Disease
Caspases
Luciferases
Treatment Failure
Proteolysis
Catalytic Domain
Parasites
Proteins
Technology
Peptides
Enzymes
Pharmaceutical Preparations

Keywords

  • Assay development
  • Chagas' disease
  • Drug discovery
  • Drug screening
  • Pharmacology
  • Proteasome
  • Trypanosoma cruzi

Cite this

Zmuda, Filip ; Sastry, Lalitha ; Shepherd, Sharon M. ; Jones, Deuan ; Scott, Alison ; Craggs, Peter D. ; Cortes, Alvaro ; Gray, David W. ; Torrie, Leah S. ; De Rycker, Manu. / Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay. In: Antimicrobial Agents and Chemotherapy. 2019 ; Vol. 63, No. 9. pp. 1-15.
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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.",
keywords = "Assay development, Chagas' disease, Drug discovery, Drug screening, Pharmacology, Proteasome, Trypanosoma cruzi",
author = "Filip Zmuda and Lalitha Sastry and Shepherd, {Sharon M.} and Deuan Jones and Alison Scott and Craggs, {Peter D.} and Alvaro Cortes and Gray, {David W.} and Torrie, {Leah S.} and {De Rycker}, Manu",
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Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay. / Zmuda, Filip; Sastry, Lalitha; Shepherd, Sharon M.; Jones, Deuan; Scott, Alison; Craggs, Peter D.; Cortes, Alvaro; Gray, David W. (Lead / Corresponding author); Torrie, Leah S.; De Rycker, Manu (Lead / Corresponding author).

In: Antimicrobial Agents and Chemotherapy, Vol. 63, No. 9, e00309-19, 09.2019, p. 1-15.

Research output: Contribution to journalArticle

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AU - Zmuda, Filip

AU - Sastry, Lalitha

AU - Shepherd, Sharon M.

AU - Jones, Deuan

AU - Scott, Alison

AU - Craggs, Peter D.

AU - Cortes, Alvaro

AU - Gray, David W.

AU - Torrie, Leah S.

AU - De Rycker, Manu

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