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 language | English |
|---|---|
| Article number | e00309-19 |
| Pages (from-to) | 1-15 |
| Number of pages | 15 |
| Journal | Antimicrobial Agents and Chemotherapy |
| Volume | 63 |
| Issue number | 9 |
| Early online date | 15 Jul 2019 |
| DOIs | |
| Publication status | Published - Sep 2019 |
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Keywords
- Assay development
- Chagas' disease
- Drug discovery
- Drug screening
- Pharmacology
- Proteasome
- Trypanosoma cruzi
Cite this
}
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 journal › Article
TY - JOUR
T1 - Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay
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
N1 - Funding: Wellcome Trust for funding (Grants 204672/Z/16/Z and 203134/Z/16/Z).
PY - 2019/9
Y1 - 2019/9
N2 - 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.
AB - 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.
KW - Assay development
KW - Chagas' disease
KW - Drug discovery
KW - Drug screening
KW - Pharmacology
KW - Proteasome
KW - Trypanosoma cruzi
UR - http://www.scopus.com/inward/record.url?scp=85071388358&partnerID=8YFLogxK
U2 - 10.1128/AAC.00309-19
DO - 10.1128/AAC.00309-19
M3 - Article
C2 - 31307977
VL - 63
SP - 1
EP - 15
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
SN - 0066-4804
IS - 9
M1 - e00309-19
ER -