Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based 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.

Challenges in Chagas Disease Drug Development

“…Probably the most prominent recent development in Chagas disease drug research has been the increased use of high-throughput phenotypic screening [ 93 , 94 , 95 ]. The requirement for large compound libraries, robotic sample handling equipment, expertise in parasite biology, and over-arching funding mechanisms has brought together both the academic and commercial sectors, with not-for-profit drug development agencies, such as the DND i .…”

Ubiquitination and the Proteasome as Drug Targets in Trypanosomatid Diseases

“…The vinylsulfone 125 I-NP-L2N-VS, labels exclusively the β2 subunit of T. brucei , but both β2 and β5 of human origin ( Wang et al, 2003 ). Furthermore, the peptide suc-Leu-Leu-Val-Tyr-AMC is cleaved exclusively by β5 in human proteasomes, but by β5 and β1 or β2 in the T. brucei proteasome ( Zmuda et al, 2019 ).…”

“…These include various peptide aldehydes, epoxomicin and its derivatives, peptidyl vinyl sulfones, peptidyl vinyl esters, and several natural γ-lactam-β-lactones ( Glenn et al, 2004 ; Steverding et al, 2005 ; Steverding et al, 2011a ; Steverding et al, 2011b ). Several studies have shown differences in the relative inhibition of β5 and β2 subunits between trypanosoma and human proteasomes, again indicative of the parasite proteasome possessing distinct catalytic activities ( Nkemgu-Njinkeng et al, 2002 ; Wang et al, 2003 ; Zmuda et al, 2019 ). However, the design or selection of trypanosomatid-selective inhibitors based on these proteolytic differences has not yet been possible ( Steverding et al, 2018 ).…”

“…LXE408 has a desirable safety profile and is now being tested in phase I clinical trials ( Nagle et al, 2020 ). GSK3494245 also showed reasonable bioavailability and a good safety margin in rat and is being tested in phase I trials as well ( Wyllie et al, 2019 ; Zmuda et al, 2019 ).…”

Chagas Disease: Perspectives on the Past and Present and Challenges in Drug Discovery

“…GNF6702 was invented by researchers working at the Genomics Institute of The Novartis Research Foundation in 2013, with activity against these diseases. Another inhibitor, the GSK3494245/DDD01305143, is a preclinical candidate for visceral leishmaniasis developed from a T. cruzi screening hit [ 50 , 51 , 52 , 53 ]. Recently, new T. cruzi proteasome inhibitors using a luminescence-based high throughput screening assay have been identified [ 52 ].…”