A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

Abstract

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

Itavastatin and Resveratrol increase triosephosphate isomerase protein (TPI) in a newly identified variant, TPIQ181P, that confers TPI deficiency

“…Using human embryonic kidney (HEK) cells expressing fluorescently tagged TPI E105D to model TPI Df, we previously screened the NIH clinical collection drug library to identify compounds that increase mutant TPI levels ( Vogt et al, 2021 ). Using this high-throughput assay, resveratrol, a natural supplement, and several statins were identified as potential therapies for TPI Df ( Vogt et al, 2021 ). We asked whether these compounds similarly increase TPI levels in compound heterozygous patient cells.…”

“…Patient and control fibroblast western blotting was performed as previously reported ( Vogt et al, 2021 ). Briefly, cells were trypsinized (using Trypsin 0.05% for 5 min), pelleted, resuspended in RIPA buffer with protease inhibitors [PMSF (100 µM), Leupeptin (1 µg/µl), Pepstatin A (0.5 µg/µl)] and pulse sonicated.…”

“…Although there is some variability in the TPI Df disease course that is not completely understood, there is a prevalence of evidence on numerous pathogenic mutations that supports the conclusions that (1) pathogenic variants retain significant activity, (2) TPI Df mutant proteins exhibit reduced stability presumably due to accelerated turnover by protein quality control (PQC) machinery, and (3) impaired stability and not activity correlates the most with disease severity. As such, we and others have proposed that impairing mutant TPI turnover is the most promising avenue for the development of TPI Df treatments ( Hrizo et al, 2021 ; Segal et al, 2019 ; Vogt et al, 2021 ). Recent studies have utilized RNA interference (RNAi) screens to identify potential therapeutic targets ( Hrizo et al, 2021 ) and exploited human cellular models to develop high-throughput optical screens to enable direct drug screening for novel TPI Df therapeutics ( Vogt et al, 2021 ).…”

“…As such, we and others have proposed that impairing mutant TPI turnover is the most promising avenue for the development of TPI Df treatments ( Hrizo et al, 2021 ; Segal et al, 2019 ; Vogt et al, 2021 ). Recent studies have utilized RNA interference (RNAi) screens to identify potential therapeutic targets ( Hrizo et al, 2021 ) and exploited human cellular models to develop high-throughput optical screens to enable direct drug screening for novel TPI Df therapeutics ( Vogt et al, 2021 ). Interestingly, these studies identified two compounds, resveratrol and itavastatin, that increase the levels of the common mutant TPI E105D protein in a human cellular TPI Df model and also in TPI Df patient cells ( Vogt et al, 2021 ).…”

“…Recent studies have utilized RNA interference (RNAi) screens to identify potential therapeutic targets ( Hrizo et al, 2021 ) and exploited human cellular models to develop high-throughput optical screens to enable direct drug screening for novel TPI Df therapeutics ( Vogt et al, 2021 ). Interestingly, these studies identified two compounds, resveratrol and itavastatin, that increase the levels of the common mutant TPI E105D protein in a human cellular TPI Df model and also in TPI Df patient cells ( Vogt et al, 2021 ). Here, we report that these compounds also increase protein levels in TPI Q181P/E105D patient fibroblasts, suggesting that these compounds should be tested for efficacy in animal models as they would represent a first-ever treatment for TPI Df.…”