Triosephosphate Isomerase deficiency (TPI-Df) is a devastating untreatable childhood metabolic disease resulting in anemia, severe locomotor impairment, and premature death. Numerous single amino acid substitutions in TPI are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI-Df mutations result in a protein that retains function, and pathogenesis is known to result from decreased steady state levels of the functioning protein. There are no small molecule therapies for TPI-Df; current treatments are limited to symptomatic support and dietary interventions. We reasoned that a phenotypic screen was most appropriate to capture agents that stabilize mutant TPI and developed a human cellular TPI-Df assay based on a cellular model of the "common" TPIE105D mutant protein fused with a GFP and a fluorescent ROS biosensor. The assay was implemented for high-content, high-throughput imaging, optimized to full HTS standards, and used to screen a 2,560 compound pilot library and the 220,700 compound NIH MLSMR compound collection to identify candidate compounds for development into small molecule TPI-Df therapies. Hits were validated in dose-response, TPI-Df patient cells, and various orthogonal assays. Limited SAR revealed three promising compound series, which were evaluated for potential mechanisms of action. The lead series had previously been identified as inducers of HIF1 alpha, spawning a novel hypothesis that HIF1 alpha activation might be a potential avenue to treat TPI-Df patients. A lead molecule was moved into preliminary mouse studies to evaluate pharmacokinetics and tissue distribution and was shown to be moderately brain-penetrant. The lead compound is now positioned for target identification studies and efficacy testing in vivo TPI Df models, including a newly validated mouse model.

Background/Objectives: Triosephosphate Isomerase (TPI) is a glycolytic enzyme known to be associated with TPI deficiency, a severe form of childhood-onset glycolytic enzymopathy associated with hemolytic anemia, neuromuscular impairment and early death. Most often the disease results from the common TPI1E105D mutation, which can be either homozygous or compound heterozygous with another allele. Methods: We purified TPIR5G protein, studied mutant protein biochemistry, established and characterized TPIR5G/f.s.patient cells, and investigated newly identified compounds for their efficacy in vitro using Western blot and TPI activity assays. Results: We identified novel TPI1 alleles that result in TPI Deficiency with an atypical presentation lacking anemia and with more slowly developing neurologic and locomotor impairment. The patient was found to be compound heterozygous with a missense mutation resulting in an R5G amino acid substitution and a frameshift mutation that is a predicted null allele. To better understand disease pathogenesis in this patient, we expressed and purified the TPIR5G human protein and studied it biochemically in addition to studying TPIR5G/f.s.patient cells. We discovered that purified TPIR5G protein has wildtype activity with modestly increased dimer stability. We also discovered that steady-state TPI protein levels were markedly reduced, suggesting that the instability of the mutant protein underlies disease pathogenesis. We tested compounds recently identified in a screen for novel TPI Df therapies for their efficacy in TPIR5G/f.s.patient cells. All three compounds significantly increased TPI protein levels in patient cells. As expected, since the mutant protein retains essentially wild type activity, the increase in TPI protein levels also resulted in a significant increase in TPI activity. Conclusions: These results establish TPIR5G as a TPI Df allele, demonstrate that reduced stability of the mutant protein underlies pathogenesis akin to other disease-causing alleles, and suggest that recently discovered developing therapies will likely function broadly and should be developed as potential TPI Df therapies.

Triosephosphate isomerase (TPI) is a ubiquitously expressed enzyme encoded by the TPI1 gene. It catalyzes the interconversion of the triose phosphate isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate in the fifth step of glycolysis. TPI deficiency (TPI Df; MIM# 615512) is an autosomal recessive disorder due to biallelic pathogenic variants in TPI1. In keeping with other glycolytic enzymopathies, severe hemolytic anemia is a common finding. Additionally, many individuals with TPI Df develop neuromuscular symptoms, which is unusual for a glycolytic enzymopathy. There appears to be a genotype-phenotype correlation between a TPI1 p.Glu105Asp/null genotype and a severe life-limiting neuromuscular phenotype. Tpi1-deficient mice with a p.Glu105Asp/null genotype recapitulate the life-limiting neuromuscular phenotype seen in humans, but the exact pathomechanism remains unclear. Here we describe a 2-month-old male proband who presented with failure to thrive, respiratory failure, seizures, and severe hemolytic anemia, who passed away at 3 months of age. Trio whole genome sequencing showed compound heterozygous variants with the common p.Glu105Asp variant in trans to a newly described likely pathogenic splice site c.324 + 1G > C variant, predicted to cause nonsense mediated decay. Here we review our case as well as the literature to hypothesize a mechanism by which TPI Df due to a p.Glu105Asp/null genotype causes severe disease. Given the overall fatal nature of this condition, novel therapeutic approaches are urgently needed. Currently, treatments are experimental. Ketogenic diet and triheptanoin were effective in treating seizures in a TPI mutant Drosophila, known as TPIsugarkill, although clinical data in humans is lacking. Additionally, bone marrow transplant has been shown to improve the hematologic phenotype in mice and has been done in an isolated number of patients. While there are no proven therapies available at this time, we hope this review will lead the discussion to consider future therapeutic options.

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme's structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI's susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Triosephosphate isomerase deficiency (TPI Df) is a rare multisystem disorder with severe neuromuscular symptoms which arises exclusively from mutations within the TPI1 gene. Studies of TPI Df have been limited due to the absence of mammalian disease models and difficulties obtaining patient samples. Recently, we developed a novel murine model of TPI Df which models the most common disease-causing mutation in humans, TPI1E105D. Using our model in the present study, the underlying pathogenesis of neuromuscular symptoms has been elucidated. This is the first report detailing studies of neuromuscular pathology within a murine model of TPI Df. We identified several contributors to neuromuscular symptoms, including neurodegeneration in the brain, alterations in neurotransmission at the neuromuscular junction, and reduced muscle fiber size. TPI Df mice also exhibited signs of cardiac pathology and displayed a deficit in vascular smooth muscle functionality. Together, these findings provide insight into pathogenesis of the neuromuscular symptoms in TPI Df and can guide the future development of therapeutics.