First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target

Abstract

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

“…From the chromatogram obtained for the markers (gel filtration standard # 151-1901 from Bio-Rad), we found that protein mass of the FoxTPI protein corresponds to the native dimers with a relative molecular weigth (MW) of 56 kDa ( Figure 4B), and which is in accordance with the MW expected from the amino acid sequence (27,000 × 2 = 54 kDa), whereas the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a unique band of around 27 kDa (Figure 3). This result is consistent with those reported for other TPIs, such as of Saccharomyces cerevisiae [44], and parasites as Entamoeba histolytica (EhTPI), Trypanosoma brucei (TbTPI), T. cruzy (TcTPI), and Encephalitozoon intestinalis (EiTPI), which were reported with a molecular mass around 27 kDa [45][46][47][48].…”

“…The result obtained show that the FoxTPI protein is a well-folded protein with a secondary structure like that of a canonical TPI barrel. It is important to mention that the intensity pattern in the far UV region shown for FoxTPI is similar to to other secondary structures of TPIs previously purified and characterized, as in G. lamblia (GlTPI) [17], human (HuTPI) [50], Trichomonas vaginalis (TvTPI) [55], Saccharomyces cerevisiae (TPI) [44], and Encephalitozoon intestinalis (EiTPI) [48], between which no significant differences were observed.…”

“…The temperature increases induced denaturation of the protein. In the denaturation profile, the protein remained at its baseline (native structure) until approximately 43 • C ( Figure 7B) and, above this temperature, the enzyme was denatured cooperatively, completing the process at approximately 60 • C. The calculated T m was 51.4 • C, which is slightly inferior to the values reported for other TPI enzymes previously characterized from E. intestinalis (EiTPI; 52.8 • C) [48], T. cruzi (TcTPI; 57.3 • C) [52], Entamoeba histolytica (EhTPI; 58.6 • C) [59], G. lamblia (GlTPI; 57.5 • C) [17], and Homo sapiens (HsTPI; 61.2 • C) [50].…”

Potential Site to Direct Selective Compounds in the Triosephosphate Isomerase for the Development of New Drugs

“…In this study, we propose a potential site as a therapeutic target against the enzyme triosephosphate isomerase for drug development, and that this potential new drug could be safe to be used at humans. We determined the K in position 214 (near the sequence YGGSV−K214) is indispensable for the interaction with the tested compounds and it helps the interaction in the active site from TIM.…”

Prevalence and Genotyping of Microsporidian Parasites in Dogs in Turkey: Zoonotic Concerns

“…The infection prevalence in human populations varies from 0% to 60% worldwide, including ranges of 2.5% to 42%, 1.38% to 13%, 5.2% to 58.1%, and 1.5% to 60% in Europe, Asia, Africa, and the United States respectively (García‐Torres et al. ; Jimenez‐Gonzalez et al. ).…”