Repurposing of rabeprazole as an anti-Trypanosoma cruzi drug that targets cellular triosephosphate isomerase.

Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis.

Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target.

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Epsilon-aminocaproic acid prevents high glucose and insulin induced-invasiveness in MDA-MB-231 breast cancer cells, modulating the plasminogen activator system.

Obesity and type II diabetes mellitus have contributed to the increase of breast cancer incidence worldwide. High glucose concentration promotes the proliferation of metastatic cells, favoring the activation of the plasminogen/plasmin system, thus contributing to tumor progression. The efficient formation of plasmin is dependent on the binding of plasminogen to the cell surface. We studied the effect of ε-aminocaproic acid (EACA), an inhibitor of the binding of plasminogen to cell surface, on proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and plasminogen activation system, in metastatic MDA-MB-231 breast cancer cells grown in a high glucose microenvironment and treated with insulin. MDA-MB-231 cells were treated with EACA 12.5 mmol/L under high glucose 30 mmol/L (HG) and high glucose and insulin 80 nmol/L (HG-I) conditions, evaluating: cell population growth, % of viability, migratory, and invasive abilities, as well as the expression of uPA, its receptor (uPAR), and its inhibitor (PAI-1), by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, MMP-2 and MMP-9 mRNAs were evaluated by RT-PCR. Markers of EMT were evaluated by Western blot. Additionally, the presence of active uPA was studied by gel zymography, using casein-plasminogen as substrates. EACA prevented the increase in cell population, migration and invasion induced by HG and insulin, which was associated with the inhibition of EMT and the attenuation of HG- and insulin-dependent expression of uPA, uPAR, PAI-1, MMP-2, MMP-9, α-enolase (ENO A), and HCAM. The interaction of plasminogen to the cell surface and plasmin formation are mediators of the prometastasic action of hyperglycemia and insulin, potentially, EACA can be employed in the prevention and as adjuvant treatment of breast tumorigenesis promoted by hyperglycemia and insulin.

High glucose and insulin enhance uPA expression, ROS formation and invasiveness in breast cancer-derived cells.

BACKGROUND: Accumulating evidence indicates that type 2 diabetes is associated with an increased risk to develop breast cancer. This risk has been attributed to hyperglycemia, hyperinsulinemia and chronic inflammation. As yet, however, the mechanisms underlying this association are poorly understood. Here, we studied the effect of high glucose and insulin on breast cancer-derived cell proliferation, migration, epithelial-mesenchymal transition (EMT) and invasiveness, as well as its relationship to reactive oxygen species (ROS) production and the plasminogen activation system. METHODS: MDA-MB-231 cell proliferation, migration and invasion were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), scratch-wound and matrigel transwell assays, respectively. ROS production was determined using 2' 7'-dichlorodihydrofluorescein diacetate. The expression of E-cadherin, vimentin, fibronectin, urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) were assessed using qRT-PCR and/or Western blotting assays, respectively. uPA activity was determined using gel zymography. RESULTS: We found that high glucose stimulated MDA-MB-231 cell proliferation, migration and invasion, together with an increased expression of mesenchymal markers (i.e., vimentin and fibronectin). These effects were further enhanced by the simultaneous administration of insulin. In both cases, the invasion and growth responses were found to be associated with an increased expression of uPA, uPAR and PAI-1, as well as an increase in active uPA. An osmolality effect of high glucose was excluded by using mannitol at an equimolar concentration. We also found that all changes induced by high glucose and insulin were attenuated by the anti-oxidant N-acetylcysteine (NAC) and, thus, depended on ROS production. CONCLUSIONS: From our data we conclude that hyperglycemia and hyperinsulinemia can promote breast cancer cell proliferation, migration and invasion. We found that these features were associated with increased expression of the mesenchymal markers vimentin and fibronectin, as well as increased uPA expression and activation through a mechanism mediated by ROS.