Development of Novel Drugs for the Treatment of Chagas Disease

Chagas disease, or American trypanosomiasis, is a zoonosis caused by the hemoflagellate parasite Trypanosoma cruzi. It is mainly transmitted by the bite of blood-sucking insects. It is endemic in Latin America and emerging in the rest of the world, affecting approximately six million people. The drugs Benznidazole and Nifurtimox currently used for its treatment are not totally effective in the chronic phase of the disease. In addition, they are toxic, and there are many resistant Trigonoscuta cruzi strains. Therefore, developing new drugs for the treatment of Chagas disease is necessary. This chapter describes the development of drugs that inhibit α-hydroxy acid dehydrogenase isoenzyme II, a key enzyme in parasite energy metabolism. These drugs have shown more significant trypanocidal activity than the currently used drugs, and they have also prevented the development of chronic Chagas disease in infected mice

Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site

The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The kcat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability

Las quinoproteínas alcohol deshidrogenasas en los sistemas bacterianos: distribución, clasificación, estructura y función

Existe una gran diversidad de alcohol deshidrogenasas (ADHs) microbianas; las cuales son divididas en tres grandes grupos: (a) Las que son dependientes de las coenzimas NAD o NADP, (b) Las que son independientes de estas coenzimas; sin embargo, utilizan pirroloquinolina quinona (PQQ) y hemo tipo C como grupo prostético y (c) Las oxidasas dependientes de FAD que catalizan la reacción irreversible de alcoholes. Las ADHs que utilizan el PQQ, se encuentran a su vez divididas en tres tipos. Las ADHs tipo I que contienen sólo PQQ como grupo prostético y se les conoce como quinoproteínas; mientras que las ADHs tipo II y tipo III además del PQQ contienen hemo tipo C y se les conoce como quinohemoproteínas. Las ADHs tipo II son enzimas solubles que se encuentran en el espacio periplásmico y están presentes en proteobacterias como Pseudomonas putida, Ralstonia eutropha y Comamonas testosteroni. Las ADHs tipo III son enzimas que se encuentran ancladas a la membrana y trabajan orientadas hacia el espacio periplásmico. Se les ha identificado y caracterizado únicamente en bacterias ácido acéticas. Las ADH tipo III, por lo general contienen tres subunidades. El transporte intramolecular de electrones en las ADHs tipo II y IIII se propone que es del PQQ al hemo C de la primera subunidad y de ahí, de hemo en hemo en la segunda subunidad hasta llegar a la quinona endógena. Los tres tipos de PQQ-ADHs son discutidas en esta revisión

Hypoxia as a Modulator of Inflammation and Immune Response in Cancer

A clear association between hypoxia and cancer has heretofore been established; however, it has not been completely developed. In this sense, the understanding of the tumoral microenvironment is critical to dissect the complexity of cancer, including the reduction in oxygen distribution inside the tumoral mass, defined as tumoral hypoxia. Moreover, hypoxia not only influences the tumoral cells but also the surrounding cells, including those related to the inflammatory processes. In this review, we analyze the participation of HIF, NF-κB, and STAT signaling pathways as the main components that interconnect hypoxia and immune response and how they modulate tumoral growth. In addition, we closely examine the participation of the immune cells and how they are affected by hypoxia, the effects of the progression of cancer, and some innovative applications that take advantage of this knowledge, to suggest potential therapies. Therefore, we contribute to the understanding of the complexity of cancer to propose innovative therapeutic strategies in the future

Purification, concentration and recovery of small fragments of DNA from Giardia lamblia and their use for other molecular techniques

Purification of nucleic acids is an essential procedure for most experiments in molecular biology. In this paper, the freeze-squeeze method with some modifications is proposed as an alternative methodology for the purification, concentration and recovery of small DNA fragments from agarose gels. The advantage of this alternative methodology is that it enables the recovery of fragments that are less than 100 bp in length and enables suspension of products in smaller volumes compared to several commercially available kits. In addition, the purified fragments were re-amplified by PCR and used for cloning and sequencing. Moreover, this protocol was used to perform the isolation and identification of microRNAs from Giardia lamblia, as previously reported. This protocol has the advantage of being inexpensive and easy and can be employed for various molecular applications. The advantages of this protocol include • A modified classical method was used for purification of small DNA fragments from G. lamblia. • The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits. • The modified method allows concentration and recovery of fragments up to 60 bp in length. • The modified freeze-squeeze method allows re-suspension of the products in volumes of up to 2.5 μL

New Alternatives in the Fight against Tuberculosis: Possible Targets for Resistant Mycobacteria

Tuberculosis (TB) is a bacterial disease that remains a global health threat due to the millions of deaths attributed to it each year. The emergence of drug resistance has exacerbated and further increased the challenges in the fight against this illness. Despite the preventive measures using the application of the Bacillus Calmette-Guérin vaccine, the desired immunization outcome is not as high as expected. Conventional TB treatments exhibit serious limitations, such as adverse effects and prolonged duration, leading to a pressing need for alternative and more effective treatment options. Despite significant efforts, it took nearly four decades for diarylquinoline to become the most recently approved medicine for this disease. In addition, various possibilities, such as the usage of medications used for many other conditions (repurposed drugs), have been explored in order to speed up the process of achieving faster outcomes. Natural compounds derived from various sources (microorganisms, plants, and animals) have emerged as potential candidates for combating TB due to their chemical diversity and their unique modes of action. Finally, efforts towards the generation of novel vaccines have received considerable attention. The goal of this paper was to perform an analysis of the current state of treating drug-resistant TB and to evaluate possible approaches to this complicated challenge. Our focus is centered on highlighting new alternatives that can be used to combat resistant strains, which have potentiated the health crisis that TB represents

Evaluation of Immunomodulatory Activities of the Heat-Killed Probiotic Strain Lactobacillus casei IMAU60214 on Macrophages In Vitro

Most Lactobacillus species have beneficial immunological (“immunoprobiotic”) effects in the host. However, it is unclear how probiotic bacteria regulate immune responses. The present study investigated the effects of heat-killed Lactobacillus casei IMAU60214 on the activity of human monocyte-derived macrophages (MDMs). Human MDMs were treated with heat-killed L. casei at a ratio (bacteria/MDM) of 50:1, 100:1, 250:1, and 500:1, and then evaluated for the following: NO production, by Griess reaction; phagocytosis of FITC-labeled Staphylococcus aureus particles; cytokine secretion profile (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-12p70, IL-10, and transforming growth factor (TGF)-β) by ELISA; and costimulatory molecule (CD80 and CD86) surface expression, by flow cytometry. Heat-killed L. casei IMAU60214 enhanced phagocytosis, NO production, cytokine release, and surface expression of CD80 and CD86 in a dose-dependent manner. All products were previously suppressed by pretreatment with a Toll-like receptor 2 (TLR2)-neutralizing antibody. Overall, our findings suggest that this probiotic strain promotes an M1-like pro-inflammatory phenotype through the TLR2 signaling pathway. These effects on macrophage phenotype help explain the probiotic efficacy of Lactobacillus and provide important information for the selection of therapeutic targets and treatments compatible with the immunological characteristics of this probiotic strain

Impact of Heat-Killed Lactobacillus casei Strain IMAU60214 on the Immune Function of Macrophages in Malnourished Children

Malnutrition is commonly associated with immunological deregulation, increasing the risk of infectious illness and death. The objective of this work was to determine the in vitro effects of heat-killed Lactobacillus casei IMAU60214 on monocyte-derived macrophages (MDMs) from well-nourished healthy children, well-nourished infected children and malnourished infected children, which was evaluated by an oxygen-dependent microbicidal mechanism assay of luminol-increase chemiluminescence and the secretion of tumor necrosis factor (TNF-α), interleukin (IL-1β), IL-6 and IL-10, as well as phagocytosis using zymosan and as its antibacterial activity against Salmonella typhimurium, Escherichia coli and Staphylococcus aureus. We found that reactive oxygen species (ROS), secretion cytokines (TNFα, IL-1β, IL-6 and IL-10 levels), phagocytosis and bactericidal capacity increased in all groups after pre-treatment with heat-killed L. casei IMAU60214 at a ratio of 500:1 (bacteria:MDM) over 24 h compared with MDM cells without pre-treatment. The results could indicate that heat-killed L. casei IMAU60214 is a potential candidate for regulating the immune function of macrophages