Parkinson's disease (PD) is characterized by progressive nigrostriatal degeneration and striatal dysfunction, yet its metabolic remodeling remains incompletely defined. Here, untargeted GC-MS metabolomics was used to investigate the effects of standardized Brazilian green propolis on the striatal metabolic profile in the 6-hydroxydopamine (6-OHDA) rat model. Discriminant metabolites, including suberic acid, gluconic acid, heptadecane, and tartaric acid, distinguished experimental groups, capturing key features of the metabolic response to dopaminergic injury and treatment. Suberic acid emerged as a prominently modulated metabolite, potentially linked to alterations in lipid catabolism associated with mitochondrial-peroxisomal pathways. Propolis treatment attenuated the elevation of suberic acid, accompanied by a reduction in gluconic acid levels, suggesting a metabolic profile linked to pathways involved in redox balance and glucose handling. Given previous reports identifying heptadecane as a hydrocarbon constituent of volatile propolis fractions, complementary GC-Q-TOF analyses demonstrated that heptadecane was absent from the administered extract, despite its consistent association with propolis-treated groups. Metabolic changes were accompanied by attenuation of nigrostriatal dopaminergic neurodegeneration and improved motor performance. Together, these findings delineate a striatal metabolic signature associated with Brazilian green propolis and identify suberic acid as a key metabolite linked to neuroprotection in experimental Parkinsonism.

Background and Objectives: Sickle cell trait (SCT) persons are significant donors, and discarding these blood units reduces their supplies, mainly in the third-world countries. This work focused on 12 metabolites associated with the red blood cell (RBC) storage lesion and 23 amino acids in the supernatants of packed RBC units from SCT and reference (non-SCT) donors stored in the same conditions. Materials and Methods: All samples of RBC concentrates were collected and separated from the storage of Colsan (Beneficient Association of Blood Collection), where they were routinely processed and separated as packed RBC units and stored in the refrigerator (2 degrees-6 degrees C). The supernatant samples of each packed RBC bag were separated by centrifugation at days 1, 7, 14, 21, 28 and 35 of storage and kept at -80 degrees C till the metabolite analysis together. Results: The quantitation of metabolites and amino acids examined in the supernatant of SCT and reference donors showed no statistical differences along the cold storage. Lactic acid and malic acid releases occur in three phases during RBC storage. Basic and acid amino acids and corresponding amides have low and stable values during the first 14 days of storage, followed by a steep increase. Conclusion: Our metabolomic results give elements that seem not to contraindicate the transfusion of RBC with SCT, besides its more structural fragility.

The purpose of this review was to present the basic concepts of metabolomics methodology and the use of saliva for diagnostic, prognostic, and predictive strategies. This review followed the focus in: “saliva metabolomics” and “oral diseases”. The authors searched studies on PubMed database. The inclusion criteria were original studies and reviews that assessed metabolomics techniques. A descriptive analysis was performed considering the study design, approach system, clinical steps, and tools for the determination of profile or biomarkers metabolites, and the advantages and disadvantages. Metabolomic analyses use a combination of analytical instrumentation and informatic tools to provide information on metabolite characteristics. In this review we described different technologies applied and the advantages and limitations of each technique. Furthermore, in the literature search, we retrieved 25 studies that investigated saliva metabolites in oral diseases: 8 studies used targeted analysis and 17 untargeted metabolomics approaches. Most studies included patients with periodontal diseases, oral squamous cell carcinoma, and Sjögren Syndrome. The most frequently reported metabolites were glycine, leucine, phenylalanine, dipeptides, linoleic acid, arachidonic acid, tyrosine, choline, taurine, lactate, valine, and proline. Metabolomics analysis has emerged as a powerful tool for tumor diagnosis and to enhance tumor classification, including salivary gland tumors (SGTs). It also holds promise for developing personalized treatment plans and defining more precise prognostic categories. Metabolomics is the most functional and comprehensive technique for monitoring and understanding gene functions and identifying the biochemical state of an organism in response to genetic and environmental changes.

Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease. The parasite has developed various mechanisms to get through its intricate life cycle and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector's digestive tract as an epimastigote form, encountering fluctuating nutrient availability and oxidative stress caused by the digestion of red blood cells from the mammalian host blood meal. To unravel how the parasite's metabolism adapts to these changing conditions, we conducted an analysis of the chemical species present in epimastigote forms. This involved comparing cultured parasites with those subjected to nutritional deficiency or oxidative stress using untargeted metabolomics. We looked at 21 samples: seven biological copies of parasites that were actively growing, seven samples that were put in a medium without nutrients for 3 h, and seven samples that were treated with glucose oxidase for 30 min to make H2O2 continuously. Importantly, in all conditions, parasite viability was maintained when the samples were collected. Upon nutrient removal, we observed a substantial decrease in amino acids and carbohydrate metabolites, accompanied by the accumulation of fatty acids and steroids, with the predominance of inositol and sphingolipid metabolism, along with a simultaneous decrease in the levels of H2O2. In the presence of H2O2, a significant rise in components of the pentose pathway and specific amino acids such as methionine and serine occurred, along with pathways related to an increase in antioxidant species metabolism such as ribulose 5-phosphate and glyceric acid. Conversely, fatty acid and steroid levels decrease. We found no common increase in metabolites or lipids. In contrast, eight species (succinic acid, glutamic acid, valine, 2-hydroxyisocaproic acid, alanine, indolelactic acid, proline, and lanosterol) were consumed under both stresses. These findings underscore the rapid and distinct enrichment responses in amino acids, lipids, and carbohydrates required to cope with each different environmental condition. We concluded that T. cruzi presents a flexible metabolism that rapidly adapts to variable changes in the environment.

Objective Sjögren's Syndrome (SS) is a chronic inflammatory autoimmune exocrinopathy, and although, the role of metabolism in the autoimmune responses has been discussed in diseases such as lupus erythematosus, rheumatoid arthritis, psoriasis and scleroderma. There is a lack of information regarding the metabolic implications of SS. Considering that the disease affects primarily salivary glands; the aim of this study is to evaluate the metabolic changes in the salivary glands' microenvironment using a targeted metabolomics approach. Methods The saliva from 10 patients diagnosed with SS by the American-European consensus and 10 healthy volunteers was analyzed in an Ultra-high Performance Liquid Chromatograph Coupled Mass Spectrometry (UPLC-MS). Results The results showed an increased concentration in SS of metabolites involved in oxidative stress such as lactate, alanine and malate, and amino acids involved in the growth and proliferation of T-cells, such as arginine, leucine valine and isoleucine. Conclusions These results revealed that is possible to differentiate the metabolic profile of SS and healthy individuals using a small amount of saliva, which in its turn may reflect the cellular changes observed in the microenvironments of damaged salivary glands from these patients.

Parkinson’s-disease (PD) is an incurable, age-related neurodegenerative disease, and its global prevalence of disability and death has increased exponentially. Although motor symptoms are the characteristic manifestations of PD, the clinical spectrum also contains a wide variety of non-motor symptoms, which are the main cause of disability and determinants of the decrease in a patient’s quality of life. Noteworthy in this regard is the stress on the cardiac system that is often observed in the course of PD; however, its effects have not yet been adequately researched. Here, an untargeted metabolomics approach was used to assess changes in cardiac metabolism in the 6-hydroxydopamine model of PD. Beta-sitosterol, campesterol, cholesterol, monoacylglycerol, α-tocopherol, stearic acid, beta-glycerophosphoric acid, o-phosphoethanolamine, myo-inositol-1-phosphate, alanine, valine and allothreonine are the metabolites that significantly discriminate parkinsonian rats from sham counterparts. Upon analysis of the metabolic pathways with the aim of uncovering the main biological pathways involved in concentration patterns of cardiac metabolites, the biosynthesis of both phosphatidylethanolamine and phosphatidylcholine, the glucose-alanine cycle, glutathione metabolism and plasmalogen synthesis most adequately differentiated sham and parkinsonian rats. Our results reveal that both lipid and energy metabolism are particularly involved in changes in cardiac metabolism in PD. These results provide insight into cardiac metabolic signatures in PD and indicate potential targets for further investigation.

While there is sustained growth of the older population worldwide, ageing is a consistent risk factor for neurodegenerative diseases, such as Parkinson's-disease (PD). Considered an emblematic movement disorder, PD comprises a miscellany of non-motor symptoms, for which effective management remains an unfulfilled need in clinical practice. Highlighted are the cardiovascular abnormalities, that cause significant burden in PD patients. Evidence suggests that key biological processes underlying PD pathophysiology can be modulated by diet-derived bioactive compounds, such as green propolis, a natural functional food with biological and pharmacological properties. The effects of propolis on cardiac affection associated to PD have received little coverage. In this study, a metabolomics approach and Positron Emission Tomography (PET) imaging were used to assess the metabolic response to diet supplementation with green propolis on heart outcomes of rats with Parkinsonism induced by 6-hydroxydopamine (6-OHDA rats). Untargeted metabolomics approach revealed four cardiac metabolites (2-hydroxybutyric acid, 3-hydroxybutyric acid, monoacylglycerol and alanine) that were significantly modified between animal groups (6-OHDA, 6-OHDA + Propolis and sham). Propolis-induced changes in the level of these cardiac metabolites suggest beneficial effects of diet intervention. From the metabolites affected, functional analysis identified changes in propanoate metabolism (a key carbohydrate metabolism related metabolic pathway), glucose-alanine cycle, protein and fatty acid biosynthesis, energy metabolism, glutathione metabolism and urea cycle. PET imaging detected higher glucose metabolism in the 17 areas of the left ventricle of all rats treated with propolis, substantially contrasting from those rats that did not consume propolis. Our results bring new insights into cardiac metabolic substrates and pathways involved in the mechanisms of the effects of propolis in experimental PD and provide potential novel targets for research in the quest for future therapeutic strategies.

Background Uterine leiomyomas are benign monoclonal tumors originating from the myometrium. Little information exists concerning metabolomics and the presence of leiomyomas. Objective The present study evaluated circulating metabolites in the plasma and their correlation with the presence and size of leiomyomas. Study Design Cross-sectional observational study, including women divided into 3 groups: 37 with leiomyomas and uterus >500 cm(3), 17 with leiomyomas and uterus <= 150 cm(3), and 21 leiomyoma-free. Patients underwent peripheral blood collection using untargeted metabolic assessment by gas chromatography coupled to mass spectrometer. Results There was no statistical difference between patients' anthropometric and demographic features and laboratory tests. Statistical differences in uterus volume (P < 0.0001) were found. Forty-six metabolites were identified (35% amino acids and derivatives, 22% fatty acids, and 18% carbohydrates). Statistically significant metabolic distinction (P < 0.05, false discovery rate< 0.05) was observed for 14 metabolites. Most amino acids (L-isoleucine, L-valine, and pyroglutamic acid) were significantly reduced in plasma levels of patients with large leiomyomas. The only exception was L-glutamine, with a significant increase. Fatty acids (arachidonic acid, alfa-tocopherol, palmitic acid, and stearic acid) were similarly reduced in large leiomyomas patients, except for alpha-linolenic acid, which increased. For carbohydrates (myo-inositol, D-threitol, and D-ribose), there was a decrease in the plasma of patients with leiomyomas. Conclusion There are different plasma metabolites levels of amino acids, fatty acids, and carbohydrates among patients with leiomyomas, most of them reduced, but some significantly increased in large leiomyomas, compared to leiomyoma-free patients.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0–2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.

Balloon catheter endothelial denudation in New Zealand white rabbits fed high cholesterol diet is a validated atherosclerosis model. Well-characterized in terms of atherosclerosis induction and progression, the metabolic changes associated with the atherosclerosis progression remain indeterminate. Non-targeted metabolomics permits to develop such elucidation and allows to evaluate the metabolic consequences of colchicine treatment, an anti-inflammatory drug that could revert these changes. 16 rabbits underwent 18 weeks of atherosclerosis induction by diet and aortic denudation. Thereafter animals were randomly assigned to colchicine treatment or placebo for 18 weeks while on diet. Plasma samples were obtained before randomization and at 36 weeks. Multiplatform (GC/MS, CE/MS, RP-HPLC/MS) metabolomics was applied. Plasma fingerprints were pre-processed, and the resulting matrixes analyzed to unveil differentially expressed features. Different chemical annotation strategies were accomplished for those significant features. We found metabolites associated with either atherosclerosis progression, or colchicine treatment, or both. Atherosclerosis was profoundly associated with an increase in circulating bile acids. Most of the changes associated with sterol metabolism could not be reverted by colchicine treatment. However, the variations in lysine, tryptophan and cysteine metabolism among others, have shown new potential mechanisms of action of the drug, also related to atherosclerosis progression, but not previously described.

The leishmanicidal potential of benzophenones has been described, some of them highlighting their potential as cysteine protease inhibitors. Therefore, this work described leishmanicidal activity of nine benzophenone derivatives (1a–c;2a–c;3a–c) against intramacrophage amastigote forms of Leishmania(L.) amazonensis (IC50) and the cytotoxic effect on murine peritoneal macrophages (CC50). The derivative 1c exhibited a selectivity index SI (CC50/IC50) of 6.7, besides cytotoxicity lower than Amphotericin B (p < 0.05). Moreover it showed inhibitory activity against papain (42.8 ± 0.3, p < 0.05), and when tested on trypanosomatids cysteine proteases 1c also proved to be a potent inhibitor of rCPB2.8, rCPB3.0 and cruzain, showing non-competitive inhibition mechanism by enzymatic assays in vitro.So, benzophenone 1c is interesting drug candidate prototype, with a multi-target directed mode of action, inhibiting rCPB2.8, rCPB3.0 and cruzain.

ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. UsingT. cruziCL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the majorT. cruzicysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase ofT. cruzimetacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion.

Metrodorea stipularis stem extracts were studied in the search for possible antichagastic, antimalarial, and antitumoral compounds using cruzain from Trypanosoma cruzi, Plasmodium falciparum, and cathepsins B and L, as molecular targets, respectively. Dihydrochalcones 1, 2, 3, and 4 showed significant inhibitory activity against all the targets. Compounds 1-4 displayed IC50 values ranging from 7.7 to 21.6 μM against cruzain; dihydrochalcones 2 and 4 inhibited the growth of three different strains of P. falciparum in low micromolar concentrations; and against cathepsins B and L these compounds presented good inhibitory activity with IC50 values ranging from 1.0 to 14.9 μM. The dihydrochalcones showed good selectivity in their inhibitory activities against the cysteine proteases.

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K(I) of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver-Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C' in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.

Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lb(pro)) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lb(pro) cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lb(pro) possesses specific binding sites at the non prime side from S(1) down to S(7) [Santos et al. (2009) Biochemistry, 48, 7948-7958]. Here, we demonstrate that Lb(pro) has high prime side specificity at least down to the S'(5) site. Lb(pro) is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I ↔ EI→ E - I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lb(pro) that show for the reversible step (E + I ↔ EI) K(i) = 3.4 μM and 11.6 μM, and for the irreversible step (EI→E-I) k(4) = 0.16 and 0.06 min(-1), respectively. Knowledge of the Lb(pro) specificity led us to extend E64 by addition of the dipeptide R-P. This compound, termed E64-R-P-NH(2), irreversibly inhibited Lb(pro) with a K(i) = 30 nM and k(4) = 0.01 min(-1) and can serve as the basis for design of specific inhibitors of FMDV replication.

Here we report a detailed analysis of magnesium (Mg²+) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg²+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR↓FAGV-Q-EDDnp (from measles virus fusion protein F₀ and Abz-RERRRKKR↓GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl₂. It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg²+ is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg²+ ions, which bind to furin with a K(d) value of 1.1 mM.

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC50) for NF and NFOH presented values of 22.83 +/- 1.2 mu M and 10.55 +/- 0.81 mu M, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins, The first cleavage is Performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P-7. Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P-1-P-3 in an extended conformation, whereas residues P-4-P-7 are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P-5. Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes

We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K+ ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P1′, and V at P2′, at P3′ the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P6; basic amino acids at P5; and no clear tendency at P3. Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.

Some unexpected promiscuous inhibitors were observed in a virtual screening protocol applied to select cruzain inhibitors from the ZINC database. Physical-chemical and pharmacophore model filters were used to reduce the database size. The selected compounds were docked into the cruzain active site. Six hit compounds were tested as inhibitors. Although the compounds were designed to be nucleophilically attacked by the catalytic cysteine of cruzain, three of them showed typical promiscuous behavior, revealing that false positives are a prevalent concern in VS programs.