The GLP-1 Receptor Agonist Liraglutide Decreases Primary Bile Acids and Serotonin in the Colon Independently of Feeding in Mice.

Liraglutide, a glucagon-like peptide 1 analog used to treat type 2 diabetes and obesity, is a potential new treatment modality for bile acid (BA) diarrhea. Here, we show that administration of liraglutide significantly decreased total BAs, especially the primary BAs, including cholic acid, chenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, glycocholic acid, and ß-muricholic acid, in the liver and feces. In addition, liraglutide significantly decreased tryptophan metabolites, including L-tryptophan, serotonin, 5-hydroxy indole-3-acetic acid, L-kynurenine, and xanthurenic acid, in the colon, whereas it significantly increased indole-3-propionic acid. Moreover, the administration of liraglutide remarkably decreased the expression of apical sodium-dependent bile acid transporter, which mediates BA uptake across the apical brush border member in the ileum, ileal BA binding protein, and fibroblast growth factor 15 in association with decreased expression of the BA-activated nuclear receptor farnesoid X receptor and the heteromeric organic solute transporter Ostα/ß, which induces BA excretion, in the ileum. Liraglutide acutely decreased body weight and blood glucose levels in association with decreases in plasma insulin and serotonin levels in food-deprived mice. These findings suggest the potential of liraglutide as a novel inhibitor of primary BAs and serotonin in the colon.

Indoleamine 2,3-Dioxygenase Inhibitor Suppresses Colon Cancer Cell Migration, Invasion, and Epithelial-Mesenchymal Transition.

BACKGROUND/AIM: Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme in tryptophan metabolism and plays an important role in immunosuppression. The effects of IDO1 on tumor invasion and metastasis have been studied in several types of malignancies. However, the role of IDO1 in these steps in colorectal cancer (CRC) has not been elucidated. Therefore, we aimed to investigate the effects of IDO1 on invasion, migration, and epithelial-mesenchymal transition (EMT) in CRC cells. MATERIALS AND METHODS: All experiments were performed using the DLD-1 colon cancer cell line that expresses IDO1. We conducted a scratch wound healing assay and Boyden chamber assay to investigate the impact of IDO1 on DLD-1 cell migration and invasion, respectively, in the presence and absence of the IDO1 inhibitor L-1-methyl-tryptophan (L-1-MT). Additionally, western blotting was performed to analyze alterations in the expression of EMT-related markers caused by L-1-MT. RESULTS: High expression of IDO1 was confirmed in the cytoplasm of DLD-1 by immunofluorescence staining. In the scratch wound healing assay, the invasion ability of DLD-1 cells decreased to 62% after treatment with L-1-MT at 1,000 µM for 24 h. In the Boyden chamber assay, the migration of DLD-1 cells was suppressed by 85% after treatment with L-1-MT at 2,500 µM for 24 h. L-1-MT treatment increased the expression level of E-cadherin and decreased the expression levels of vimentin, Snail, and Slug. CONCLUSION: IDO1 inhibition reduced the invasion and migration ability of IDO1-expressing DLD-1 colon cancer cells, which was accompanied by altered expression of EMT-related proteins. IDO1 could be a potential target for the treatment of advanced CRC.

Epigallocatechin-3-gallate mitigates cadmium-induced intestinal damage through modulation of the microbiota-tryptophan-aryl hydrocarbon receptor pathway.

Early studies have shown that the gut microbiota is a critical target during cadmium exposure. The prebiotic activity of epigallocatechin-3-gallate (EGCG) plays an essential role in treating intestinal inflammation and damage. However, the exact intestinal barrier protection mechanism of EGCG against cadmium exposure remains unclear. In this experiment, four-week-old mice were exposed to cadmium (5 mg kg-1) for four weeks. Through 16 S rDNA analysis, we found that cadmium disrupted the gut microbiota and inhibited the indole metabolism pathway of tryptophan (TRP), which serves as the principal microbial production route for endogenous ligands to activate the aryl hydrocarbon receptor (AhR). Additionally, cadmium downregulated the intestinal AhR signaling pathway and harmed the intestinal barrier function. Treatment with EGCG (20 mg kg-1) and the AhR agonist 6-Formylindolo[3,2-b] carbazole (FICZ) (1 µg/d) significantly activated the AhR pathway and alleviated intestinal barrier injury. Notably, EGCG partially restored the gut microbiota and upregulated the TRP-indole metabolism pathway to increase the level of indole-related AhR agonists. Our findings demonstrate that cadmium dysregulates common gut microbiota to disrupt TRP metabolism, impairing the AhR signaling pathway and intestinal barrier. EGCG reduces cadmium-induced intestinal functional impairment by intervening in the intestinal microbiota to metabolize AhR agonists. This study offers insights into the toxic mechanisms of environmental cadmium and a potential mechanism to protect the intestinal barrier with EGCG.

Identification of 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids as promising DNMT1 inhibitors.

DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintaining DNA methylation patterns during cellular division, crucial for cancer development by suppressing tumor suppressor genes. In this study, we retained the phthalimide structure of N-phthaloyl-l-tryptophan (RG108) and substituted its indole ring with nitrogen-containing aromatic rings of varying sizes. We synthesized 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids and confirmed them as DNMT1 inhibitors through protein affinity testing, radiometric method using tritium labeled SAM, and MTT assay. Preliminary structure-activity relationship analysis revealed that introducing substituents on the carbazole ring could enhance inhibitory activity, with S-configuration isomers showing greater activity than R-configuration ones. Notably, S-3-(3,6-di-tert-butyl-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7r-S) and S-3-(1,3,6-trichloro-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7t-S) exhibited significant DNMT1 enzyme inhibition activity, with IC50 values of 8.147 µM and 0.777 µM, respectively (compared to RG108 with an IC50 above 250 µM). Moreover, they demonstrated potential anti-proliferative activity on various tumor cell lines including A2780, HeLa, K562, and SiHa. Transcriptome analysis and KEGG pathway enrichment of K562 cells treated with 7r-S and 7t-S identified differentially expressed genes (DEGs) related to apoptosis and cell cycle pathways. Flow cytometry assays further indicated that 7r-S and 7t-S induce apoptosis in K562 cells and arrest them in the G0/G1 phase in a concentration-dependent manner. Molecular docking revealed that 7t-S may bind to the methyl donor S-adenosyl-l-methionine (SAM) site in DNMT1 with an orientation opposite to RG108, suggesting potential for deeper penetration into the DNMT1 pocket and laying the groundwork for further modifications.

Acidity-responsive polyphenol-coordinated nanovaccines for improving tumor immunotherapy via bidirectional reshaping of the immunosuppressive microenvironment and controllable release of antigens.

The tumor immunosuppressive microenvironment (TIME) and uncontrollable release of antigens can lower the efficacy of nanovaccine-based immunotherapy (NBI). Therefore, it is necessary to develop a new strategy for TIME reshaping and controllable release of antigens to improve the NBI efficacy. Herein, an acidity-responsive Schiff base-conjugated polyphenol-coordinated nanovaccine was constructed for the first time to realize bidirectional TIME reshaping and controllable release of antigens for activating T cells. In particular, an acidity-responsive tannic acid-ovalbumin (TA-OVA) nanoconjugate was prepared via a Schiff base reaction. FeIII was coordinated with TA-OVA to produce a FeIII-TA-OVA nanosystem, and 1-methyltryptophan (1-MT) as an indoleamine 2,3-dioxygenase inhibitor was loaded to form a polyphenol-coordinated nanovaccine. The coordination between FeIII and TA could cause photothermal ablation of primary tumors, and the acidity-triggered Schiff base dissociation of TA-OVA could controllably release OVA to realize lysosome escape, initiating the body's immune response. More importantly, oxidative stress generated by a tumor-specific Fenton reaction of Fe ions could promote the polarization of tumor-associated macrophages from the M2 to M1 phenotype, resulting in the upregulation of cytotoxic T cells and helper T cells. Meanwhile, 1-MT could downregulate immunosuppressive regulatory T cells. Overall, such skillful combination of bidirectional TIME reshaping and controllable antigen release into one coordination nanosystem could effectively enhance the NBI efficacy of tumors.

1-Methyltryptophan treatment ameliorates high-fat diet-induced depression in mice through reversing changes in perineuronal nets.

Depression and obesity are prevalent disorders with significant public health implications. In this study, we used a high-fat diet (HFD)-induced obese mouse model to investigate the mechanism underlying HFD-induced depression-like behaviors. HFD-induced obese mice exhibited depression-like behaviors and a reduction in hippocampus volume, which were reversed by treatment with an indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-MT). Interestingly, no changes in IDO levels were observed post-1-MT treatment, suggesting that other mechanisms may be involved in the anti-depressive effect of 1-MT. We further conducted RNA sequencing analysis to clarify the potential underlying mechanism of the anti-depressive effect of 1-MT in HFD-induced depressive mice and found a significant enrichment of shared differential genes in the extracellular matrix (ECM) organization pathway between the 1-MT-treated and untreated HFD-induced depressive mice. Therefore, we hypothesized that changes in ECM play a crucial role in the anti-depressive effect of 1-MT. To this end, we investigated perineuronal nets (PNNs), which are ECM assemblies that preferentially ensheath parvalbumin (PV)-positive interneurons and are involved in many abnormalities. We found that HFD is associated with excessive accumulation of PV-positive neurons and upregulation of PNNs, affecting synaptic transmission in PV-positive neurons and leading to glutamate-gamma-aminobutyric acid imbalances in the hippocampus. The 1-MT effectively reversed these changes, highlighting a PNN-related mechanism by which 1-MT exerts its anti-depressive effect.

Liquid Chromatographic Enantioseparation of Newly Synthesized Fluorinated Tryptophan Analogs Applying Macrocyclic Glycopeptides-Based Chiral Stationary Phases Utilizing Core-Shell Particles.

Due to the favorable features obtained through the incorporation of fluorine atom(s), fluorinated drugs are a group with emerging pharmaceutical importance. As their commercial availability is still very limited, to expand the range of possible candidates, new fluorinated tryptophan analogs were synthesized. Control of enantiopurity during the synthesis procedure requires that highly efficient enantioseparation methods be available. In this work, the enantioseparation of seven fluorinated tryptophans and tryptophan was studied and compared systematically to (i) develop analytical methods for enantioselective separations and (ii) explore the chromatographic features of the fluorotrytophans. For enantioresolution, macrocyclic glycopeptide-based selectors linked to core-shell particles were utilized, applying liquid chromatography-based methods. Application of the polar-ionic mode resulted in asymmetric and broadened peaks, while reversed-phase conditions, together with mobile-phase additives, resulted in baseline separation for all studied fluorinated tryptophans. The marked differences observed between the methanol and acetonitrile-containing eluent systems can be explained by the different solvation abilities of the bulk solvents of the applied mobile phases. Among the studied chiral selectors, teicoplanin and teicoplanin aglycone were found to work effectively. Under optimized conditions, baseline separations were achieved within 6 min. Ionic interactions were semi-quantitatively characterized and found to not influence enantiorecognition. Interestingly, fluorination of the analytes does not lead to marked changes in the chromatographic characteristics of the methanol-containing eluents, while larger differences were noticed when the polar but aprotic acetonitrile was applied. Experiments conducted on the influence of the separation temperature indicated that the separations are enthalpically driven, with only one exception. Enantiomeric elution order was found to be constant on both teicoplanin and teicoplanin aglycone-based chiral stationary phases (L < D) under all applied chromatographic conditions.

Significance of nitrosative stress and glycoxidation products in the diagnosis of COVID-19.

Nitrosative stress promotes protein glycoxidation, and both processes can occur during an infection with the SARS-CoV-2 virus. Therefore, the aim of this study was to assess selected nitrosative stress parameters and protein glycoxidation products in COVID-19 patients and convalescents relative to healthy subjects, including in reference to the severity of COVID-19 symptoms. The diagnostic utility of nitrosative stress and protein glycoxidation biomarkers was also evaluated in COVID-19 patients. The study involved 218 patients with COVID-19, 69 convalescents, and 48 healthy subjects. Nitrosative stress parameters (NO, S-nitrosothiols, nitrotyrosine) and protein glycoxidation products (tryptophan, kynurenine, N-formylkynurenine, dityrosine, AGEs) were measured in the blood plasma or serum with the use of colorimetric/fluorometric methods. The levels of NO (p = 0.0480), S-nitrosothiols (p = 0.0004), nitrotyrosine (p = 0.0175), kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan fluorescence was significantly (p < 0.0001) lower in COVID-19 patients than in the control group. Significant differences in the analyzed parameters were observed in different stages of COVID-19. In turn, the concentrations of kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan levels were significantly (p < 0.0001) lower in convalescents than in healthy controls. The ROC analysis revealed that protein glycoxidation products can be useful for diagnosing infections with the SARS-CoV-2 virus because they differentiate COVID-19 patients (KN: sensitivity-91.20%, specificity-92.00%; NFK: sensitivity-92.37%, specificity-92.00%; AGEs: sensitivity-99,02%, specificity-100%) and convalescents (KN: sensitivity-82.22%, specificity-84.00%; NFK: sensitivity-82,86%, specificity-86,00%; DT: sensitivity-100%, specificity-100%; AGE: sensitivity-100%, specificity-100%) from healthy subjects with high sensitivity and specificity. Nitrosative stress and protein glycoxidation are intensified both during and after an infection with the SARS-CoV-2 virus. The levels of redox biomarkers fluctuate in different stages of the disease. Circulating biomarkers of nitrosative stress/protein glycoxidation have potential diagnostic utility in both COVID-19 patients and convalescents.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

C-Nitrosation, C-Nitration, and Coupling of Flavonoids with N-Acetyltryptophan Limit This Amine N-Nitrosation in a Simulated Cured and Cooked Meat.

Nitrite is a common additive in cured meat formulation that provides microbiological safety, lipid oxidation management, and typical organoleptic properties. However, it is associated with the formation of carcinogenic N-nitrosamines. In this context, the antinitrosating capacity of selected flavonoids and ascorbate was evaluated in a simulated cooked and cured meat under formulation and digestion conditions. N-Acetyltryptophan was used as a secondary amine target. (-)-Epicatechin, rutin, and quercetin were all able to limit the formation of N-acetyl-N-nitrosotryptophan (NO-AcTrp) at pH 2.5 and pH 5 although (-)-epicatechin was 2 to 3-fold more efficient. Kinetics for the newly identified compounds allowed us to unravel common mechanistic pathways, which are flavonoid oxidation by nitrite followed by C-nitration and an original covalent coupling between NO-AcTrp and flavonoids or their nitro and nitroso counterparts. C-nitrosation of the A-ring was evidenced only for (-)-epicatechin. These major findings suggest that flavonoids could help to manage N-nitrosamine formation during cured meat processing, storage, and digestion.

5-Methoxytryptophan enhances the sensitivity of sorafenib on the inhibition of proliferation and metastasis for lung cancer cells.

BACKGROUND: Lung cancer is a leading cause of cancer-related mortality worldwide, and effective therapies are limited. Lung cancer is a leading cause of cancer-related mortality worldwide with limited effective therapy. Sorafenib is a multi-tyrosine kinase inhibitor frequently used to treat numerous types of malignant tumors. However, it has been demonstrated that sorafenib showed moderate antitumor activity and is associated with several side effects in lung cancer, which restricted its clinical application. This study aimed to examine the antitumor effect of the combination treatment of sorafenib and 5-methoxytryptophan (5-MTP) on cell growth and metastasis of Lewis lung carcinoma (LLC) cells. METHOD: The anticancer effect of the combination treatment of sorafenib and 5-MTP was determined through cytotoxicity assay and colony forming assays. The mechanism was elucidated using flow cytometry and western blotting. Wound healing and Transwell assays were conducted to evaluate the impact of the combination treatment on migration and invasion abilities. An in vivo model was employed to analyze the effect of the combination treatment on the tumorigenic ability of LLC cells. RESULT: Our results demonstrated that the sorafenib and 5-MTP combination synergistically reduced viability and proliferation compared to sorafenib or 5-MTP treatment alone. Reduction of cyclin D1 expression was observed in the sorafenib alone or combination treatments, leading to cell cycle arrest. Furthermore, the sorafenib-5-MTP combination significantly increased the inhibitory effect on migration and invasion of LLC cells compared to the single treatments. The combination also significantly downregulated vimentin and MMP9 levels, contributing to the inhibition of metastasis. The reduction of phosphorylated Akt and STAT3 expression may further contribute to the inhibitory effect on proliferation and metastasis. In vivo, the sorafenib-5-MTP combination further reduced tumor growth and metastasis compared to the treatment of sorafenib alone. CONCLUSIONS: In conclusion, our data indicate that 5-MTP sensitizes the antitumor activity of sorafenib in LLC cells in vitro and in vivo, suggesting that sorafenib-5-MTP has the potential to serve as a therapeutic option for patients with lung cancer.

Design, synthesis, and activity evaluation of novel multitargeted l-tryptophan derivatives with powerful antioxidant activity against Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurological disease, and the multitarget directed ligand (MTDL) strategy may be an effective approach to delay its progression. Based on this strategy, 27 derivatives of l-tryptophan, 3a-1-3d-1, were designed, synthesized, and evaluated for their biological activity. Among them, IC50 (inhibitor concentration resulting in 50% inhibitory activity) values of compounds 3a-18 and 3b-1 were 0.58 and 0.44 µM for human serum butyrylcholinesterase (hBuChE), respectively, and both of them exhibited more than 30-fold selectivity for human serum acetylcholinesterase. Enzyme kinetics studies showed that these two compounds were mixed inhibitors of hBuChE. In addition, these two derivatives possessed extraordinary antioxidant activity in OH radical scavenging and oxygen radical absorption capacity fluorescein assays. Meanwhile, these compounds could also prevent ß-amyloid (Aß) self-aggregation and possessed low toxicity on PC12 and AML12 cells. Molecular modeling studies revealed that these two compounds could interact with the choline binding site, acetyl binding site, and peripheral anionic site to exert submicromolar BuChE inhibitory activity. In the vitro blood-brain barrier permeation assay, compounds 3a-18 and 3b-1 showed enough blood-brain barrier permeability. In drug-likeness prediction, compounds 3a-18 and 3b-1 showed good gastrointestinal absorption and a low risk of human ether-a-go-go-related gene toxicity. Therefore, compounds 3a-18 and 3b-1 are potential multitarget anti-AD lead compounds, which could work as powerful antioxidants with submicromolar selective inhibitory activity for hBuChE as well as prevent Aß self-aggregation.

Análise do transcriptoma de diferentes tipos de células únicas do sangue periférico de pacientes infectados com o vírus da Dengue para detalhamento da massiva geração de células secretoras de anticorpos / Evaluation of the tryptophan metabolism in the massive generation of antibody-secreting cells in Dengue patients through the transcriptome of single blood cells

A Dengue induz uma resposta exacerbada e transitória das células secretoras de anticorpos (ASCs) no sangue de pacientes cerca de sete dias após o início dos sintomas. A frequência dessas ASCs chega a representar mais de 50% de todas as células B circulantes neste período. No entanto, ainda é desconhecido se a magnitude dessa resposta tem relação com a gravidade da Dengue. Nosso grupo de pesquisa já mostrou que a cultura de células nucleares do sangue periférico (PBMCs) de indivíduos saudáveis com partículas do vírus da Dengue (DENV), por 7 dias, levava a diferenciação de células B em ASCs em magnitude similar àquelas estimuladas por mitógenos. Essas culturas apresentavam um consumo significativamente maior de triptofano (TRP), associado à maior expressão das enzimas IDO1 e IDO2, e, consequentemente, maior síntese de quinurenina (KYN) em relação ao estímulo por mitógenos. Considerando que as concentrações de TRP e KYN detectadas nos sobrenadantes dessas culturas eram diretamente proporcionais ao aumento de ASCs, decidimos investigar o papel desse metabolismo do TRP e de seus respectivos metabólitos na diferenciação das ASCs. Para isso, análises do transcriptoma público de células únicas do sangue periférico de pacientes com Dengue (estudo E-MTAB- 9467) foram realizadas para inferir a real participação do metabolismo do TRP na geração de ASCs. Com o programa R foram executadas análises de Downstream. Identificamos um aumento massivo das ASCs nas amostras dos pacientes infectados com Dengue. No entanto, os principais genes desencadeadores da ativação do metabolismo do TRP (IDO1 e IDO2) não foram expressos nas subpopulações de células B, mas sim em células dendríticas e monócitos CD14+ respectivamente. Isso sugeriria que esta via não seria ativada nos linfócitos B. Por outro lado, genes codificadores de outros participantes da via do TRP (HSD17B10, ECHS1 e SIRT3) foram detectados em células B e podem estar relacionados com a proliferação das ASCs. Além disso, a análise de enriquecimento mostrou uma aumentada expressão de genes associados com moléculas de MHC de classe II em plasmablastos e plasmócitos de pacientes com Dengue. Porém, com a expressão aumentada de ENTPD1 nessas células durante a fase sintomatológica, nossos dados sugerem também que um eventual papel de plasmablastos e plasmócitos como apresentadoras de antígenos na Dengue poderia induzir uma resposta supressora de células T

Dengue can cause an exacerbated and transient antibody-secreting cell (ASC) response in the blood of patients nearly 7 days of symptomatology. The ASC frequency reaches more than 50% of all circulating B cells during that period. However, it is still unknown whether the magnitude of this response may be directly related to the severity of Dengue. Our research group has already shown that the culture of peripheral blood mononuclear cells (PBMCs) from healthy individuals with Dengue virus (DENV) particles for 7 days led to a differentiation of B cells into ASCs to a magnitude similar to those stimulated by mitogens. These cultures showed significantly higher consumption of tryptophan (TRP), associated with higher expression of enzymes IDO1 and IDO2, and consequently, higher synthesis of quinurenine (KYN) compared to mitogen stimulation. The concentrations of TRP and KYN detected in the supernatants of these cultures were directly proportional to ASC frequency increase. Thus, we have decided to investigate the role of TRP metabolism and its respective metabolites in ASC differentiation. For this, we performed an analysis of single-cell transcriptome with peripheral blood from Dengue patients (dataset from E-MTAB-9467 study). Downstream analyses were performed with R software. Corroborating with literature, we identified a massive increase in ASC frequency of Dengue infected patients. However, the main genes triggering TRP metabolism activation (IDO1 and IDO2) were not expressed in B-cell subsets, but in dendritic cells and CD14+ monocytes, respectively. This would suggest that this pathway would not be activated in B lymphocytes. Nevertheless, genes encoding other participants in the TRP pathway (HSD17B10, ECHS1, and SIRT3) were detected in B cells and may be related to ASC proliferation. Futhermore, a Gene Ontology analysis showed an increased expression of genes associated with MHC class II molecules in plasmablasts and plasma cells of Dengue patients. As these cells also presented an increased expression of ENTPD1 during the symptomatic phase, our data suggest a potential role of plasmablasts and plasma cells as antigen-presenting cells associated with a suppressive T cell response in Dengue

Synthetic Analogs of 6-Bromohypaphorine, a Natural Agonist of Nicotinic Acetylcholine Receptors, Reduce Cardiac Reperfusion Injury in a Rat Model of Myocardial Ischemia.

The data available to date indicate that the activation of nicotinic acetylcholine receptors (nAChR) of α7 type can reduce heart damage resulting from ischemia and subsequent reperfusion. We have studied two new synthetic D-analogs of 6-bromohypaphorine, which are selective agonists of α7 nAChR, in a rat model of myocardial ischemia. Acute myocardial infarction in animals was induced by occlusion of the left coronary artery with its subsequent reperfusion under mechanical lung ventilation. It was found that one of the analogs was more active, and treatment with it at the onset of reperfusion statistically reduced infarct size. This analog also prevented changes in the concentration of potassium and sodium ions in the blood, occurring during occlusion/reperfusion injury. The data obtained indicate that hypaphorine analogs are promising for the development of drugs that reduce the adverse effects of myocardial infarction.

Polymeric indoximod based prodrug nanoparticles with doxorubicin entrapment for inducing immunogenic cell death and improving the immunotherapy of breast cancer.

Immunotherapy based on host immunity has emerged as a powerful therapeutic strategy for tumor treatment. However, utilizing the immune system against tumors often fails to result in a durable immune response due to insufficient immunogenicity and the immunosuppressive conditions in the tumor microenvironment. Herein, we developed prodrug-based nanoparticles (DOX/IND@NPs) for the codelivery of indoximod (IND), an indoleamine 2,3-dioxygenase (IDO) inhibitor that can block the IDO pathway and generate antitumor immunity, and doxorubicin, a DNA-damaging therapeutic agent that can induce tumor immunogenic cell death (ICD). The nanocarrier was designed for tumor chemoimmunotherapy, synergistically promoting immunogenicity and modulating the immunosuppressive tumor microenvironment (ITME). Our data showed that DOX induced tumor immunogenicity and increased the infiltration of CD8 + T cells into the tumor microenvironment; nevertheless, immunosuppressive immune cell components, such like regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs), hindered the antitumor efficacy. The introduction of IND reduced the levels of these protumor immune cells within the tumor microenvironment and further enhanced CD8 + T cell infiltration and the CD8 +/Treg cell ratio. Moreover, significant reductions in vascular endothelial growth factor (VEGF), MMP9, and CD31 (a vascular marker) expression levels were observed after DOX-IND nanoparticle treatment. This resulted in obvious tumor regression in a murine breast cancer model compared to reference formulations, indicating that the codelivery of DOX and IND is a potent potential strategy for breast cancer chemoimmunotherapy.

Substitution of l-Tryptophan by α-Methyl-l-Tryptophan in 177Lu-RM2 Results in 177Lu-AMTG, a High-Affinity Gastrin-Releasing Peptide Receptor Ligand with Improved In Vivo Stability.

Theranostic applications targeting the gastrin-releasing peptide receptor (GRPR) have shown promising results. When compared with other peptide ligands for radioligand therapy, the most often used GRPR ligand, DOTA-Pip5-d-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2 (RM2), may be clinically impacted by limited metabolic stability. With the aim of improving the metabolic stability of RM2, we investigated whether the metabolically unstable Gln7-Trp8 bond within the pharmacophore of RM2 can be stabilized via substitution of l-Trp8 by α-methyl-l-tryptophan (α-Me-l-Trp) and whether the corresponding DOTAGA analog might also be advantageous. A comparative preclinical evaluation of 177Lu-α-Me-l-Trp8-RM2 (177Lu-AMTG) and its DOTAGA counterpart (177Lu-AMTG2) was performed using 177Lu-RM2 and 177Lu-NeoBOMB1 as reference compounds. Methods: Peptides were synthesized by solid-phase peptide synthesis and labeled with 177Lu. Lipophilicity was determined at pH 7.4 (logD 7.4). Receptor-mediated internalization was investigated on PC-3 cells (37°C, 60 min), whereas GRPR affinity (half-maximal inhibitory concentration) was determined on both PC-3 and T-47D cells. Stability toward peptidases was examined in vitro (human plasma, 37°C, 72 ± 2 h) and in vivo (murine plasma, 30 min after injection). Biodistribution studies were performed at 24 h after injection, and small-animal SPECT/CT was performed on PC-3 tumor-bearing mice at 1, 4, 8, 24, and 28 h after injection. Results: Solid-phase peptide synthesis yielded 9%-15% purified labeling precursors. 177Lu labeling proceeded quantitatively. Compared with 177Lu-RM2, 177Lu-AMTG showed slightly improved GRPR affinity, a similar low internalization rate, slightly increased lipophilicity, and considerably improved stability in vitro and in vivo. In vivo, 177Lu-AMTG exhibited the highest tumor retention (11.45 ± 0.43 percentage injected dose/g) and tumor-to-blood ratio (2,702 ± 321) at 24 h after injection, as well as a favorable biodistribution profile. As demonstrated by small-animal SPECT/CT imaging, 177Lu-AMTG also revealed a less rapid clearance from tumor tissue. Compared with 177Lu-AMTG, 177Lu-AMTG2 did not show any further benefits. Conclusion: The results of this study, particularly the superior metabolic stability of 177Lu-AMTG, strongly recommend a clinical evaluation of this novel GRPR-targeted ligand to investigate its potential for radioligand therapy of GRPR-expressing malignancies.

Imaging of cerebral tryptophan metabolism using 7-[18F]FTrp-PET in a unilateral Parkinsonian rat model.

Degradation products of the essential amino acid tryptophan (Trp) are important signaling molecules in the mammalian brain. Trp is metabolized either through the kynurenine pathway or enters serotonin and melatonin syntheses. The aim of the present work was to examine the potential of the novel PET tracer 7-[18F]fluorotryptophan ([18F]FTrp) to visualize all three pathways in a unilateral 6-OHDA rat model. [18F]FDOPA-PET scans were performed in nine 6-OHDA-injected and six sham-operated rats to assess unilateral dopamine depletion severity four weeks after lesion placement. Afterwards, 7-[18F]FTrp-PET scans were conducted at different timepoints up to seven months after 6-OHDA injection. In addition, two 6-OHDA-injected rats were examined for neuroinflammation using [18F]DAA1106-PET. 7-[18F]FTrp-PET showed significantly increased tracer uptake at the 6-OHDA injection site which was negatively correlated to time after lesion placement. Accumulation of [18F]DAA1106 at the injection site was increased as well, suggesting that 7-[18F]FTrp uptake in this region may reflect kynurenine pathway activity associated with inflammation. Bilaterally in the dorsal hippocampus, 7-[18F]FTrp uptake was significantly decreased and was inversely correlated to dopamine depletion severity, indicating that it reflects reduced serotonin synthesis. Finally, 7-[18F]FTrp uptake in the pineal gland was significantly increased in relation with dopamine depletion severity, providing evidence that melatonin synthesis is increased in the 6-OHDA rat model. We conclude that 7-[18F]FTrp is able to detect alterations in both serotonin/melatonin and kynurenine metabolic pathways, and can be applied to visualize pathologic changes related to neurodegenerative processes.

Discovery of glyantrypine-family alkaloids as novel antiviral and antiphytopathogenic-fungus agents.

BACKGROUND: Plant diseases caused by viruses and fungi have caused great losses to crop quality and yield. The discovery of novel and efficient antiviral and antiphytopathogenic-fungus agents is urgently needed. It is the most important pesticide innovation strategy to find active compounds from natural products. Here, glyantrypine-family alkaloids were taken as the parent structures and a series of their derivatives were designed through molecular splicing, ring expansion, and ring contraction strategies, and synthesized. The anti-tobacco mosaic virus (TMV) activities and antifungal activities of these alkaloids were systematically investigated for the first time. RESULT: The antiviral activities of compounds 7bb, 7bc, 11c, 18b, 18d, 28d, and 28e are equivalent to or better than that of ribavirin (inhibitory rates 39%, 37%, and 40% at 500 µg mL-1 for inactivation, curative, and protection activity in vivo, respectively). Compounds 18d and 28d with good antiviral activities were selected for antiviral mode of action studies, which indicated that these alkaloids could achieve good antiviral effects by inhibiting TMV particle extension during assembly. These compounds also exhibited broad-spectrum fungicidal activities. CONCLUSION: Glyantrypine-family alkaloids and their derivatives were synthesized and evaluated for anti-TMV and fungicidal activities for the first time. Compounds 18d and 28d with excellent antiviral activities and compound 7bc with remarkable fungicidal activity emerged as novel lead compounds. This study lays a foundation for the application of glyantrypine alkaloids in plant protection.

Mycetoindole, an N-acyl dehydrotryptophan with plant growth inhibitory activity from an actinomycete of the genus Actinomycetospora.

A rare actinomycetal strain of the genus Actinomycetospora was found to produce a new tryptophan derivative, designated mycetoindole (1). The structure of 1 was determined to be N-3-methylcrotonoyl (Z)-dehydrotryptophan by NMR and MS analytical methods. Compound 1 reduced the root growth of lettuce Lactuca sativa seedlings at concentrations above 0.1 µM and almost completely inhibited seed germination at 10 µM.