Enzyme Assay Research Articles

Synthesis and Anticancer Activity of Novel Indole Derivatives as Dual EGFR/SRC Kinase Inhibitors.

Recent studies showed that the cooperation between c-SRC and EGFR is responsible for more aggressive phenotype in diverse tumors, including glioblastomas and carcinomas of the colon, breast, and lung. Studies show that combination of SRC and EGFR inhibitors can induce apoptosis and delay the acquired resistance to chemotherapy. Therefore, such combination may lead to a new therapeutic strategy for the treatment of EGFR-mutant lung cancer. Osimertinib was developed as a third-generation EGFR-TKI to combat the toxicity of EGFR mutant inhibitors. Due to the resistance and adverse reaction of osimertinib and other kinase inhibitors, 12 novel compounds structurally similar to osimertinib were designed and synthesized. Compounds were synthesized by developing novel original synthesis methods and receptor interactions were evaluated through a molecular docking study. To evaluate their inhibitory activities against EGFR and SRC kinase, in vitro enzyme assays were used. Anticancer potencies were determined using lung, breast, prostate (A549, MCF6, PC3) cancer cell lines. Compounds were also tested against normal (HEK293) cell line to evaluate their cyctotoxic effects. Although, none of compounds showed stronger inhibition compared to osimertinib in the EGFR enzyme inhibition studies, compound 16 showed the highest efficacy with an IC50 of 1.026 μM. It also presented potent activity against SRC kinase with an IC50 of 0.002 μM. Among the tested compounds, the urea containing derivatives 6-11 exhibited a strong inhibition profile (80.12-89.68%) against SRC kinase in comparison to the reference compound dasatinib (93.26%). Most of the compounds caused more than 50% of cell death in breast, lung and prostate cancer cell lines and weak toxicity for normal cells in comparison to reference compounds osimertinib, dasatinib and cisplatin. Compound 16 showed strong cytotoxicity on lung and prostate cancer cells. Treatment of prostate cancer cell lines with the most active compound, 16, significantly increased the caspase-3 (8-fold), caspase-8 (6-fold) and Bax (5.7-fold) levels and decreased the Bcl-2 level (2.3-fold) compared to the control group. These findings revealed that the compound 16 strongly induces apoptosis in the prostate cancer cell lines. Overall kinase inhibition, cytotoxicity and apoptosis assays presented that compound 16 has dual inhibitory activity against SRC and EGFR kinases while maintaining low toxicity against normal cells. Other compounds also showed considerable activity profiles in kinase and cell culture assays.

Pyruvate kinase deficiency in 29 Turkish patients with two novel intronic variants.

Pyruvate kinase (PK) is a key enzyme of anaerobic glycolysis. The genetic heterogeneity of PK deficiency (PKD) is high, and over 400 unique variants have been identified. Twenty-nine patients who had been diagnosed as PKD genetically in seven distinct paediatric haematology departments were evaluated. Fifteen of 23 patients (65.2%) had low PK levels. The PK:hexokinase ratio had 100% sensitivity for PKD diagnosis, superior to PK enzyme assay. Two novel intronic variants (c.695-1G>A and c.694+43C>T) have been described. PKD should be suspected in patients with chronic non-spherocytic haemolytic anaemia, even if enzyme levels are falsely normal. Total PKLR gene sequencing is necessary for the characterization of patients with PKD and for genetic counselling.

Shiikuwasha leaf and peel extracts inhibit allergic reactions by suppressing degranulation in RBL-2H3 rat basophilic leukemia cells and immunoglobulin production in mouse spleen lymphocytes.

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced β-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced β-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.

Detoxifying enzyme profiles in pesticide tolerant strains of Trichogramma chilonis Ishii, a hymenopteran parasitoid

Investigation on enzyme assays were carried out for carboxyl esterase (á and â esterase) and glutathione s-transferase in the susceptible and pesticide tolerant Trichogramma chilonis Ishii strains such as TCT1, TCT4, TCT5 and TCCb tolerant to endosulfan, spinosad, lamda-cyhalothrin and indoxycarb respectively. The electrophoretic profile of á-esterase enzyme in the susceptible Trichogramma strain showed 3 alleles in comparison with tolerant Trichogramma strains TcT5, TCT1, TCCb and TCT4 which depicted 5, 4, 3 and 2 alleles respectively. Similarly, â-esterases profile in the susceptible indicated 4 alleles compared to TCT5, TCT1, TCCb and TCT4 strains which showed 6, 5, 3 and 3 alleles respectively. The results of the quantitative analysis showed an increased enzyme activity in the tolerant strains. The increase in á-esterase activity in the tolerant strains TCT1, TCT4 and TCT5 was 2.07, 1.53 and 1.51 times more than the susceptible one and the mean difference was statistically significant and â-esterase activity in the tolerant strains TCT1, TCT4 and TCT5 was 1.41, 1.69 and 1.16 times more than the susceptible one. In the glutathione s-transferase enzyme activity, the mean value of the susceptible strain was 0.029?M and in tolerant strains, TCT1, TCT4 and TCT5 the values were 0.0243, 0.0289 and 0.023?M respectively indicating that the tolerant strains had lesser activity than the susceptible one. Such an increase in enzyme activity indicated the elevated production of detoxifying enzymes such as carboxylesterase to sustain the parasitoid in the field.

Dual‐Targeting of the HER2 Cancer Receptor with an Antibody‐Directed Enzyme and a Nanobody‐Guided MMAE Prodrug Scaffold

Antibody‐enzyme conjugates have shown potential as tissue‐specific prodrug activators by antibody‐directed enzyme prodrug therapy (ADEPT), but the approach met challenges clinically due to systemic drug release. Here, we report a novel dual‐targeting ADEPT system (DuADEPT) which is based on active cancer receptor targeting of both a trastuzumab‐sialidase conjugate (Tz‐Sia) and a highly potent sialidase‐activated monomethyl auristatin E (MMAE) prodrug scaffold. The scaffold is based on a four‐way junction of the artificial nucleic acid analog acyclic (L)‐threoninol nucleic acid ((L)‐aTNA) which at the ends of its four arms carries one nanobody targeting HER2 and three copies of the prodrug. Dual‐targeting of the constructs to two proximal epitopes of HER2 was shown by flow cytometry, and a dual‐targeted enzymatic drug release assay revealed cytotoxicity upon prodrug activation specifically for HER2‐positive cancer cells. The specific delivery and activation of prodrugs in this way could potentially be used to decrease systemic side effects and increase drug efficacy, and utilization of Tz‐Sia provides an opportunity to combine the local chemotherapeutic effect of the DuADEPT with an anticancer immune response.

Association of Breastfeeding with Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis in mothers: A cross sectional survey

Objective: To determine the association of Breastfeeding with Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis in mothers.Methodology: A cross sectional study on randomly selected 280 mothers presented in the outpatient department of Sheikh Zayed Hospital, Rahim Yar Khan for different medical reasons from July to Dec 2023. Data collection commenced after taking ethical approval from the Institutional Review Board of the institute and taking informed verbal consent from study subjects. These mothers were divided into 2 groups. Group 1 which never breastfed their children and group 2 which breastfed their children for more than 6 months. Each group had 140 participants. Subject having history of alcohol consumption or used medicines like glucocorticoids, amiodarone, tamoxifen, and methotrexate were excluded from the study. Biodata of all the participants along with demographic profiles were obtained, who then went through an LFT and Lipid profile blood sampling and an USG abdomen. Results were recorded on a predesigned proforma. Serum ALT, AST, Serum bilirubin, serum LDL, HDL, Triglycerides, and Serum Cholesterol were determined by enzymatic assay and high-performance liquid chromatography methods. Data was analysed using SPSS v.26. Descriptive statistics was used to summarize the data. Mean and standard deviations were reported for continuous variables. Chi square test was used to compare the qualitative variables, with p value of less than 0.05 taken as significant.Results: Mean age ± SD of participants was 40±4 years, with group-1 having mean age of 38.1±5 years and group-2 having 40.4±3 years (p=0.4). The findings showed that frequency of NASH in patients who have not breastfed their children was higher 19 out of 140(13.6%) whereas the mothers who breastfed their children only have 5% frequency of NASH i.e. 7 out of 140 (P value = 0.01). Out of 140 patients who did not breastfeed their children 56 (40.0%) developed NAFLD, contrary to 140 patients who breastfed their children only 20 (14.3%) (P value = 0.00) got NAFLD. Elevated ALT and AST levels are significant for NAFLD and NASH. Lipid profile is a major indicator of NAFL and NASH.Conclusion: The frequency of NAFLD and NASH is significantly low in mothers who breastfed their children. The frequency further decreases if the period of breastfeeding was greater.

Molecular and geochemical basis of microbially induced carbonate precipitation for treating acid mine drainage: The case of a novel Sporosarcina genomospecies from mine tailings

Microbially induced carbonate precipitation (MICP) immobilizes toxic metals and reduces their bioavailability in aqueous systems. However, its application in the treatment of acid mine drainage (AMD) is poorly understood. In this study, the genomes of Sporosarcina sp. UB5 and UB10 were sequenced. Urease, carbonic anhydrases, and metal resistance genes were identified and enzymatic assays were performed for their validation. The geochemical mechanism of precipitation in AMD was elucidated through geo-mineralogical analysis. Sporosarcina sp. UB5 was shown to be a new genomospecies, with an average nucleotide identity < 95 % (ANI) and DNA-DNA hybridization < 70 % (DDH) whereas UB10 is close to S. pasteurii. UB5 contained two urease operons, whereas only one was identified in UB10. The ureolytic activities of UB5 and UB10 were 122.67 ± 15.74 and 131.70 ± 14.35 mM NH4+ min−1, respectively. Both strains feature several carbonic anhydrases of the α, β, or γ families, which catalyzed the precipitation of CaCO3. Only Sporosarcina sp. UB5 was able to immobilize metals and neutralize AMD. Geo-mineralogical analyses revealed that UB5 directly immobilized Fe (1–23 %), Mn (0.65–1.33 %) and Zn (0.8–3 %) in AMD via MICP and indirectly through adsorption to calcite and binding to bacterial cell walls. The MICP-treated AMD exhibited high removal rates (>67 %) for Ag, Al, As, Ca, Cd, Co, Cu, Fe, Mn, Pb, and Zn, and a removal rate of 15 % for Mg. This study provides new insights into the MICP process and its applications to AMD treatment using autochthonous strains.

3D-QSAR model-oriented optimization of Pyrazole β-Ketonitrile derivatives with diphenyl ether moiety as novel potent succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitor (SDHI) fungicides play important roles in the control of plant fungal diseases. However, they are facing serious challenges from issues with resistance and cross-resistance, primarily attributed to their frequent application and structural similarities. There is an urgent need to design and develop SDHI fungicides with novel structures. Aiming to discover novel potent SDHI fungicides, 31 innovative pyrazole β-ketonitrile derivatives with diphenyl ether moiety were rationally designed and synthesized, which were guided by a 3D-QSAR model from our previous study. The optimal target compound A23 exhibited not only outstanding in vitro inhibitory activities against Rhizoctonia solani with a half-maximal effective concentration (EC50) value of 0.0398 μg mL-1 comparable to that for fluxapyroxad (EC50 = 0.0375 μg mL-1), but also a moderate protective efficacy in vivo against rice sheath blight. Porcine succinate dehydrogenase (SDH) enzymatic inhibitory assay revealed that A23 is a potent inhibitor of SDH, with a half-maximal inhibitory concentration of 0.0425 μm. Docking study within R. solani SDH indicated that A23 effectively binds into the ubiquinone site mainly through hydrogen-bonds, and cation-π and π-π interactions. The identified β-ketonitrile compound A23 containing diphenyl ether moiety is a potent SDH inhibitor, which might be a good lead for novel fungicide research and optimization. © 2024 Society of Chemical Industry.

Steroidal scaffold decorations in Solanum alkaloid biosynthesis

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and “anti-nutritional” effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology.

Tunicamycins from Marine-Derived Streptomyces bacillaris Inhibit MurNAc-Pentapeptide Translocase in Staphylococcus aureus.

Four tunicamycin class compounds, tunicamycin VII (1), tunicamycin VIII (2), corynetoxin U17a (3), and tunicamycin IX (4), were isolated from the culture broth of the marine-derived actinomycete Streptomyces sp. MBTG32. The strain was identified using the 16S rDNA sequencing technique, and the isolated strain was closely related to Streptomyces bacillaris. The structures of the isolated compounds were elucidated based on spectroscopic data and comparisons with previously reported NMR data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria, especially Staphylococcus aureus, with MIC values of 0.13-0.25 µg/mL. Through a recombinant enzyme assay and overexpression analysis, we found that the isolated compounds exerted potent inhibitory effects on S. aureus MurNAc-pentapeptide translocase (MraY), with IC50 values of 0.08-0.21 µg/mL. The present results support that the underlying mechanism of action of tunicamycins isolated from marine-derived Streptomyces sp. is also associated with the inhibition of MraY enzyme activity in S. aureus.

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding.

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.

The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity

BackgroundN-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells. MethodsUsing AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity. ResultsOur data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases. ConclusionsThe slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity. General significanceOur findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.

Functional characterization of a cystatin A from the bat Myotis davidii

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

Lipoprotein-Associated Phospholipase A2 and hs-CRP are Correlated with Anti-TPO Antibodies in Jordanian Non-Pregnant Women with Hashimoto’s Thyroiditis

Hashimoto’s thyroiditis (HT) is an autoimmune disorder characterized by elevated thyroid-stimulating hormone (TSH) levels. This research investigates the complex interaction between HT and cardiovascular risk in adult Jordanian non-pregnant women aged 20-50. Through a study involving 50 HT subjects and 40 healthy subjects, the levels of lipoprotein-associated phospholipase A2 (PLA2), high sensitivity C-reactive protein (hs-CRP), and anti-thyroid peroxidase (anti-TPO) antibodies were compared using ELISA methods and enzymatic colorimetric assays for lipid profiles. The results revealed significantly higher serum levels of hs-CRP, PLA2, and Anti-TPO in Hashimoto's patients, coupled with elevated cholesterol, triglyceride, and low-density lipoprotein (LDL) levels. Conversely, reduced levels of high-density lipoprotein (HDL) were observed in Hashimoto’s patients compared to healthy subjects. The study establishes a noteworthy correlation between thyroid autoimmunity, thyroid disease, PLA2, hs-CRP, and lipid profile, underscoring an increased cardiovascular risk in individuals with Hashimoto’s thyroiditis. The findings emphasize the prevalence of Anti-TPO antibodies in adult Jordanian non-pregnant women with Hashimoto’s thyroiditis.

Evaluation of Fluorescence-Based Screening Assays for the Detection and Quantification of Silyl Hydrolase Activity.

This study reports the development of fluorometric assays for the detection and quantification of silyl hydrolase activity using silicatein as a model enzyme. These assays employed a series of organosilane substrates containing either mycophenolate or umbelliferone moieties, which become fluorescent upon hydrolysis of a scissile Si-O bond. Among these substrates, the mycophenolate-derived molecule MycoF, emerged as the most promising candidate due to its relative stability in aqueous media, which resulted in good differentiation between the enzyme-catalyzed and uncatalyzed background hydrolysis. The utility of MycoF was also demonstrated in the detection of enzyme activity in cell lysates and was found to be capable of qualitative identification of positive "hit" candidates in a high-throughput format. These fluorogenic substrates were also suitable for use in quantitative kinetic assays, as demonstrated by the acquisition of their Michaelis-Menten parameters.

Eugenol transport and biosynthesis through grafting in aromatic plants of the Ocimum genus.

Aromatic compounds play essential roles in plant physiology and various industries because of their unique fragrances and beneficial properties. In this study, we investigated the transport and biosynthesis of eugenol, a prominent aromatic compound, within the Ocimum genus, using grafting experiments. Grafting sweet basil (Ocimum basilicum) scions onto diverse rootstocks, including tobacco (Nicotiana benthamiana) and thyme (Thymus vulgaris), revealed that eugenol is transported from the shoot to the root across distinct plant species. Furthermore, grafting within the Ocimum genus, which includes O. basilicum, O. tenuiflorum, and O. americanum, resulted in variations in eugenol transport and accumulation. The eugenol content in the shoots remained constant across all combinations, whereas the root eugenol levels varied depending on the scion-rootstock pair. To elucidate the biosynthetic capabilities of eugenol in Ocimum roots, we performed in vitro enzyme assays using crude protein extracts from roots, which revealed that eugenol can be synthesized in roots in addition to being transported. Expression analysis of eugenol synthase (EGSs) genes showed that EGS4 expression was influenced by grafting in O. basilicum roots, suggesting compensation by other EGSs. Our results suggest that eugenol transport and biosynthesis are multifaceted processes influenced by the interactions between different species and tissues. The potential to engineer eugenol levels in rootstocks lacking biosynthetic capacity has potential applications in agriculture and industry. This study reveals the dynamic interplay between eugenol transport and biosynthesis in the Ocimum genus, providing insights into the manipulation of aromatic compound production in plants.

Cystathionine γ-lyase-derived H2S negatively regulates thymic egress via allosteric inhibition of sphingosine-1-phosphate lyase.

Thymic egress is a crucial process for thymocyte maturation, strictly regulated by sphingosine-1-phosphate lyase (S1PL). Recently, cystathionine γ-lyase (CSE), one of the enzymes producing hydrogen sulfide (H2S), has emerged as a vital immune process regulator. However, the molecular connection between CSE, H2S and thymic egress remains largely unexplored. In this study, we investigated the regulatory function of CSE in the thymic egress of immune cells. We showed that genetic knockout of CSE or pharmacological inhibition by CSE enzyme inhibitor NSC4056 or D,L-propargylglycine (PAG) significantly enhanced the migration of mature lymphocytes and monocytes from the thymus to the peripheral blood, and this redistribution effect could be reversed by treatment with NaHS, an exogenous donor of H2S. In addition, the CSE-generated H2S significantly increased the levels of S1P in the peripheral blood, thymus and spleen of mice, suppressed the production of proinflammatory cytokines and rescued pathogen-induced sepsis in cells and in vivo. Notably, H2S or polysulfide inhibited S1PL activity in cells and an in vitro purified enzyme assay. We found that this inhibition relied on a newly identified C203XC205 redox motif adjacent to the enzyme's active site, shedding light on the biochemical mechanism of S1PL regulation. In conclusion, this study uncovers a new function and mechanism for CSE-derived H2S in thymic egress and provides a potential drug target for treating S1P-related immune diseases.

Machine learning-based QSAR and LB-PaCS-MD guided design of SARS-CoV-2 main protease inhibitors

The global outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 virus had led to profound respiratory health implications. This study focused on designing organoselenium-based inhibitors targeting the SARS-CoV-2 main protease (Mpro). The ligand-binding pathway sampling method based on parallel cascade selection molecular dynamics (LB-PaCS-MD) simulations was employed to elucidate plausible paths and conformations of ebselen, a synthetic organoselenium drug, within the Mpro catalytic site. Ebselen effectively engaged the active site, adopting proximity to H41 and interacting through the benzoisoselenazole ring in a π-π T-shaped arrangement, with an additional π-sulfur interaction with C145. In addition, the ligand-based drug design using the QSAR with GFA-MLR, RF, and ANN models were employed for biological activity prediction. The QSAR-ANN model showed robust statistical performance, with an r2training exceeding 0.98 and an RMSEtest of 0.21, indicating its suitability for predicting biological activities. Integration the ANN model with the LB-PaCS-MD insights enabled the rational design of novel compounds anchored in the ebselen core structure, identifying promising candidates with favorable predicted IC50 values. The designed compounds exhibited suitable drug-like characteristics and adopted an active conformation similar to ebselen, inhibiting Mpro function. These findings represent a synergistic approach merging ligand and structure-based drug design; with the potential to guide experimental synthesis and enzyme assay testing.

Sex-Related Differences in Oxidant and Antioxidant Profiles of Murine Kidney and Brain: A Focus on Sirt3-Mediated Regulation

Background: Sirt3 is a mitochondrial deacetylase with an important role in maintainance of cellular redox and metabolic homeostasis and mitochondrial function. As growing evidence support the existence of sex-specific responses to metabolic and oxidative stress, we aimed to investigate sex- and organ-specific effects of Sirt3 loss. Materials and methods: Expression of Sirt3, PGC-1a, CuZnSOD, MnSOD and Cat proteins in kidneys and brains of Sirt3-wild type (Sirt3 WT) and Sirt3-knockout (Sirt3 KO) mice was assessed by Western blotting. Protein carbonylation and lipid peroxidation levels were measured using ELISA and fluorometric assays, respectively. SOD and Cat activities were determined using standard enzymatic assays. Results: Significant sex- and organ- specific differences in response to Sirt3 loss were detected. Sirt3 knockout affected kidneys more than brain tissue, with females showing lower levels PC and LPO. In kidneys, female KO showed higher MnSOD, but lower CuZnSOD and Cat activity compared to males. In brains, WT females show higher activities of these enzymes than males, suggesting a sex-specific protection mechanism, but female KO brains show a larger decrease in these parameters. Conclusion: Our study provides comprehensive insights into the complex interplay of Sirt3, oxidative stress, and antioxidant defenses in murine kidney and brain. The observed differences between the two organs and the impact of sex highlight the need for studying Sirt3 function in diverse physiological contexts. The tissue-specific responses and sex-related variations underscore the importance of considering these factors in the development of therapeutic strategies targeting mitochondrial function and redox homeostasis.

Tandem mass spectrometric enzyme assay for simultaneous detection of Tay-Sachs and Sandhoff diseases in dried blood spots for newborn screening

GM2 gangliosidosis is a group of rare lysosomal storage disorders (LSDs) including Tay-Sachs disease (TSD) and Sandhoff disease (SD), caused by deficiency in activity of either β-hexosaminidase A (HexA) or both β-hexosaminidase A and β-hexosaminidase B (Hex B). Methods for screening and diagnosis of TSD and SD include measurement and comparison of the activity of these two enzymes. Here we report a novel method for duplex screening of dried blood spots (DBS) for TSD and SD by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method requires incubation of a single 3 mm DBS punch with the assay cocktail followed by the injection into the LC-MS/MS. The performance of the method was evaluated by comparing the confirmed TSD and SD patient DBS to random healthy newborn DBS which showed easy discrimination between the three cohorts. The method is multiplexable with other LSD MS/MS enzyme assays which is critical to the continued expansion of the NBS panels.