Inhibitory Compounds Research Articles

Abstract B028: Antiproliferative effect of a cathepsin L inhibitor on hepatocellular carcinoma

Abstract Liver cancer has emerged as one of the fastest-growing malignancies in the United States, despite an overall decline in cancer incidence and mortality rates. Hepatocellular carcinoma (HCC) exhibits significant gender and race/ethnicity differences in incidence and mortality. The need to address this escalating problem necessitates the development of novel and effective chemotherapeutic agents for HCC. The antiproliferative actions of two cathepsin L inhibitors (compounds 1 and 2) using in vitro and mice HCC xenograft models were investigated in this study. In addition, the ability of the compounds to inhibit CatB and generate ROS in HCC cell lines was investigated. The inhibitors have antiproliferative effects on Hep G2 and Hep 3B cell lines with low micromolar IC 50 values. Compound 1 preferentially inhibits both recombinant and endogenous CatL in a time-dependent manner compared to CatB. The levels of ROS produced when cells were treated with compound 1 were non-significant compared with vehicle-treated controls . Given the superior efficacy of compound 1 over its analog in antiproliferative studies, it was advanced to in vitro ADME studies. The inhibitor has bidirectional cellular permeability and is not a substrate for the P-glycoprotein efflux pump (P-gp). In mice, it produced a significant reduction in the sizes of subcutaneous tumors with no gross toxicity. Transcriptomics analysis using RNA-Seq revealed that the cell migration pathway was enriched in a time and dose-dependent manner. In addition, genes responsible for apoptosis and cell structural organization were overexpressed. Overall, the CatL inhibitors under investigation appear as promising starting points for investigation as potential chemotherapy against HCC. Citation Format: Olamide O. Crown, Ifedayo V. Ogungbe, Felicite K. Noubissi. Antiproliferative effect of a cathepsin L inhibitor on hepatocellular carcinoma [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B028.

Identification of non-steroidal aromatase inhibitors via in-silico and in-vitro studies.

Breast cancer is the most common cancer affecting women worldwide, including Pakistan. More than half of breast cancer patients have hormone-dependent breast cancer, which is developed due to the over-production of estrogen (the main hormone in breast cancer). The biosynthesis of estrogen is catalyzed by the aromatase enzyme, which thus serves as a target for the treatment of breast cancer. During the current study, biochemical, computational, and STD-NMR methods were employed to identify new aromatase inhibitors. A series of phenyl-3-butene-2-one derivatives 1-9 were synthesized and evaluated for human placental aromatase inhibitory activity. Among them, four compounds 2, 3, 4, and 8 showed a moderate to weak inhibitory activity (IC50 = 22.6 - 47.9 µM), as compared to standard aromatase inhibitory drugs, letrozole (IC50 = 0.0147 ± 1.45 µM), anastrozole (IC50 = 0.0094 ± 0.91 µM), and exemestane (IC50 = 0.2 ± 0.032 µM). Kinetic studies on two moderate inhibitors, 4 and 8, revealed a competitive- and mixed-type of inhibition, respectively. Docking studies on all active compounds indicated their binding adjacent to the heme group and interaction with Met374, a critical residue of aromatase. STD-NMR further highlighted the interactions of these ligands with the aromatase enzyme. STD-NMR-based epitope mapping indicated close proximity of the alkyl chain followed by an aromatic ring with the receptor (aromatase). These compounds were also found to be non-cytotoxic against human fibroblast cells (BJ cells). Thus, the current study has identified new aromatase inhibitors (compounds 4, and 8) for further pre-clinical and clinical research.

Effect-Directed Assays and Biological Detection Approaches Coupled with Thin-Layer Chromatography as an Evolving Hyphenated Technique: A Comprehensive Review.

Bioautography is a technique for the detection of biological activity that combines the elements of planar chromatography. Its hyphenated variants are widely used in the screening of natural products possessing biological activity. It can be used in the activity-based screening of phytochemical ingredients by employing various enzyme processes and reactions and facilitates the rapid determination of bioactive compounds in pant samples. To give a comprehensive overview of effect-directed assays and biological detection approaches used in conjugation with thin layer chromatography technique. The present review article attempts to throw light on the various aspects of bioautography, including its types and applications, thereby giving its concise overview and its relevance in the field of natural product screening. Various search engines were used for the literature survey, including Google Scholar, Semantic Scholar, PubMed, ResearchGate and Scopus. Bioautography has wide-ranging uses in the screening of compounds such as antioxidants, antifungals, antimicrobials, estrogenic, antitumors, and various enzyme inhibitors compounds like α and β-glucosidase inhibitors and α-amylase inhibitors. Bioautography serves to be an effective tool for the isolation of bioactive phytochemicals, thereby allowing us to scientifically validate the biological activities of various compounds, which can then be utilized for making potent medications for various diseases.

Abstract A044: The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer

Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.

Advancements in virtual screening techniques for study of enzyme inhibitor compounds

Enzymes are closely associated with the onset and progression of numerous diseases, making enzymes a primary target in innovative drug development. However, the challenge remains in identifying compounds that exhibit potent inhibitory effects on the target enzymes. With the continuous expansion of the total number of natural products and increasing difficulty in isolating and enriching new compounds, traditional high-throughput screening methods are finding it increasingly challenging to meet the demands of new drug development. Virtual screening, characterized by its high efficiency and low cost, has gradually become an indispensable technology in drug development. It represents a prominent example of the integration of artificial intelligence with biopharmaceuticals and is an inevitable trend in the rapid development of innovative drug screening in the future. Therefore, this article primarily focused on systematically reviewing the recent applications of virtual screening technology in the development of enzyme inhibitors and explored the prospects and advantages of using this technology in developing new drugs, aiming to provide essential theoretical insights and references for the application of related technologies in the field of new drug development.

Investigation of Best QSPR-Based Machine Learning Model to Predict Corrosion Inhibition Performance of Pyridine-Quinoline Compounds

Corrosion is a major concern for the industrial and academic sectors because it causes significant losses in many fields. Currently, there is a great deal of interest in the topic of material damage control using organic chemicals. Pyridine and quinoline are potential corrosion inhibitors because they are non-toxic, inexpensive, and efficient in various corrosive conditions. Experimental studies in searching for candidate corrosion inhibitor candidates require a lot of time, cost, and labor intensive. Using a machine learning (ML) strategy based on a quantitative structure-property relationship (QSPR) model, we evaluate gradient boosting regressor (GBR), support vector regression (SVR), and k-nearest neighbor (KNN) algorithms as predictive models to investigate corrosion inhibition efficiency (CIE) of pyridine-quinoline compounds in this study. We found that the GBR model, when compared with the SVR and KNN models as well as models from the literature for the pyridine-quinoline compound dataset, has the best predictive performance based on the metric coefficient of determination (R2) and root mean square error (RMSE). Overall, our study provides a new perspective on how the ML model can estimate the effectiveness of corrosion prevention on iron surfaces by organic inhibitor compounds.

OFC neurons do not represent the negative value of a conditioned inhibitor

The orbitofrontal cortex (OFC) is often proposed to function as a value integrator; however, alternative accounts focus on its role in representing associative structures that specify the probability and sensory identity of future outcomes. These two accounts make different predictions about how this area should respond to conditioned inhibitors of reward, since in the former, neural activity should reflect the negative value of the inhibitor, whereas in the latter, it should track the estimated probability of a future reward based on all cues present. Here, we assessed these predictions by recording from small groups of neurons in the lateral OFC of rats during training in a conditioned inhibition design. Rats showed negative summation when the inhibitor was compounded with a novel excitor, suggesting that they learned to respond to the conditioned inhibitor appropriately. Against this backdrop, we found unit and population responses that scaled with expected reward value on excitor + inhibitor compound trials. However, the responses of these neurons did not differentiate between the conditioned inhibitor and a neutral cue when both were presented in isolation. Further, when the ensemble patterns were analyzed, activity to the conditioned inhibitor did not classify according to putative negative value. Instead, it classified with a same-modality neutral cue when presented alone and as a unique item when presented in compound with a novel excitor. This pattern of results supports the notion that OFC encodes a model of the causal structure of the environment rather than either the modality or the value of cues.

Dependence of IL-7R-Mediated Signaling on Sphingosine Kinase Activity in Acute Lymphoblastic Leukemia, but Not Healthy Lymphoid Cells, Is an Exploitable Therapeutic Vulnerability

Acute lymphoblastic leukemia (ALL) is an aggressive cancer affecting B or T cell precursors. Interleukin 7 (IL-7) is essential for normal lymphoid development but shown also to contribute to ALL development and resistance to glucocorticoids. IL7R (the gene that encodes for IL-7 receptor α) is a bona fide lymphoid oncogene that is mutated in around 10% T-ALL and 3% B-ALL patients. Mutational activation of IL7R or high levels of wild type IL7R can drive ALL development in vivo. Sphingosine kinases, of which there are two isoforms (SK1 and SK2), generate sphingosine-1-phosphate (S1P) from sphingosine, thus promoting cell survival. We previously identified SKs as potential targets for therapeutic intervention in ALL with IL7R gain-of-function mutations. However, how IL-7R-mediated signaling crosstalks with SKs, and whether there is a therapeutic window for the use of SK inhibitors in ALL without provoking excessive side effects, remains to be determined. Our thorough immunophenotypic characterization of CD2Cre.SK1 fl/fl.SK2 -/- mice, which display SK complete ablation exclusively in the lymphoid compartment, revealed no major impact on lymphoid development. Since, on the contrary, IL7R-null mice display severe B- and T-cell deficiency, this indirectly indicates that SKs are not involved in IL-7R-mediated signaling during lymphoid development. Accordingly, we found that healthy thymocytes and B-cell precursors did not require SK activity for IL-7-mediated signaling, as measured by the levels of STAT5 phosphorylation. In sharp contrast, we found compelling evidence that SKs are involved in IL-7R-mediated signaling and functional impact in the context of leukemia. First, SK deletion in the lymphoid compartment significantlydelayed the development of B-ALL in mice with IL7R mutation in lymphoid precursors (CD2Cre.Il7r cpt/wt mice). Second, SK deletion impaired both basal and IL-7-mediated JAK/STAT5 pathway activation in leukemia cells from the same mice. In agreement, CRISPR-Cas9 SK1/SK2 gene editing in the human IL-7R-responsive HPB-ALL cell line prevented IL-7-induced JAK/STAT5 pathway activation and leukemia cell viability. Finally, pharmacological inhibition of SKs blocked IL-7-triggered viability of human patient-derived xenograft T-ALL cells, without significantly impacting healthy thymocytes. We next sought to understand the reasons underlying IL-7R-mediated signaling aberrant dependence on SK activity in leukemia cells. Besides direct intracellular roles, S1P is exported to the extracellular compartment and able to induce autocrine or paracrine signaling via S1P receptors expressed at the cell surface. We found that S1PR4 transcript levels were upregulated in diagnostic T-ALL patient samples. Inhibition of S1PR4 downregulated IL-7-mediated JAK/STAT5 pathway activation in leukemia cells but not in healthy thymocytes. In agreement, S1P stimulation partially mimicked the effects of IL-7 exclusively in T-ALL cells. In addition, our analyses demonstrated that SK1 and SK2 are overexpressed in B- and T-ALL diagnostic patient samples. Importantly, we further report that SK1 co-immunoprecipitated with IL-7Rα in ALL cells and that SK1 overexpression upregulates IL-7R-mediated signaling in HeLa cells transfected with the IL-7R signaling machinery. Our results indicate that overexpression of SKs and/or S1PR4 may justify the augmented reliance of IL-7R-mediated signaling on SK activity displayed by ALL cells. Finally, we demonstrated the potential therapeutic impact of our findings. The SK pharmacological inhibitor compound 49 reversed IL-7-dependent pro-survival effects in human B- and T-ALL samples in vitro. We then conducted a phase 2-like preclinical trial with 7 distinct IL-7-responsive T-ALL patient samples xenografted into immunodeficient mice, which showed that compound 49 significantly reduced disease burden (p=0.0156, Wilcoxon matched pairs signed rank test), with highest effects in the samples that displayed higher IL-7 signaling responsiveness. Overall, our studies demonstrate that SKs are key players in oncogenic, but not in physiological, IL-7/IL-7R-mediated signaling. We uncover the importance of the SK-IL7R signaling crosstalk in ALL and pinpoint the therapeutic potential of SK inhibitors for IL-7R-dependent ALL.

ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation.

Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.

Polystyrene nanoplastic exposure induces excessive mitophagy by activating AMPK/ULK1 pathway in differentiated SH-SY5Y cells and dopaminergic neurons in vivo

BackgroundMicroplastics and nanoplastics (MNPs) are emerging environmental contaminants detected in human samples, and have raised concerns regarding their potential risks to human health, particularly neurotoxicity. This study aimed to investigate the deleterious effects of polystyrene nanoplastics (PS-NPs, 50 nm) and understand their mechanisms in inducing Parkinson's disease (PD)-like neurodegeneration, along with exploring preventive strategies.MethodsFollowing exposure to PS-NPs (0.5–500 μg/mL), we assessed cytotoxicity, mitochondrial integrity, ATP levels, and mitochondrial respiration in dopaminergic-differentiated SH-SY5Y cells. Molecular docking and dynamic simulations explored PS-NPs' interactions with mitochondrial complexes. We further probed mitophagy's pivotal role in PS-NP-induced mitochondrial damage and examined melatonin's ameliorative potential in vitro. We validated melatonin's intervention (intraperitoneal, 10 mg/kg/d) in C57BL/6 J mice exposed to 250 mg/kg/d of PS-NPs for 28 days.ResultsIn our in vitro experiments, we observed PS-NP accumulation in cells, including mitochondria, leading to cell toxicity and reduced viability. Notably, antioxidant treatment failed to fully rescue viability, suggesting reactive oxygen species (ROS)-independent cytotoxicity. PS-NPs caused significant mitochondrial damage, characterized by altered morphology, reduced mitochondrial membrane potential, and decreased ATP production. Subsequent investigations pointed to PS-NP-induced disruption of mitochondrial respiration, potentially through interference with complex I (CI), a concept supported by molecular docking studies highlighting the influence of PS-NPs on CI. Rescue experiments using an AMPK pathway inhibitor (compound C) and an autophagy inhibitor (3-methyladenine) revealed that excessive mitophagy was induced through AMPK/ULK1 pathway activation, worsening mitochondrial damage and subsequent cell death in differentiated SH-SY5Y cells. Notably, we identified melatonin as a potential protective agent, capable of alleviating PS-NP-induced mitochondrial dysfunction. Lastly, our in vivo experiments demonstrated that melatonin could mitigate dopaminergic neuron loss and motor impairments by restoring mitophagy regulation in mice.ConclusionsOur study demonstrated that PS-NPs disrupt mitochondrial function by affecting CI, leading to excessive mitophagy through the AMPK/ULK1 pathway, causing dopaminergic neuron death. Melatonin can counteract PS-NP-induced mitochondrial dysfunction and motor impairments by regulating mitochondrial autophagy. These findings offer novel insights into the MNP-induced PD-like neurodegenerative mechanisms, and highlight melatonin's protective potential in mitigating the MNP’s environmental risk.

Optimizing environmental viral surveillance: bovine serum albumin increases RT-qPCR sensitivity for high pathogenicity avian influenza H5Nx virus detection from dust samples

ABSTRACT The latest outbreaks of high pathogenicity avian influenza viruses (HPAIVs) were the most widespread ever seen in Europe, entailing considerable economic costs and raising public health concerns. Virological surveillance protocols involve reverse transcription quantitative polymerase chain reactions (RT-qPCR) from tracheal and cloacal swabs, which are laborious to perform on a large scale and require special skills. Environmental sampling, and especially dust collection, may represent a relevant alternative, as it is cheap, non-invasive for animals, simpler, and quicker to carry out. The main drawback of dust samples is that they may contain high amounts of organic substances that can inhibit RT-qPCR reactions. Bovine serum albumin (BSA) is a molecule known to facilitate DNA polymerization in the presence of numerous inhibitors, including those from feces, litter, or food. We tested its use on dust samples collected on 107 farms localized in areas affected by epizootics of clade 2.3.4.4b HPAIV H5N1. We used a latent class modeling approach to compare the performance of three detection protocols: (i) BSA addition to the RT-qPCR reaction mix, (ii) dilution of template RNA, and (iii) a control protocol. Our results indicate that BSA addition to the RT-qPCR reaction mix improved the sensitivity of the method, by neutralizing inhibitors’ effect. Indeed, for hemagglutinin (HA) or matrix (M) RNA detection, the sensitivity of the BSA protocol was the highest, followed by that of the control protocol, and that of the dilution protocol. Our results suggest that the use of BSA could be routinely implemented in HPAIV dust monitoring RT-qPCR protocols. IMPORTANCE With the circulation of high pathogenicity avian influenza viruses having intensified considerably in recent years, the European Union is considering the vaccination of farmed birds. A prerequisite for this vaccination is the implementation of drastic surveillance protocols. Environmental sampling is a relevant alternative to animal sampling. However, environmental samples often contain inhibitory compounds in large enough quantities to inhibit RT-qPCR reactions. As bovine serum albumin is a molecule used in many fields to overcome this inhibitory effect, we tested its use on dust samples from poultry farms in areas heavily affected by HPAIV epizootics. Our results show that its use significantly increases the sensitivity of the method.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1–5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 μM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between −5.7 to −7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

Canagliflozin ameliorates high glucose-induced apoptosis in NRK-52E cells via inhibiting oxidative stress and activating AMPK/mTOR-mediated autophagy.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors, as a new type of hypoglycemic drug, can prevent proximal renal tubule injury related to glucose toxicity and play a renoprotective role. Canagliflozin, a recognized SGLT-2 inhibitor, has been proved to have potential protection in diabetic nephropathy (DN), but its mechanism has not been fully elucidated. In this study, the protective effect of canagliflozin against high glucose (HG)-induced renal tubular epithelial cell (NRK-52E) injury in vitro was assessed. The viability and apoptosis of NRK-52E cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, oxidative stress-related proteins (NOX4 and Nrf2), autophagy marker light chain 3 (LC3) I/II, and adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway-related proteins were evaluated by Western blot. Reactive oxygen species (ROS) level was evaluated by dihydroethidium (DHE) reactive oxygen species assay, the activities of SOD, CAT, GSH-Px and MDA were analyzed using kits. The changes of morphology and red fluorescent protein (RFP)-LC3 fluorescence were observed under microscopy. Canagliflozin significantly ameliorated HG-induced NRK-52E cell apoptosis and caspase-3 cleavage. Furthermore, canagliflozin markedly ameliorated HG-induced NRK-52E cell oxidative stress. Moreover, canagliflozin significantly increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Finally, canagliflozin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C abolished canagliflozin-induced autophagy activation, as well as the anti-apoptotic effect of canagliflozin. Canagliflozin effectively ameliorate HG-induced apoptosis of NRK-52E cells in vitro that involved its antioxidant effect and induction of autophagy through the AMPK/mTOR pathway.

Ghrelin alleviates hypoxia/reoxygenation-induced H9C2 injury by activating autophagy and AMPK/ULK1 pathway.

The research aims to explore the protective effects of ghrelin and its underlying molecular mechanisms in an H9C2 hypoxia/reoxygenation model. H9C2 cells were transfected with ghrelin overexpression lentiviral vector. The hypoxia/reoxygenation H9C2 model was constructed. The expression of ghrelin was analyzed by qRT-PCR and Western Blotting. CCK8, flow cytometry and TUNEL assay were used to analyze the impact of ghrelin on the survival and apoptosis of H9C2 injured by hypoxia/reoxygenation. The levels of autophagy-related proteins in H9C2 cells were evaluated through Western blotting. ELISA was utilized to assess how ghrelin affects the inflammatory response triggered by hypoxia/reoxygenation. Western blotting was utilized to investigate the regulatory role of ghrelin on the AMPK/ULK1 pathway. Additionally, the AMPK inhibitor Compound C was introduced to delve further into the associated mechanism. Hypoxia/reoxygenation injury decreased the expression of ghrelin. Transfection of ghrelin overexpression lentiviral vector significantly increased the expression of ghrelin in H9C2 cells. Ghrelin overexpression can significantly promote cell survival, reduce apoptosis, activate AMPK, ULK1 and AMBRA1, promote autophagy, increase the expression of LC3BII/LC3BI and Beclin-1, reduce the expression of P62, and reduce inflammatory response. Ghrelin inhibited apoptosis of H9C2 caused by hypoxia/reoxygenation and reduced inflammatory response, which mechanism is related to activation of AMPK/ULK1 pathway and autophagy.

In Vitro and In Silico Evaluation of Amylase, Tyrosinase, and Pancreatic Lipase Inhibitions of Novel Benzothiazole‐Sulfonate Derivatives

AbstractThis paper provides a comprehensive account of the synthesis and assessment of newly developed aryl sulfonate derivatives based on benzothiazole as enzyme inhibitors, specifically focusing on their ability to target tyrosinase, amylase, and pancreatic lipase. The assessments were performed utilizing experimental (in vitro) and computational (in silico) methodologies. For this aim, nine different aryl sulfonate derivatives were synthesized. The synthesized compounds were subjected to structural characterizations by utilizing Nuclear Magnetic Resonance (1H NMR, 13C NMR) and High‐Resolution Mass Spectrometry (HRMS) studies, which provided confirmation of their structural properties. The inhibitory efficiency of the compounds was determined by measuring their 50 % inhibitory concentration (IC50) values and comparing them with the standard inhibitory compounds. According to the in vitro amylase activity, benzo[d]thiazol‐2‐yl 4‐methylbenzenesulfonate showed the best inhibition with the lowest IC50 value (43.31±4.3 μM) which was calculated to be at a close level to the IC50 value of standard acarbose (38.50±3.8 μM). Also, benzo[d]thiazol‐2‐yl 4‐bromobenzenesulfonate (22.73±4.15 μM) and benzo[d]thiazol‐2‐yl 4‐chlorobenzenesulfonate (25.28±1.95 μM) were calculated to have the lowest IC50 values and the most effective inhibition capacities against pancreatic lipase. Benzo[d]thiazol‐2‐yl 4‐fluorobenzenesulfonate and benzo[d]thiazol‐2‐yl 4‐methylbenzenesulfonate significantly exhibited superior tyrosinase inhibition compared to other derivatives and conventional kojic acid. In silico molecular docking affirmed that benzo[d]thiazol‐2‐yl naphthalene‐2‐sulfonate presented the highest binding affinities to the studied enzymes, with values of −7.8 kcal/mol for tyrosinase, −10.7 kcal/mol for pancreatic lipase, and −9.4 kcal/mol for α‐amylase. The pharmacokinetic characteristics and drug‐likeness of the synthesized sulfonate derivatives were also evaluated using absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction. Density Functional Theory (DFT) calculations were employed at the DFT/B3LYP/6‐311 g(d,p) level of theory to investigate the electronic characteristics of the compounds, therefore facilitating the comprehension of the reported enzyme inhibitory actions. In summary, our research highlights the potential of benzothiazole aryl sulfonate derivatives as effective enzyme inhibitors that hold promise for therapeutic applications.

PSXII-29 Development of an in Vitro Rumen Fermentation Model to Assess Fusobacterium Necrophorum Growth Inhibition

Abstract This experiment aimed to determine the impact of growth-inhibitory compounds on F. necrophorum subsp. necrophorum (F.n.n.), a primary etiologic agent of liver abscesses in feedlot cattle, in an in vitro rumen fermentation system. The assessment of inhibition was by quantitative PCR. We hypothesized that the addition of growth inhibitory compounds would reduce F.n.n. concentrations. A 2 x 2 x 6 factorial arrangement of treatments was used. Factors included: 1) sodium lactate (0.0 or 100 mM); 2) bacteria (0 or 1x108 CFU/ml of F.n.n.); and 3) 1 mL of test compound [Control (solvent with no additive); Tylosin: 5 μg/mL; Compound A; Compound B; Compound C; and Compound D]. Prior to morning feeding, rumen contents were collected from a single ruminally canulated steer fed a grain-based finishing diet. Rumen fluid was strained through 4 layers of cheesecloth, mixed with McDougall’s buffer (1:1 ratio; RFB), and 30 mL of the RFB was added into 100 mL-serum bottles containing the appropriate treatments. Serum bottles (n = 3 replicates per treatment) were flushed with CO2 closed with rubber stoppers, crimped with aluminum seals, and incubated at 39ºC for 0, 6, 12, and 24 h. Following each incubation time point, a 2-mL aliquot was removed from each serum bottle under a stream of CO2 to maintain anaerobic conditions. Immediately after sample collection, pH was determined, and DNA was extracted by boiling the sample, centrifugation, and purifying with a GeneClean Turbo DNA extraction kit. The concentration of F.n.n was determined by a quantitative PCR assay targeting the promoter region of the leukotoxin genes. Concentrations of F.n.n. from the RFB were used to adjust F.n.n. concentrations for all treatments at all time points. There was a lactate x test compound x incubation time interaction (P < 0.05) for F.n.n. concentration. In the absence of lactate, serum bottles containing tylosin or test compound treatments had decreased (P < 0.05) F.n.n. concentrations by 24 h post incubation. However, when sodium lactate was added, no treatments had F.n.n. concentrations less than the control treatment at 24 h post incubation (P > 0.05). These data suggest that compounds used in this experiment reduced the concentrations of F.n.n by 24 h post incubation in the absence of additional lactate. However, when lactate, a substrate for F.n.n, was added to the in vitro system, the reduction in F.n.n. growth was not observed.

Auraptene Enhances AMP-Activated Protein Kinase Phosphorylation and Thereby Inhibits the Proliferation, Migration and Expression of Androgen Receptors and Prostate-Specific Antigens in Prostate Cancer Cells.

Citrus hassaku extract reportedly activates AMPK. Because this extract contains an abundance of auraptene, we investigated whether pure auraptene activates AMPK and inhibits proliferation using prostate cancer cell lines. Indeed, auraptene inhibited the proliferation and migration of LNCaP cells and induced phosphorylation of AMPK or its downstream ACC in LNCaP, PC3, and HEK-293 cells, but not in DU145 cells not expressing LKB1. In addition, the mTOR-S6K pathway, located downstream from activated AMPK, was also markedly suppressed by auraptene treatment. Importantly, it was shown that auraptene reduced androgen receptor (AR) and prostate-specific antigen (PSA) expressions at both the protein and the mRNA level. This auraptene-induced downregulation of PSA was partially but significantly reversed by treatment with AMPK siRNA or the AMPK inhibitor compound C, suggesting AMPK activation to, at least partially, be causative. Finally, in DU145 cells lacking the LKB1 gene, exogenously induced LKB1 expression restored AMPK phosphorylation by auraptene, indicating the essential role of LKB1. In summary, auraptene is a potent AMPK activator that acts by elevating the AMP/ATP ratio, thereby potentially suppressing prostate cancer progression, via at least three molecular mechanisms, including suppression of the mTOR-S6K pathway, reduced lipid synthesis, and AR downregulation caused by AMPK activation.

Autophagic and apoptotic proteins in goat corpus luteum and the effect of Adiponectin/AdipoRon on luteal cell autophagy and apoptosis

The most abundant adipokine Adiponectin (APN) is present in ovaries. AdipoRon is a small molecule oral APN receptor agonist that binds and activates APN receptors. However, the function of APN/AdipoRon in regulation of luteal cell processes has not been elucidated. To investigate autophagic and apoptotic proteins in goat CLs and effects of APN/AdipoRon on goat luteal autophagy and apoptosis, goat CLs were collected during the early, mid and late luteal stages of the estrous cycle to evaluate autophagic and apoptotic protein patterns. LC3B, Beclin 1, Caspase-3 and Bax/Bcl-2 as well as p-AMPK were differentially abundant at different stages of CL development. All these proteins were primarily localized in large and small luteal steroidogenic cells. Then, isolated luteal steroidogenic cells were evaluated to ascertain the functions and mechanism of APN/AdipoRon in luteal autophagy and apoptosis. Treatment with AdipoRon (25 and 50 μM) and APN (1 μg/mL) for 48 h resulted in a decrease in cell viability and P4 level, increased autophagic and apoptotic proteins. Treatment with AdipoRon (25 μM) led to rapid and transient p-AMPK activation, with p-AMPK elevated at 30 min to 1 h with there being a return to a basal concentration at 2 h post-treatment. Moreover, treatment with AdipoRon led to an increase in autophagy by activating AMPK, which was markedly reduced with treatment with an AMPK inhibitor Compound C and siAMPK, however, abundances of apoptotic proteins were not affected by these treatments. In conclusion, autophagy and apoptosis are involved in the structural regression of goat CL. APN/AdipoRon led to a lesser cell viability and P4 concentration, and activated autophagy through induction of the AMPK while there was induction of apoptosis through an AMPK - independent pathway in goat luteal cells.

Novel Water-soluble quinoxaline-2,3-dione-6-sulfohydrazide derivatives as efficient acid corrosion inhibitors: Design, characterization, experimental, and theoretical studies

BackgroundThe corrosion of metals results in significant financial losses for the nation and poses a substantial threat to human safety. Few corrosion inhibitors on the market today are water-soluble, so they can't keep up with the growing need for efficient inhibitors. So, many researchers seek to construct efficient and low-cost materials for metal protection. Methods: In the present article, two novel water-soluble quinoxaline-2,3-dione-6-sulfohydrazide derivatives, Qs-HP and Qs-HI, were constructed, synthesized, characterized, and evaluated their activity as anticorrosion inhibitors. The authors confirmed the capacity of both organic compounds to act as corrosion inhibitors for CS alloy by using Potentiodynamic polarization (PP), weight loss, and electrochemical impedance polarization (EIS) studies in 1.0 M hydrochloric acid solution. Significant findingsThe inhibitors, Qs-HP and Qs-HI, demonstrated the highest corrosion protection of 92.06 % and 95.93 %, respectively, at 1000 ppm by the PP method. The results also demonstrated that a higher inhibitor concentration resulted in a more effective inhibition. The PP effect on the condition of mixed adsorption is demonstrated as physical-chemical adsorption (mixed type). Also, Langmuir's adsorption isotherm explains how the CS alloy's surface absorbed the inhibitory materials. EIS results continuously showed that increasing the quantity of injected inhibitor compounds increased charge transfer resistance (Rct), and the electrochemical double layer (Cdl) decreased. Moreover, theoretical studies like EHOMO, ELUMO, diploe moment (µ), and suitable inhibition mechanisms were discussed in detail and used to confirm the donor-acceptor (D-A) system. Finally, all the experimental and theoretical data demonstrate that these derivatives can successfully build a blocking layer and regulate corrosion.