Dopamine modification of glycolytic enzymes impairs glycolysis: possible implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons. Dopamine (DA), the neurotransmitter produced by dopaminergic neurons, and its metabolites can covalently modify proteins, and dysregulation of this process has been implicated in neuronal loss in PD. However, much remains unknown about the protein targets. METHODS: In the present work, we designed and synthesized a dopamine probe (DA-P) to screen and identify the potential protein targets of DA using activity-based protein profiling (ABPP) technology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In situ pull-down assays, cellular thermal shift assays (CETSAs) and immunofluorescence were performed to confirm the DA modifications on these hits. To investigate the effects of DA modifications, we measured the enzymatic activities of these target proteins, evaluated glycolytic stress and mitochondrial respiration by Seahorse tests, and systematically analyzed the changes in metabolites with unbiased LC-MS/MS-based non-targeted metabolomics profiling. RESULTS: We successfully identified three glycolytic proteins, aldolase A, α-enolase and pyruvate kinase M2 (PKM2), as the binding partners of DA. DA bound to Glu166 of α-enolase, Cys49 and Cys424 of PKM2, and Lys230 of aldolase A, inhibiting the enzymatic activities of α-enolase and PKM2 and thereby impairing ATP synthesis, resulting in mitochondrial dysfunction. CONCLUSIONS: Recent research has revealed that enhancing glycolysis can offer protection against PD. The present study identified that the glycolytic pathway is vulnerable to disruption by DA, suggesting a promising avenue for potential therapeutic interventions. Safeguarding glycolysis against DA-related disruption could be a potential therapeutic intervention for PD.

Multi-omics dissection of stage-specific artemisinin tolerance mechanisms in Kelch13-mutant Plasmodium falciparum.

AIMS: We investigated the stage-specific mechanisms of partial resistance to artemisinin (ART, an antimalarial drug) in Plasmodium falciparum (P. falciparum) carrying the Kelch13 C580Y mutation. METHODS: Using fluorescence labeling and activity-based protein profiling, we systematically profile the ART activation levels in P. falciparum during the entire intra-erythrocytic developmental cycle (IDC), and determined the ART-targets profile of the ART-sensitive and -resistant strains at different stages. We retrieved and integrated datasets of single-cell transcriptomics and label-free proteomics across three IDC stages of wild-type P. falciparum. We also employed lipidomics to validate lipid metabolic reprogramming in the resistant strain. RESULTS: The activation and expression patterns of genes and proteins of ART-targets in both ART-sensitive and resistant strains varied at different stages and periods of P. falciparum development, with the late trophozoite stage harboring the largest number of ART targets. We identified and validated 36 overlapping targets, such as GAPDH, EGF-1a, and SpdSyn, during the IDC stages in both strains. We revealed the ART-insensitivity of fatty acid-associated activities in the partially resistant strain at both the early ring and early trophozoite stages. CONCLUSIONS: Our multi-omics strategies provide novel insights into the mechanisms of ART partial resistance in Kelch13 mutant P. falciparum, demonstrating the stage-specific interaction between ART and malaria parasites.

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann-Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal-Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples.

Single-cell RNA sequencing deciphers the mechanism of sepsis-induced liver injury and the therapeutic effects of artesunate.

Liver, as an immune and detoxification organ, represents an important line of defense against bacteria and infection and a vulnerable organ that is easily injured during sepsis. Artesunate (ART) is an anti-malaria agent, that also exhibits broad pharmacological activities including anti-inflammatory, immune-regulation and liver protection. In this study, we investigated the cellular responses in liver to sepsis infection and ART hepatic-protective mechanisms against sepsis. Cecal ligation and puncture (CLP)-induced sepsis model was established in mice. The mice were administered ART (10 mg/kg, i.p.) at 4 h, and sacrificed at 12 h after the surgery. Liver samples were collected for preparing single-cell RNA transcriptome sequencing (scRNA-seq). The scRNA-seq analysis revealed that sepsis-induced a dramatic reduction of hepatic endothelial cells, especially the subtypes characterized with proliferation and differentiation. Macrophages were recruited during sepsis and released inflammatory cytokines (Tnf, Il1b, Il6), chemokines (Ccl6, Cd14), and transcription factor (Nfkb1), resulting in liver inflammatory responses. Massive apoptosis of lymphocytes and abnormal recruitment of neutrophils caused immune dysfunction. ART treatment significantly improved the survival of CLP mice within 96 h, and partially relieved or reversed the above-mentioned pathological features, mitigating the impact of sepsis on liver injury, inflammation, and dysfunction. This study provides comprehensive fundamental proof for the liver protective efficacy of ART against sepsis infection, which would potentially contribute to its clinical translation for sepsis therapy. Single cell transcriptome reveals the changes of various hepatocyte subtypes of CLP-induced liver injury and the potential pharmacological effects of artesunate on sepsis.

O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells.

Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis (FAS) activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and FAS in cancer therapy.

Recent pharmacological advances in the repurposing of artemisinin drugs.

Artemisinins are a family of sesquiterpene lactones originally derived from the sweet wormwood (Artemisia annua). Beyond their well-characterized role as frontline antimalarial drugs, artemisinins have also received increased attention for other potential pharmaceutical effects, which include antiviral, antiparsitic, antifungal, anti-inflammatory, and anticancer activities. With concerted efforts in further preclinical and clinical studies, artemisinin-based drugs have the potential to be viable treatments for a great variety of human diseases. Here, we provide a comprehensive update on recent reports of pharmacological actions and applications of artemisinins outside of their better-known antimalarial role and highlight their potential therapeutic viability for various diseases.

Study towards improving artemisinin-based combination therapies.

Covering: Up to 2020 Artemisinin has made a significant contribution towards global malaria control since its initial discovery. Countless lives have been saved by this unique and miraculous molecule. In 2006, artemisinin-based combination therapies (ACTs) were recommended by the World Health Organization (WHO) as the first-line treatment for uncomplicated malaria infection and have since remained as the mainstays of the antimalarial treatment. Even so, substantial efforts to pursue better curative effects for the treatment of malaria have never ceased, particularly with regards to the circumstances surrounding the appearance of delayed clearance of malaria parasites by 3 day ACT treatments in South-East Asian countries. Strategies to further optimize artemisinin-based therapies, including synthesizing better artemisinin derivatives, developing advanced drug delivery systems, and diversifying artemisinin partner drugs, have been proposed over the past few years. Here, we provide an updated account of the continuous efforts in improving ACTs for better efficacy in curing malarial infection.

Targeting autophagy enhances the anticancer effect of artemisinin and its derivatives.

Artemisinin and its derivatives, with their outstanding clinical efficacy and safety, represent the most effective and impactful antimalarial drugs. Apart from its antimalarial effect, artemisinin has also been shown to exhibit selective anticancer properties against multiple cancer types both in vitro and in vivo. Specifically, our previous studies highlighted the therapeutic effects of artemisinin on autophagy regulation. Autophagy is a well-conserved degradative process that recycles cytoplasmic contents and organelles in lysosomes to maintain cellular homeostasis. The deregulation of autophagy is often observed in cancer cells, where it contributes to tumor adaptation to nutrient-deficient tumor microenvironments. This review discusses recent advances in the anticancer properties of artemisinin and its derivatives via their regulation of autophagy, mitophagy, and ferritinophagy. In particular, we will discuss the mechanisms of artemisinin activation in cancer and novel findings regarding the role of artemisinin in regulating autophagy, which involves changes in multiple signaling pathways. More importantly, with increasing failure rates and the high cost of the development of novel anticancer drugs, the strategy of repurposing traditional therapeutic Chinese medicinal agents such as artemisinin to treat cancer provides a more attractive alternative. We believe that the topics covered here will be important in demonstrating the potential of artemisinin and its derivatives as safe and potent anticancer agents.

What has traditional Chinese medicine delivered for modern medicine?

The field of Traditional Chinese Medicine (TCM) represents a vast and largely untapped resource for modern medicine. Exemplified by the success of the antimalarial artemisinin, the recent years have seen a rapid increase in the understanding and application of TCM-derived herbs and formulations for evidence-based therapy. In this review, we summarise and discuss the developmental history, clinical background and molecular basis of an action for several representative TCM-derived medicines, including artemisinin, arsenic trioxide, berberine and Salvia miltiorrhiza or Danshen. Through this, we highlight important examples of how TCM-derived medicines have already contributed to modern medicine, and discuss potential avenues for further research.

Target identification with quantitative activity based protein profiling (ABPP).

As many small bioactive molecules fulfill their functions through interacting with protein targets, the identification of such targets is crucial in understanding their mechanisms of action (MOA) and side effects. With technological advancements in target identification, it has become possible to accurately and comprehensively study the MOA and side effects of small molecules. While small molecules with therapeutic potential were derived solely from nature in the past, the remodeling and synthesis of such molecules have now been made possible. Presently, while some small molecules have seen successful application as drugs, the majority remain undeveloped, requiring further understanding of their MOA and side effects to fully tap into their potential. Given the typical promiscuity of many small molecules and the complexity of the cellular proteome, a high-flux and high-accuracy method is necessary. While affinity chromatography approaches combined with MS have had successes in target identification, limitations associated with nonspecific results remain. To overcome these complications, quantitative chemical proteomics approaches have been developed including metabolic labeling, chemical labeling, and label-free methods. These new approaches are adopted in conjunction with activity-based protein profiling (ABPP), allowing for a rapid process and accurate results. This review will briefly introduce the principles involved in ABPP, then summarize current advances in quantitative chemical proteomics approaches as well as illustrate with examples how ABPP coupled with quantitative chemical proteomics has been used to detect the targets of drugs and other bioactive small molecules including natural products.

Artemisinin as an anticancer drug: Recent advances in target profiling and mechanisms of action.

Artemisinin and its derivatives (collectively termed as artemisinins) are among the most important and effective antimalarial drugs, with proven safety and efficacy in clinical use. Beyond their antimalarial effects, artemisinins have also been shown to possess selective anticancer properties, demonstrating cytotoxic effects against a wide range of cancer types both in vitro and in vivo. These effects appear to be mediated by artemisinin-induced changes in multiple signaling pathways, interfering simultaneously with multiple hallmarks of cancer. Great strides have been taken to characterize these pathways and to reveal their anticancer mechanisms of action of artemisinin. Moreover, encouraging data have also been obtained from a limited number of clinical trials to support their anticancer property. However, there are several key gaps in knowledge that continue to serve as significant barriers to the repurposing of artemisinins as effective anticancer agents. This review focuses on important and emerging aspects of this field, highlighting breakthroughs in unresolved questions as well as novel techniques and approaches that have been taken in recent studies. We discuss the mechanism of artemisinin activation in cancer, novel and significant findings with regards to artemisinin target proteins and pathways, new understandings in artemisinin-induced cell death mechanisms, as well as the practical issues of repurposing artemisinin. We believe these will be important topics in realizing the potential of artemisinin and its derivatives as safe and potent anticancer agents.

Artesunate Activates the Intrinsic Apoptosis of HCT116 Cells through the Suppression of Fatty Acid Synthesis and the NF-κB Pathway.

The artemisinin compounds, which are well-known for their potent therapeutic antimalarial activity, possess in vivo and in vitro antitumor effects. Although the anticancer effect of artemisinin compounds has been extensively reported, the precise mechanisms underlying its cytotoxicity remain under intensive study. In the present study, a high-throughput quantitative proteomics approach was applied to identify differentially expressed proteins of HCT116 colorectal cancer cell line with artesunate (ART) treatment. Through Ingenuity Pathway Analysis, we discovered that the top-ranked ART-regulated biological pathways are abrogation of fatty acid biosynthetic pathway and mitochondrial dysfunction. Subsequent assays showed that ART inhibits HCT116 cell proliferation through suppressing the fatty acid biosynthetic pathway and activating the mitochondrial apoptosis pathway. In addition, ART also regulates several proteins that are involved in NF-κB pathway, and our subsequent assays showed that ART suppresses the NF-κB pathway. These proteomic findings will contribute to improving our understanding of the underlying molecular mechanisms of ART for its therapeutic cytotoxic effect towards cancer cells.

Andrographolide Suppresses MV4-11 Cell Proliferation through the Inhibition of FLT3 Signaling, Fatty Acid Synthesis and Cellular Iron Uptake.

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR's anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

Chemoproteomics Reveals Glaucocalyxin A Induces Mitochondria-Dependent Apoptosis of Leukemia Cells via Covalently Binding to VDAC1.

Chronic myelogenous leukemia (CML) that is resistant to tyrosine kinase inhibitors is one of the deadliest hematologic malignancies, and the T315I mutation in the breakpoint cluster region-Abelson (BCR-ABL) kinase domain is the most prominent point mutation responsible for imatinib resistance in CML. Glaucocalyxin A (GLA), a natural bioactive product derived from the Rabdosia rubescens plant, has strong anticancer activity. In this study, the effect and molecular mechanism of GLA on imatinib-sensitive and imatinib-resistant CML cells harboring T315I mutation via a combined deconvolution strategy of chemoproteomics and label-free proteomics is investigated. The data demonstrated that GLA restrains proliferation and induces mitochondria-dependent apoptosis in both imatinib-sensitive and resistant CML cells. GLA covalently binds to the cysteine residues of mitochondrial voltage-dependent anion channels (VDACs), resulting in mitochondrial damage and overflow of intracellular apoptotic factors, eventually leading to apoptosis. In addition, the combination of GLA with elastin, a mitochondrial channel VDAC2/3 inhibitor, enhances mitochondria-dependent apoptosis in imatinib-sensitive and -resistant CML cells, representing a promising therapeutic approach for leukemia treatment. Taken together, the results show that GLA induces mitochondria-dependent apoptosis via covalently targeting VDACs in CML cells. GLA may thus be a candidate compound for the treatment of leukemia.

Single-Cell Transcriptomics Reveals the Ameliorative Effect of Oridonin on Septic Liver Injury.

Sepsis is a life-threatening syndrome leading to hemodynamic instability and potential organ dysfunction. Oridonin, commonly used in Traditional Chinese Medicine (TCM), exhibits significant anti-inflammation activity. To explore the protective mechanisms of oridonin against the pathophysiological changes, the authors conducted single-cell transcriptome (scRNA-seq) analysis on septic liver models induced by cecal ligation and puncture (CLP). They obtained a total of 63,486 cells, distributed across 11 major cell clusters, and concentrated their analysis on four specific clusters (hepatocytes/Heps, macrophages, endothelial/Endos and T/NK) based on their changes in proportion during sepsis and under oridonin treatment. Firstly, biological changes in Hep, which are related to metabolic dysregulation and pro-inflammatory signaling, are observed during sepsis. Secondly, they uncovered the dynamic profiles of macrophage's phenotype, indicating that a substantial number of macrophages exhibited a M1-skewed phenotype associated with pro-inflammatory characteristics in septic model. Thirdly, they detected an upregulation of both inflammatory cytokines and transcriptomic factor Nfkb1 expression within Endo, along with slight capillarization during sepsis. Moreover, excessive accumulation of cytotoxic NK led to an immune imbalance. Though, oridonin ameliorated inflammatory-related responses and improved the liver dysfunction in septic mice. This study provides fundamental evidence of the protective effects of oridonin against sepsis-induced cytokine storm.

Photoaffinity probe-based antimalarial target identification of artemisinin in the intraerythrocytic developmental cycle of Plasmodium falciparum.

Malaria continues to pose a serious global health threat, and artemisinin remains the core drug for global malaria control. However, the situation of malaria resistance has become increasingly severe due to the emergence and spread of artemisinin resistance. In recent years, significant progress has been made in understanding the mechanism of action (MoA) of artemisinin. Prior research on the MoA of artemisinin mainly focused on covalently bound targets that are alkylated by artemisinin-free radicals. However, less attention has been given to the reversible noncovalent binding targets, and there is a paucity of information regarding artemisinin targets at different life cycle stages of the parasite. In this study, we identified the protein targets of artemisinin at different stages of the parasite's intraerythrocytic developmental cycle using a photoaffinity probe. Our findings demonstrate that artemisinin interacts with parasite proteins in vivo through both covalent and noncovalent modes. Extensive mechanistic studies were then conducted by integrating target validation, phenotypic studies, and untargeted metabolomics. The results suggest that protein synthesis, glycolysis, and oxidative homeostasis are critically involved in the antimalarial activities of artemisinin. In summary, this study provides fresh insights into the mechanisms underlying artemisinin's antimalarial effects and its protein targets.

STEP: profiling cellular-specific targets and pathways of bioactive small molecules in tissues via integrating single-cell transcriptomics and chemoproteomics.

Identifying the cellular targets of bioactive small molecules within tissues has been a major concern in drug discovery and chemical biology research. Compared to cell line models, tissues consist of multiple cell types and complicated microenvironments. Therefore, elucidating the distribution and heterogeneity of targets across various cells in tissues would enhance the mechanistic understanding of drug or toxin action in real-life scenarios. Here, we present a novel multi-omics integration pipeline called Single-cell TargEt Profiling (STEP) that enables the global profiling of protein targets in mammalian tissues with single-cell resolution. This pipeline integrates single-cell transcriptome datasets with tissue-level protein target profiling using chemoproteomics. Taking well-established classic drugs such as aspirin, aristolochic acid, and cisplatin as examples, we confirmed the specificity and precision of cellular drug-target profiles and their associated molecular pathways in tissues using the STEP analysis. Our findings provide more informative insights into the action modes of bioactive molecules compared to in vitro models. Collectively, STEP represents a novel strategy for profiling cellular-specific targets and functional processes with unprecedented resolution.