Gambogic acid exhibits promising anticancer activity by inhibiting the pentose phosphate pathway in lung cancer mouse model.

BACKGROUND: The pentose phosphate pathway (PPP) plays a crucial role in the material and energy metabolism in cancer cells. Targeting 6-phosphogluconate dehydrogenase (6PGD), the rate-limiting enzyme in the PPP metabolic process, to inhibit cellular metabolism is an effective anticancer strategy. In our previous study, we have preliminarily demonstrated that gambogic acid (GA) induced cancer cell death by inhibiting 6PGD and suppressing PPP at the cellular level. However, it is unclear whether GA could suppress cancer cell growth by inhibiting PPP pathway in mouse model. PURPOSE: This study aimed to confirm that GA as a covalent inhibitor of 6PGD protein and to validate that GA suppresses cancer cell growth by inhibiting the PPP pathway in a mouse model. METHODS: Cell viability was detected by CCK-8 assays as well as flow cytometry. The protein targets of GA were identified using a chemical probe and activity-based protein profiling (ABPP) technology. The target validation was performed by in-gel fluorescence assay, the Cellular Thermal Shift Assay (CETSA). A lung cancer mouse model was constructed to test the anticancer activity of GA. RNA sequencing was performed to analyze the global effect of GA on gene expression. RESULTS: The chemical probe of GA exhibited high biological activity in vitro. 6PGD was identified as one of the binding proteins of GA by ABPP. Our findings revealed a direct interaction between GA and 6PGD. We also found that the anti-cancer activity of GA depended on reactive oxygen species (ROS), as evidenced by experiments on cells with 6PGD knocked down. More importantly, GA could effectively reduce the production of the two major metabolites of the PPP in lung tissue and inhibit cancer cell growth in the mouse model. Finally, RNA sequencing data suggested that GA treatment significantly regulated apoptosis and hypoxia-related physiological processes. CONCLUSION: These results demonstrated that GA was a covalent inhibitor of 6PGD protein. GA effectively suppressed cancer cell growth by inhibiting the PPP pathway without causing significant side effects in the mouse model. Our study provides in vivo evidence that elucidates the anticancer mechanism of GA, which involves the inhibition of 6PGD and modulation of cellular metabolic processes.

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.

Quantitative Chemical Proteomics Reveals Triptolide Selectively Inhibits HCT116 Human Colon Cancer Cell Viability and Migration Through Binding to Peroxiredoxin 1 and Annexin A1.

Triptolide (TPL), a natural product extracted from Tripterygium wilfordii Hook F, exerts potential anti-cancer activity. Studies have shown that TPL is involved in multiple cellular processes and signal pathways; however, its pharmaceutical activity in human colorectal cancer (CRC) as well as the underlying molecular mechanism remain elusive. In this study, the effects of TPL on HCT116 human colon cancer cells and CCD841 human colon epithelial cells are first evaluated. Next, the protein targets of TPL in HCT116 cells are identified through an activity-based protein profiling approach. With subsequent in vitro experiments, the mode of action of TPL in HCT116 cells is elucidated. As a result, TPL is found to selectively inhibit HCT116 cell viability and migration. A total of 54 proteins are identified as the targets of TPL in HCT116 cells, among which, Annexin A1 (ANXA1) and Peroxiredoxin I/II (Prdx I/II) are picked out for further investigation due to their important role in CRC. The interaction between TPL and ANXA1 or Prdx I is confirmed, and it is discovered that TPL exerts inhibitory effect against HCT116 cells through binding to ANXA1 and Prdx I. The study reinforces the potential of TPL in the CRC therapy, and provides novel therapeutic targets for the treatment of CRC.

Advanced Strategies for Overcoming Endosomal/Lysosomal Barrier in Nanodrug Delivery.

Nanocarriers have therapeutic potential to facilitate drug delivery, including biological agents, small-molecule drugs, and nucleic acids. However, their efficiency is limited by several factors; among which, endosomal/lysosomal degradation after endocytosis is the most important. This review summarizes advanced strategies for overcoming endosomal/lysosomal barriers to efficient nanodrug delivery based on the perspective of cellular uptake and intracellular transport mechanisms. These strategies include promoting endosomal/lysosomal escape, using non-endocytic methods of delivery to directly cross the cell membrane to evade endosomes/lysosomes and making a detour pathway to evade endosomes/lysosomes. On the basis of the findings of this review, we proposed several promising strategies for overcoming endosomal/lysosomal barriers through the smarter and more efficient design of nanodrug delivery systems for future clinical applications.

Advanced strategies for nucleic acids and small-molecular drugs in combined anticancer therapy.

Cancer has been considered as complex malignant consequence of genetic mutations that control the cellular proliferation, differentiation and homeostasis, thus making tumor treatment extremely challenging. To date, a variety of cargo molecules, including nucleic acids drugs (pDNA, miRNA and siRNA), therapeutic drugs (doxorubicin, paclitaxel, daunomycin and gefitinib) and imaging agents (radioisotopes, fluorescence dyes, and MRI contrast agents) have been regarded as the potential medicines in clinical application. However, non-single therapeutic drug could induce the satisfied clinical results because of tumor heterogeneity and multiple drug resistance and the nanotechnology-based combined therapy is becoming an advanced important mode for enhanced anticancer effects. The review gathers the current advanced development to co-deliver small-molecular drugs and nucleic acids for the anticancer therapy with nanomedicine-based combination. Furthermore, the superiority is definitely presented and the barriers are detail discussed to surmount the clinical challenges. In final, future perspectives in rational direction for combined tumor therapy of drugs and nucleic acids are exhibited.

Preparation and application of calcium phosphate nanocarriers in drug delivery.

Calcium phosphate nanoparticles represent promising materials for drug delivery because of its favorable properties, including biocompatibility, biodegradability and strong affinity for binding to nucleic acids (pDNA, siRNA, miRNA, etc.) and therapeutic drugs (cisplatin, carboplatin, paclitaxel, gefitinib, doxorubicin, etc.). Various strategies to prepare the size-controllable, stable, targeting and pH-responsive CaP nanocarriers have been extensively developed as the potential candidates in clinic. This review discusses the mostly recent developments in the design of calcium phosphate nanocarriers as drug delivery systems and therapeutic agents. Additionally, the advantage is unquestionably demonstrated and the obstacles are thoroughly examined in order to overcome future clinical issues.

Oridonin acts as a novel senolytic by targeting glutathione S-transferases to activate the ROS-p38 signaling axis in senescent cells.

Most of the known senolytics are anti-cancer drugs or their derivative molecules. However, senolytics derived from the active ingredients of traditional Chinese medicine (TCM) are rarely reported. Here, we identified oridonin as a novel senolytic and further revealed that it might target a class of glutathione S-transferases to activate ROS-p38 signaling and induce apoptosis in senescent cells.

A formal [4 + 2] annulation of diamines and prop-2-ynyl sulfonium salts for the synthesis of tetrahydroquinoxalines.

A formal [4 + 2] annulation of diamines and prop-2-ynyl sulfonium salts was developed. This strategy enables efficient access to tetrahydroquinoxalines in excellent yields.

18beta-glycyrrhetinic acid induces ROS-mediated apoptosis to ameliorate hepatic fibrosis by targeting PRDX1/2 in activated HSCs.

Hepatic stellate cells (HSCs) are essential drivers of fibrogenesis. Inducing activated-HSC apoptosis is a promising strategy for treating hepatic fibrosis. 18beta-glycyrrhetinic acid (18ß-GA) is a natural compound that exists widely in herbal medicines, such as Glycyrrhiza uralensis Fisch, which is used for treating multiple liver diseases, especially in Asia. In the present study, we demonstrated that 18ß-GA decreased hepatic fibrosis by inducing the apoptosis in activated HSCs. 18ß-GA inhibited the expression of α-smooth muscle actin and collagen type I alpha-1. Using a chemoproteomic approach derived from activity-based protein profiling, together with cellular thermal shift assay and surface plasmon resonance, we found that 18ß-GA covalently targeted peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2) proteins via binding to active cysteine residues and thereby inhibited their enzymatic activities. 18ß-GA induced the elevation of reactive oxygen species (ROS), resulting in the apoptosis of activated HSCs. PRDX1 knockdown also led to ROS-mediated apoptosis in activated HSCs. Collectively, our findings revealed the target proteins and molecular mechanisms of 18ß-GA in ameliorating hepatic fibrosis, highlighting the future development of 18ß-GA as a novel therapeutic drug for hepatic fibrosis.

Neuroprotective Effects of Celastrol in Neurodegenerative Diseases-Unscramble Its Major Mechanisms of Action and Targets.

There are rarely new therapeutic breakthroughs present for neurodegenerative diseases in the last decades. Thus, new effective drugs are urgently needed for millions of patients with neurodegenerative diseases. Celastrol, a pentacyclic triterpenoid compound, is one of the main active ingredients isolated from Tripterygium wilfordii Hook. f. that has multiple biological activities. Recently, amount evidence indicates that celastrol exerts neuroprotective effects and holds therapeutic potential to serve as a novel agent for neurodegenerative diseases. This review focuses on the therapeutic efficacy and major regulatory mechanisms of celastrol to rescue damaged neurons, restore normal cognitive and sensory motor functions in neurodegenerative diseases. Importantly, we highlight recent progress regarding identification of the drug targets of celastrol by using advanced quantitative chemical proteomics technology. Overall, this review provides novel insights into the pharmacological activities and therapeutic potential of celastrol for incurable neurodegenerative diseases.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA.

BACKGROUND: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood. METHODS: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays. RESULTS: We developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photo-crosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods. CONCLUSIONS: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

Dissection of Targeting Molecular Mechanisms of Aristolochic Acid-induced Nephrotoxicity via a Combined Deconvolution Strategy of Chemoproteomics and Metabolomics.

Aristolochic acid (AA), mainly derived from herbal Aristolochia and Asarum plants, was listed as a human carcinogen class I in 2002. Aristolochic acid nephropathy (AAN) is a rapidly progressive tubulointerstitial nephritis and urothelial cancer caused by AA. However, the targeting molecular mechanisms of AAs-induced nephrotoxicity are largely unclear. This study aims to dissect targeting molecular mechanisms of AA-induced nephrotoxicity. Activity-based protein profiling (ABPP) in combination with cellular thermal shift assay (CETSA) was performed to identify the AAs binding target proteins. Our data indicated that several key enzymes in the metabolic process and mitochondrial respiration including IDH2 and MDH2 (Krebs cycle), PKM and LDH (aerobic respiration), FASN (fatty acid beta-oxidation), HK2 (glucose metabolism), and ATP synthase were identified as directly binding targets of AAs. Metabolomics and oxygen consumption rate (OCR) experiments further confirmed that AAs targeting proteins disrupted metabolic biosynthesis processes and impaired mitochondrial functions. Ultimately, AAs induced renal cells apoptosis by disturbing various biological processes. Cumulatively, AAs may directly bind to key proteins involved in the metabolic process and mitochondrial homeostasis, and finally induce aristolochic acid nephropathy. Our findings provide novel insight into underlying mechanisms of AAs-induced kidney toxicity, which may help to develop therapeutic strategies for AAN.

Discovery and repurposing of artemisinin.

Malaria is an ancient infectious disease that threatens millions of lives globally even today. The discovery of artemisinin, inspired by traditional Chinese medicine (TCM), has brought in a paradigm shift and been recognized as the "best hope for the treatment of malaria" by World Health Organization. With its high potency and low toxicity, the wide use of artemisinin effectively treats the otherwise drug-resistant parasites and helps many countries, including China, to eventually eradicate malaria. Here, we will first review the initial discovery of artemisinin, an extraordinary journey that was in stark contrast with many drugs in western medicine. We will then discuss how artemisinin and its derivatives could be repurposed to treat cancer, inflammation, immunoregulation-related diseases, and COVID-19. Finally, we will discuss the implications of the "artemisinin story" and how that can better guide the development of TCM today. We believe that artemisinin is just a starting point and TCM will play an even bigger role in healthcare in the 21st century.

A highly efficient protein corona-based proteomic analysis strategy for the discovery of pharmacodynamic biomarkers.

The composition of serum is extremely complex, which complicates the discovery of new pharmacodynamic biomarkers via serum proteome for disease prediction and diagnosis. Recently, nanoparticles have been reported to efficiently reduce the proportion of high-abundance proteins and enrich low-abundance proteins in serum. Here, we synthesized a silica-coated iron oxide nanoparticle and developed a highly efficient and reproducible protein corona (PC)-based proteomic analysis strategy to improve the range of serum proteomic analysis. We identified 1,070 proteins with a median coefficient of variation of 12.56% using PC-based proteomic analysis, which was twice the number of proteins identified by direct digestion. There were also more biological processes enriched with these proteins. We applied this strategy to identify more pharmacodynamic biomarkers on collagen-induced arthritis (CIA) rat model treated with methotrexate (MTX). The bioinformatic results indicated that 485 differentially expressed proteins (DEPs) were found in CIA rats, of which 323 DEPs recovered to near normal levels after treatment with MTX. This strategy can not only help enhance our understanding of the mechanisms of disease and drug action through serum proteomics studies, but also provide more pharmacodynamic biomarkers for disease prediction, diagnosis, and treatment.

Transcriptome and Lipid Metabolomics-Based Discovery: Glycyrrhizic Acid Alleviates Tripterygium Glycoside Tablet-Induced Acute Liver Injury by Regulating the Activities of CYP and the Metabolism of Phosphoglycerides.

Background: Glycyrrhizic acid (GA) has been reported to be liver protective; however, the characters and underlying mechanisms of GA against tripterygium glycoside tablet (TGT)-induced acute liver injury remain unelucidated. Hypothesis/Purpose: We assumed that GA could relieve TGT-induced acute liver injury by regulating liver function-related genes and lipid metabolites. Study Design: TGT-induced acute liver injury models were constructed in vivo and in vitro. Then the liver protective effect and mechanisms of GA were investigated by a combination of transcriptome, lipid metabolomics, and experimental validation. Methods: Intraperitoneal injection of GA was given in advance for six successive days. Then, the TGT-induced acute liver injury model was constructed by a single oral administration of TGT at 270 mg/kg, except for the normal group. All animals were sacrificed 18 h later. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) were quantified. Liver tissues were used to observe pathological changes through hematoxylin-eosin (HE) staining and selected for transcriptome and metabolome sequencing. The underlying mechanisms were analyzed and further validated both in vivo and in vitro. Results: Pre-administration of GA markedly decreased the serum concentrations of AST, ALT, ALP, and TBIL but increased those of SOD and GSH-Px, improving the liver morphology of mice with TGT-induced acute liver injury. In addition, GA significantly increased the gene levels of Cyp2b13, Cyp2c69, Cyp3a16, Cyp3a44, Fmo3, and Nipal1. Differentially accumulated metabolites were screened and classified as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The in vitro results indicated that pre-administration of GA markedly alleviated the inhibitory effect of TGT on BRL-3A activity. Conclusion: This study combined transcriptome, lipid metabolomics, and experimental validation to offer convincing evidence that GA alleviates TGT-induced acute liver injury partially by regulating the activities of CYP and the metabolism of PC and PE.

Calcium ion-exchange cross-linked porous starch microparticles with improved hemostatic properties.

Starch hemostatic agents have been clinically used in surgical hemostasis in recent years. Calcium ion (Ca2+)-exchange cross-linked porous starch microparticles (Ca2+CPSMs) were prepared as a new hemostatic agent to enhance the hemostatic efficacy. A series of Ca2+CPSMs with varying Ca2+ contents were prepared and characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), scanning electron microscope (SEM), and ion content analysis. The XPS and FT-IR results indicated that the surface of Ca2+CPSMs was modified by Ca2+, which might form coordination bonds with oxygen atoms of starch molecules. Ca2+CPSMs revealed a porous surface structure and a lower crystallinity degree according to SEM and XRD, which facilitated the phosphate buffer saline (PBS) uptake rate and enzymatic degradation in vitro. The fast release of Ca2+ from Ca2+CPSMs accelerated the whole blood clotting rate, shortened the activated partial thromboplastin time, and promoted platelet adhesion. The physical hemostatic mechanism benefited from the rapid PBS uptake capacity and porous surface structure of Ca2+CPSMs, in addition to the chemical activation of coagulation process by Ca2+, thus achieving a significant hemorrhage control in the mouse tail amputation model.

Pharmacokinetic Properties of Arsenic Species after Intravenous and Intragastrical Administration of Arsenic Trioxide Solution in Cynomolgus Macaques Using HPLC-ICP-MS.

A rapid and sensitive method was established for arsenic (As) speciation based on high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method was validated for the quantification of four arsenic species, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in cynomolgus macaque plasma. Separation was achieved in just 3.7 min with an alkyl reverse phase column and highly aqueous mobile phase containing 20 mM citric acid and 5 mM sodium hexanesulfonate (pH = 4.3). The calibration curves were linear over the range of 5⁻500 ng·mL-1 (measured as As), with r > 0.99. The above method was validated for selectivity, precision, accuracy, matrix effect, recovery, carryover effect and stability, and applied in a comparative pharmacokinetic study of arsenic species in cynomolgus macaque samples following intravenous and intragastrical administration of arsenic trioxide solution (0.80 mg·kg-1; 0.61 mg·kg-1 of arsenic); in addition, the absolute oral bioavailability of the active ingredient AsIII of arsenic trioxide in cynomolgus macaque samples was derived as 60.9 ± 16.1%.

[Quality of realgar and its influencing factors based on toxicity].

The results of a toxicity analysis showed differences from those of the existing experimental data. Therefore, HPLC-ICP-MS was used to analyze the soluble arsenic content at different valences in realgar prepared with water grind processing, which were collected from 3 companies. The results showed that the free arsenic of the 3 companies did not exceed the limit of Chinese Pharmacopoeia. However, if the free arsenic was calculated based on the total value of As(Ⅲ) + As(Ⅴ), free arsenic of 1 company exceeded the limit of Chinese Pharmacopoeia. The method of determining free arsenic in Chinese Pharmacopoeia. was ancient Cai's arsenic detection method, which had a certain limitation and failed to effectively avoid the toxicity of remaining arsenics except for trivalent arsenic. Then, we examined the effects of water and temperature on the content and form of soluble arsenic in realgar. The results showed that the content of soluble arsenic increased with the rise of water content, and the form of soluble arsenic did not change, there were only As (Ⅲ) and As (Ⅴ); With the simple temperature factor, there was an increasing trend in the content of soluble arsenic in the samples, the maximum increment was As (Ⅲ) 2.489 mg•g⁻¹ and As (Ⅴ) 0.546 mg•g⁻¹; When water and temperature played an synergistic effect, the increase of soluble arsenic in the samples significantly changed, the maximum increment was As (Ⅲ) 23.690 mg•g⁻¹, As (Ⅴ) 0.468 mg•g⁻¹, respectively. Through comprehensive analysis, we believed that the quality of realgar was susceptible to water content and temperature. Both of the single effect of water content and the synergistic effect of water and temperature can significantly change the content of soluble arsenic in realgar, and the water content was a high-risk factor. In the current Chinese Pharmacopoeia 2015 version, the free arsenic detection method had limitations, hence new techniques shall be introduced; At the same time, realgar does not have a water content inspection item in the current pharmacopoeia, which shall be added. However, due to the limit of water content, more in-depth studies are required.