In Vitro Ciclopirox Glucuronidation in Liver Microsomes from Humans and Various Experimental Animals.

BACKGROUND AND OBJECTIVE: Ciclopirox is a widely used antifungal drug, redisposition of which has drawn increasing attentions due to multiple promising activities. The drug undergoes extensive glucuronidation, which acts as a major obstacle in the ongoing novel application and still remains poorly understood. The current study aims to phenotype ciclopirox glucuronidation pathway and as well to decipher the related species differences. METHODS: Ciclopirox glucuronidation was investigated in liver microsomes from humans (HLM) and various experimental animals. Assays with recombinant uridine diphosphate glucuronosyltransferases (UGTs), enzyme kinetic analyses and selective inhibitors were used to determine the role of individual UGTs in ciclopirox glucuronidation. RESULTS: HLM is highly active in ciclopirox glucuronidation with Michaelis-Menten constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint) values of 139 µM, 7.89 nmol/min/mg, and 56 µL/min/mg, respectively. UGT1A9 displays by far the highest activity, whereas several other isoforms (UGT1A6, UGT1A7, and UGT1A8) catalyze formation of traced glucuronides. Further kinetic analysis demonstrates that UGT1A9 has a closed Km value (167 µM) to HLM. UGT1A9 selective inhibitor (magnolol) can potently inhibit ciclopirox glucuronidation in HLM with the IC50 value of 0.12 µM. The reaction displays remarkable differences across liver microsomes from mice, rats, cynomolgus monkey, minipig, and beagle dog, with the CLint values in the range of 26-369 µL/min/mg. In addition, ciclopirox glucuronidation activities of experimental animals' liver microsomes were less sensitive to magnolol than that of HLM. CONCLUSIONS: Ciclopirox glucuronidation displays remarkable species differences with UGT1A9 as a dominant contributor in humans. It is suggested that the pharmacological or toxicological effects of ciclopirox may be UGT1A9 and species dependent.

A Novel Fluorescence-Based Microplate Assay for High-Throughput Screening of hSULT1As Inhibitors.

Human sulfotransferase 1As (hSULT1As) play a crucial role in the metabolic clearance and detoxification of a diverse range of endogenous and exogenous substances, as well as in the bioactivation of some procarcinogens and promutagens. Pharmacological inhibiting hSULT1As activities may enhance the in vivo effects of most hSULT1As drug substrates and offer protective strategies against the hSULT1As-mediated bioactivation of procarcinogens. To date, a fluorescence-based high-throughput assay for the efficient screening of hSULT1As inhibitors has not yet been reported. In this work, a fluorogenic substrate (HN-241) for hSULT1As was developed through scaffold-seeking and structure-guided molecular optimization. Under physiological conditions, HN-241 could be readily sulfated by hSULT1As to form HN-241 sulfate, which emitted brightly fluorescent signals around 450 nm. HN-241 was then used for establishing a novel fluorescence-based microplate assay, which strongly facilitated the high-throughput screening of hSULT1As inhibitors. Following the screening of an in-house natural product library, several polyphenolic compounds were identified with anti-hSULT1As activity, while pectolinarigenin and hinokiflavone were identified as potent inhibitors against three hSULT1A isozymes. Collectively, a novel fluorescence-based microplate assay was developed for the high-throughput screening and characterization of hSULT1As inhibitors, which offered an efficient and facile approach for identifying potent hSULT1As inhibitors from compound libraries.

Discovery of baicalein derivatives as novel inhibitors against human pancreatic lipase: Structure-activity relationships and inhibitory mechanisms.

Human pancreatic lipase (hPL) is a vital digestive enzyme responsible for breaking down dietary fats in humans, inhibiting hPL is a feasible strategy for preventing and treating obesity. This study aims to investigate the structure-activity relationships (SARs) of flavonoids as hPL inhibitors, and to find potent hPL inhibitors from natural and synthetic flavonoids. In this work, the anti-hPL effects of forty-nine structurally diverse naturally occurring flavonoids were assessed and the SARs were summarized. The results demonstrated that the pyrogallol group on the A ring was a key moiety for hPL inhibition. Subsequently, a series of baicalein derivatives were synthesized, while 4'-amino baicalein (ABA) and 4'-pyrrolidine baicalein (PBA) were identified as novel potent hPL inhibitors (IC50 < 1 µM). Further investigations showed that scutellarein, ABA and PBA potently inhibited hPL in a non-competitive manner (Ki < 1 µM). Among all tested flavonoids, PBA showed the most potent anti-hPL effect in vitro, while this agent also exhibited favorable safety profiles, unique tissue distribution (high exposure level to intestinal system but low exposure levels to deep organs) and impressive in vivo effects for lowering blood triglyceride levels in mice. Collectively, this work uncovers the SARs of flavonoids against hPL, while a newly synthetic flavonoid (PBA) emerges as a potent hPL inhibitor with favorable safety profiles and impressive anti-hPL effects in vivo.

RNA binding protein RALY facilitates colorectal cancer metastasis via enhancing exosome biogenesis in m6A dependent manner.

Tumor metastasis is the leading cause of cancer-related death in patients with colorectal cancer (CRC). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins, involved in the tumorigenesis and metastasis of various cancers. However, the molecular mechanisms of hnRNPs in CRC metastasis remain unclear. This study aims to uncover the pivotal roles and molecular mechanisms of hnRNPs in CRC metastasis. Clinical database analysis suggested that the expression of hnRNP-Associated with Lethal Yellow (RALY, an important member of hnRNPs) was strongly correlated with the aggressiveness and survival of CRC patients. Gain- and loss-of-function studies demonstrated that RALY promotes the production of exosomes by increasing the formation of multivesicular bodies (MVBs) and enhancing the fusion of MVBs with the plasma membrane. Notably, RALY directly interacts with phospholipase D2 (PLD2) to enable exosome biogenesis, and cooperates with RBM15b to control PLD2 mRNA stability in an m6A-dependent manner. RALY-mediated exosome secretion activates pro-tumor macrophages and further facilitates CRC metastasis, while rescue experiments in vivo further confirmed that RALY-mediated exosome biogenesis facilitates CRC metastasis. Collectively, our findings demonstrate that RALY promotes exosome biogenesis and facilitates colorectal cancer metastasis by upregulating PLD2 and enhancing exosome production in an m6A-dependent manner, suggesting potential therapeutic strategies for combating CRC metastasis.

Design, synthesis and structure-activity relationship of 1,8-naphthalimide derivatives as highly potent hCYP1B1 inhibitors.

Human cytochrome P450 1B1 enzyme (hCYP1B1), a member of hCYP1 subfamily, plays a crucial role in multiple diseases by participating in many metabolic pathways. Although a suite of potent hCYP1B1 inhibitors have been previously reported, most of them also act as aryl hydrocarbon receptor (AhR) agonists that can up-regulate the expression of hCYP1B1 and then counteract their inhibitory potential in living systems. This study aimed to develop novel efficacious hCYP1B1 inhibitors that worked well in living cells but without AhR agonist effects. For these purposes, a series of 1,8-naphthalimide derivatives were designed and synthesized, and their structure-activity relationships (SAR) as hCYP1B1 inhibitors were analyzed. Following three rounds SAR studies, several potent hCYP1B1 inhibitors were discovered, among which compound 3n was selected for further investigations owing to its extremely potent anti-hCYP1B1 activity (IC50 = 0.040 nM) and its blocking AhR transcription activity in living cells. Inhibition kinetic analyses showed that 3n potently inhibited hCYP1B1 via a mix inhibition manner, showing a Ki value of 21.71 pM. Docking simulations suggested that introducing a pyrimidine moiety to the hit compound (1d) facilitated 3n to form two strong interactions with hCYP1B1/heme, viz., the C-Br⋯π halogen bond and the N-Fe coordination bond. Further investigations demonstrated that 3n (5 µM) could significantly reverse the paclitaxel (PTX) resistance in H460/PTX cells, evidenced by the dramatically reduced IC50 values, from 632.6 nM (PTX alone) to 100.8 nM (PTX plus 3n). Collectively, this study devised a highly potent hCYP1B1 inhibitor (3n) without AhR agonist effect, which offered a promising drug candidate for overcoming hCYP1B1-associated drug resistance.

Bioactive components and mechanisms of Pu-erh tea in improving levodopa metabolism in rats through COMT inhibition.

Catechol-O-methyltransferase (COMT) plays a central role in the metabolic inactivation of endogenous neurotransmitters and xenobiotic drugs and hormones having catecholic structures. Its inhibitors are used in clinical practice to treat Parkinson's disease. In this study, a fluorescence-based visualization inhibitor screening method was developed to assess the inhibition activity on COMT both in vitro and in living cells. Following the screening of 94 natural products, Pu-erh tea extract exhibited the most potent inhibitory effect on COMT with an IC50 value of 0.34 µg mL-1. In vivo experiments revealed that Pu-erh tea extract substantially hindered COMT-mediated levodopa metabolism in rats, resulting in a significant increase in levodopa levels and a notable decrease in 3-O-methyldopa in plasma. Subsequently, the chemical components of Pu-erh tea were analyzed using UHPLC-Q-Exactive Orbitrap HRMS, identifying 24 major components. Among them, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, and catechin gallate exhibited potent inhibition of COMT activity with IC50 values from 93.7 nM to 125.8 nM and were the main bioactive constituents in Pu-erh tea responsible for its COMT inhibition effect. Inhibition kinetics analyses and docking simulations revealed that these compounds competitively inhibit COMT-mediated O-methylation at the catechol site. Overall, this study not only explained how Pu-erh tea catechins inhibit COMT, suggesting Pu-erh tea as a potential dietary intervention for Parkinson's disease, but also introduced a new strategy for discovering COMT inhibitors more effectively.

Pectolinarigenin ameliorates osteoporosis via enhancing Wnt signaling cascade in PPARß-dependent manner.

BACKGROUND: Osteoporosis is a prevalent metabolic bone disease in older adults. Peroxisome proliferator-activated receptor ß (PPARß), the most abundant PPAR isotype expressed in bone tissues, plays a critical role in regulating the energy metabolism of osteoblasts. However, the botanical compounds targeting PPARß for the treatment of osteoporosis remain largely unexplored. PURPOSE: To discover a potent PPARß agonist from botanical compounds, as well as to investigate the anti-osteoporosis effects and to elucidate the underlying mechanisms of the newly identified PPARß agonist. METHODS: The PPARß agonist effects of botanical compounds were screened by an in vitro luciferase reporter gene assay. The PPARß agonist effects of pectolinarigenin (PEC) in bone marrow mesenchymal stromal cells (BMSCs) were validated by Western blotting. RNA-seq transcriptome analyses were conducted to reveal the underlying osteoporosis mechanisms of PEC in BMSCs. The PPARß antagonist (GSK0660) and Wnt signaling inhibitor (XAV969) were used to explore the role of the PPARß and Wnt signaling cascade in the anti-osteoporosis effects of PEC. PEC or the PEG-PLGA nanoparticles of PEC (PEC-NP) were intraperitoneally administrated in both wild-type mice and ovariectomy-induced osteoporosis mice to examine its anti-osteoporotic effects in vivo. RESULTS: PEC, a newly identified naturally occurring PPARß agonist, significantly promotes osteogenic differentiation and up-regulates the osteogenic differentiation-related genes (Runx2, Osterix, and Bmp2) in BMSCs. RNA sequencing and functional gene enrichment analysis suggested that PEC could activate osteogenic-related signaling pathways, including Wnt and PPAR signaling pathways. Further investigations suggested that PEC could enhance Wnt/ß-catenin signaling in a PPARß-dependent manner in BMSCs. Animal tests showed that PEC-NP promoted bone mass and density, increased the bone cell matrix protein, and accelerated bone formation in wild-type mice, while PEC-NP also played a preventive role in ovariectomy-induced osteoporosis mice via maintaining the expression level of bone cell matrix protein, balancing the rate of bone formation, and slowing down bone loss. Additionally, PEC-NP did not cause any organ injury and body weight loss after long-term use (11 weeks). CONCLUSION: PEC significantly promotes bone formation and reduces bone loss in both BMSCs and ovariectomy-induced osteoporosis mice via enhancing the Wnt signaling cascade in a PPARß-dependent manner, providing a new alternative therapy for preventing estrogen deficiency-induced osteoporotic diseases.

A Carbon-Caged Rhodamine Generating Nitrosoperoxycarbonate for Photoimmunotherapy.

Photoimmunotherapy is a promising cancer treatment modality. While potent 1-e- oxidative species are known to induce immunogenic cell death (ICD), they are also associated with unspecific oxidation and collateral tissue damage. This difficulty may be addressed by post-generation radical reinforcement. Namely, non-oxidative radicals are first generated and subsequently activated into powerful oxidative radicals to induce ICD. Here, we developed a photo-triggered molecular donor (NPCD565) of nitrosoperoxycarbonate (ONOOCO2 -), the first of its class to our knowledge, and further evaluated its feasibility for immunotherapy. Upon irradiation of NPCD565 by light within a broad spectral region from ultraviolet to red, ONOOCO2 - is released along with a bright rhodamine dye (RD565), whose fluorescence is a reliable and convenient build-in reporter for the localization, kinetics, and dose of ONOOCO2 - generation. Upon photolysis of NPCD565 in 4T1 cells, damage-associated molecular patterns (DAMPs) indicative of ICD were observed and confirmed to exhibit immunogenicity by induced maturation of dendritic cells. In vivo studies with a bilateral tumor-bearing mouse model showcased the potent tumor-killing capability of NPCD565 of the primary tumors and growth suppression of the distant tumors. This work unveils the potent immunogenicity of ONOOCO2 -, and its donor (NPCD565) has broad potential for photo-immunotherapy of cancer.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. AIM OF THE STUDY: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. MATERIALS AND METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-ß-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 µM, 9.05 µM, 3.87 µM, and 7.00 µM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. CONCLUSION: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Discovery of a botanical compound as a broad-spectrum inhibitor against gut microbial ß-glucuronidases from the Tibetan medicine Rhodiola crenulata.

Gut microbial ß-glucuronidases (gmß-GUS) played crucial roles in regulating a variety of endogenous substances and xenobiotics on the circulating level, thus had been recognized as key modulators of drug toxicity and human diseases. Inhibition or inactivation of gmß-GUS enzymes has become a promising therapeutic strategy to alleviate drug-induced intestinal toxicity. Herein, the Rhodiola crenulata extract (RCE) was found with potent and broad-spectrum inhibition on multiple gmß-GUS enzymes. Subsequently, the anti-gmß-GUS activities of the major constituents in RCE were tested and the results showed that 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) acted as a strong and broad-spectrum inhibitor on multiple gmß-GUS (including EcGUS, CpGUS, SaGUS, and EeGUS). Inhibition kinetic assays demonstrated that PGG effectively inhibited four gmß-GUS in a non-competitive manner, with the Ki values ranging from 0.12 µM to 1.29 µM. Docking simulations showed that PGG could tightly bound to the non-catalytic sites of various gmß-GUS, mainly via hydrogen bonding and aromatic interactions. It was also found that PGG could strongly inhibit the total gmß-GUS activity in mice feces, with the IC50 value of 1.24 µM. Collectively, our findings revealed that RCE and its constituent PGG could strongly inhibit multiple gmß-GUS enzymes, suggesting that RCE and PGG could be used for alleviating gmß-GUS associated enterotoxicity.

Xanthohumol ameliorates drug-induced hepatic ferroptosis via activating Nrf2/xCT/GPX4 signaling pathway.

BACKGROUND: As a canonical iron-dependent form of regulated cell death (RCD), ferroptosis plays a crucial role in chemical-induced liver injuries. Previous studies have demonstrated that xanthohumol (Xh), a natural prenylflavonoid isolated from hops, exhibits anti-inflammatory, anti-antioxidative and hepatoprotective properties. However, the regulatory effects of Xh on hepatic ferroptosis and the underlying mechanism have not yet been fully elucidated. PURPOSE: To investigate the hepatoprotective effects of Xh against drug-induced liver injury (DILI) and the regulatory effects of Xh on hepatic ferroptosis, as well as to reveal the underlying molecular mechanisms. METHODS/STUDY DESIGN: The hepatoprotective benefits of Xh were investigated in APAP-induced liver injury (AILI) mice and HepaRG cells. Xh was administered intraperitoneally to assess its in vivo effects. Histological and biochemical studies were carried out to evaluate liver damage. A series of ferroptosis-related markers, including intracellular Fe2+ levels, ROS and GSH levels, the levels of MDA, LPO and 4-HNE, as well as the expression levels of ferroptosis-related proteins and modulators were quantified both in vivo and in vitro. The modified peptides of Keap1 by Xh were characterized utilizing nano LC-MS/MS. RESULTS: Xh remarkably suppresses hepatic ferroptosis and ameliorates AILI both in vitro and in vivo, via suppressing Fe2+ accumulation, ROS formation, MDA generation and GSH depletion, these observations could be considerably mitigated by the ferroptosis inhibitor ferrostatin-1 (Fer-1). Mechanistically, Xh could significantly activate the Nrf2/xCT/GPX4 signaling pathway to counteract AILI-induced hepatocyte ferroptosis. Further investigations showed that Xh could covalently modify three functional cysteine residues (cys151, 273, 288) of Keap1, which in turn, reduced the ubiquitination rates of Nrf2 and prolonged its degradation half-life. CONCLUSIONS: Xh evidently suppresses hepatic ferroptosis and ameliorates AILI via covalent modifying three key cysteines of Keap1 and activating Nrf2/xCT/GPX4 signaling pathway.

CYP1A inhibitors: Recent progress, current challenges, and future perspectives.

Mammalian cytochrome P450 1A (CYP1A) are key phase I xenobiotic-metabolizing enzymes that play a distinctive role in metabolic activation or metabolic clearance of a variety of procarcinogens, drugs, and endogenous substances. Human CYP1A subfamily contains two members (hCYP1A1 and hCYP1A2), which are known to catalyze the oxidative activation of some environmental procarcinogens into carcinogenic species. Increasing evidence has demonstrated that CYP1A inhibitor therapies are promising strategies for cancer chemoprevention or overcoming CYP1A-associated drug toxicity and resistance. Herein, we reviewed recent advances in the discovery and characterization of hCYP1A inhibitors, from the discovery approaches to structural features and biomedical applications of hCYP1A inhibitors. The inhibition potentials, inhibition modes, and inhibition constants of all reported hCYP1A inhibitors are comprehensively summarized. Meanwhile, the structural features and structure-activity relationships of different classes of hCYP1A1 and hCYP1A2 inhibitors are analyzed and discussed in depth. Furthermore, the major challenges and future directions for this field are presented and highlighted. Collectively, the information and knowledge presented here will strongly facilitate the researchers to discover and develop more efficacious CYP1A inhibitors for specific purposes, such as chemo-preventive agents or as tool molecules in hCYP1A-related fundamental studies.

An Uncertainty-Guided Deep Learning Method Facilitates Rapid Screening of CYP3A4 Inhibitors.

Cytochrome P450 3A4 (CYP3A4), a prominent member of the P450 enzyme superfamily, plays a crucial role in metabolizing various xenobiotics, including over 50% of clinically significant drugs. Evaluating CYP3A4 inhibition before drug approval is essential to avoiding potentially harmful pharmacokinetic drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Despite the development of several CYP inhibitor prediction models, the primary approach for screening CYP inhibitors still relies on experimental methods. This might stem from the limitations of existing models, which only provide deterministic classification outcomes instead of precise inhibition intensity (e.g., IC50) and often suffer from inadequate prediction reliability. To address this challenge, we propose an uncertainty-guided regression model to accurately predict the IC50 values of anti-CYP3A4 activities. First, a comprehensive data set of CYP3A4 inhibitors was compiled, consisting of 27,045 compounds with classification labels, including 4395 compounds with explicit IC50 values. Second, by integrating the predictions of the classification model trained on a larger data set and introducing an evidential uncertainty method to rank prediction confidence, we obtained a high-precision and reliable regression model. Finally, we use the evidential uncertainty values as a trustworthy indicator to perform a virtual screening of an in-house compound set. The in vitro experiment results revealed that this new indicator significantly improved the hit ratio and reduced false positives among the top-ranked compounds. Specifically, among the top 20 compounds ranked with uncertainty, 15 compounds were identified as novel CYP3A4 inhibitors, and three of them exhibited activities less than 1 µM. In summary, our findings highlight the effectiveness of incorporating uncertainty in compound screening, providing a promising strategy for drug discovery and development.

Acyl-caged rhodamines: photo-controlled and self-calibrated generation of acetyl radicals for neural function recovery in early AD mice.

The biological function of radicals is a broad continuum from signaling to killing. Yet, biomedical exploitation of radicals is largely restricted to the theme of healing-by-killing. To explore their potential in healing-by-signaling, robust radical generation methods are warranted. Acyl radicals are endogenous, exhibit facile chemistry and elicit matrix-dependent biological outcomes. Their implications in health and disease remain untapped, primarily due to the lack of a robust generation method with spatiotemporal specificity. Fusing the Norrish chemistry into the xanthene scaffold, we developed a novel general and modular molecular design strategy for photo-triggered generation of acyl radicals, i.e., acyl-caged rhodamine (ACR). A notable feature of ACR is the simultaneous release of a fluorescent probe for cell redox homeostasis allowing real-time monitoring of the biological outcome of acyl radicals. With a donor of the endogenous acetyl radical (ACR575a), we showcased its capability in precise and continuous modulation of the cell redox homeostasis from signaling to stress, and induction of a local oxidative burst to promote differentiation of neural stem cells (NSCs). Upon intracerebral-injection of ACR575a and subsequent fiber-optical activation, early AD mice exhibited enhanced differentiation of NSCs toward neurons, reduced formation of Aß plaques, and significantly improved cognitive abilities, including learning and memory.

Discovery of the covalent SARS-CoV-2 Mpro inhibitors from antiviral herbs via integrating target-based high-throughput screening and chemoproteomic approaches.

The main proteases (Mpro ) are highly conserved cysteine-rich proteins that can be covalently modified by numerous natural and synthetic compounds. Herein, we constructed an integrative approach to efficiently discover covalent inhibitors of Mpro from complex herbal matrices. This work begins with biological screening of 60 clinically used antiviral herbal medicines, among which Lonicera japonica Flos (LJF) demonstrated the strongest anti-Mpro effect (IC50 = 37.82 µg/mL). Mass spectrometry (MS)-based chemical analysis and chemoproteomic profiling revealed that LJF extract contains at least 50 constituents, of which 22 exhibited the capability to covalently modify Mpro . We subsequently verified the anti-Mpro effects of these covalent binders. Gallic acid and quercetin were found to potently inhibit severe acute respiratory syndrome coronavirus 2 Mpro in dose- and time- dependent manners, with the IC50 values below 10 µM. The inactivation kinetics, binding affinity and binding mode of gallic acid and quercetin were further characterized by fluorescence resonance energy transfer, surface plasmon resonance, and covalent docking simulations. Overall, this study established a practical approach for efficiently discovering the covalent inhibitors of Mpro from herbal medicines by integrating target-based high-throughput screening and MS-based assays, which would greatly facilitate the discovery of key antiviral constituents from medicinal plants.

GeoDILI: A Robust and Interpretable Model for Drug-Induced Liver Injury Prediction Using Graph Neural Network-Based Molecular Geometric Representation.

Drug-induced liver injury (DILI) is a significant cause of drug failure and withdrawal due to liver damage. Accurate prediction of hepatotoxic compounds is crucial for safe drug development. Several DILI prediction models have been published, but they are built on different data sets, making it difficult to compare model performance. Moreover, most existing models are based on molecular fingerprints or descriptors, neglecting molecular geometric properties and lacking interpretability. To address these limitations, we developed GeoDILI, an interpretable graph neural network that uses a molecular geometric representation. First, we utilized a geometry-based pretrained molecular representation and optimized it on the DILI data set to improve predictive performance. Second, we leveraged gradient information to obtain high-precision atomic-level weights and deduce the dominant substructure. We benchmarked GeoDILI against recently published DILI prediction models, as well as popular GNN models and fingerprint-based machine learning models using the same data set, showing superior predictive performance of our proposed model. We applied the interpretable method in the DILI data set and derived seven precise and mechanistically elucidated structural alerts. Overall, GeoDILI provides a promising approach for accurate and interpretable DILI prediction with potential applications in drug discovery and safety assessment. The data and source code are available at GitHub repository (https://github.com/CSU-QJY/GeoDILI).

Rationally Engineered hCES2A Near-Infrared Fluorogenic Substrate for Functional Imaging and High-Throughput Inhibitor Screening.

Human carboxylesterase 2A (hCES2A) is an important endoplasmic reticulum (ER)-resident enzyme that is responsible for the hydrolytic metabolism or activation of numerous ester-bearing drugs and environmental toxins. The previously reported hCES2A fluorogenic substrates suffer from limited emission wavelength, low specificity, and poor localization accuracy, thereby greatly limiting the in situ functional imaging of hCES2A and drug discovery. Herein, a rational ligand design strategy was adopted to construct a highly specific near-infrared (NIR) substrate for hCES2A. Following scaffold screening and recognition group optimization, HTCF was identified as a desirable NIR fluorophore with excellent photophysical properties and high ER accumulation ability, while several HTCF esters held a high potential to be good hCES2A substrates. Further investigations revealed that TP-HTCF (the tert-pentyl ester of HTCF) was an ideal substrate with ultrahigh sensitivity, excellent specificity, and a substantial signal-to-noise ratio. Upon the addition of hCES2A, TP-HTCF could be rapidly hydrolyzed to release HTCF, a chemically stable product that emitted bright fluorescent signals at around 670 nm. A TP-HTCF-based biochemical assay was then established for the high-throughput screening of potent and cell-active hCES2A inhibitors from an in-house compound library. Furthermore, TP-HTCF displayed high imaging resolution for imaging hCES2A in living cells as well as mouse liver slices and tumor-xenograft mice. Collectively, this study demonstrates a rational strategy for developing highly specific fluorogenic substrates for an ER-resident target enzyme, while TP-HTCF can act as a practical tool for sensing hCES2A in living systems.

Uncovering the anti-obesity constituents in Ginkgo biloba extract and deciphering their synergistic effects.

Obesity has been recognized as a key risk factor for multiple metabolic disorders, including diabetes, cardiovascular diseases and many types of cancer. Herbal medicines have been frequently used for preventing and treating obesity in many countries, but in most cases, the key anti-obesity constituents in herbs and their anti-obesity mechanisms are poorly understood. This study demonstrated a case study for uncovering the anti-obesity constituents in an anti-obesity herbal medicine (Ginkgo biloba extract) and deciphering their synergistic effects via targeting human pancreatic lipase (hPL). Following screening the anti-hPL effects of eighty herbal medicines, Ginkgo biloba extract (GBE50) was found with the most potent anti-hPL activity. Global chemical profiling of herbal constituents coupling with hPL inhibition assay revealed that the bioflavonoids and several flavonoids in GBE50 were key anti-hPL constituents. Among all tested thirty-eight constituents, sciadopitysin, bilobetin, quercetin, isoginkgetin, and ginkgetin showed potent anti-hPL effects (IC50 values <2.5 µM). Inhibition kinetic analyses suggested that sciadopitysin, bilobetin, quercetin, isoginkgetin, and ginkgetin acted as non-competitive inhibitors of hPL, with the Ki values were <2 µM. Docking simulations revealed that four bioflavonoids (sciadopitysin, bilobetin, isoginkgetin, and ginkgetin) could tightly bind on hPL at cavity 2, which it is different from the binding cavity of quercetin on hPL. Further investigations demonstrated that the combinations of quercetin and one bioflavonoid-type hPL inhibitor (sciadopitysin or bilobetin) showed synergistic anti-hPL effects, suggesting that the multi-components in GBE50 may generate more potent anti-hPL effect. Collectively, our findings uncovered the anti-obesity constituents in GBE50, and explored their anti-hPL mechanisms as well as synergistic effects at molecular levels, which will be very helpful for further understanding the anti-obesity mechanisms of Ginkgo biloba.

Air-ventilated normothermic machine perfusion alleviates hepatic injury from DCD rat through CYP1A2.

BACKGROUND: Normothermic machine perfusion (NMP) could provide protection to organs from donation after circulatory death (DCD) before transplantation, and its molecular mechanism remains unclear. Our previous study discovered that the air-ventilated NMP confers a better DCD liver recovery than oxygen-ventilated NMP. The purpose in the current study was to investigate the protective mechanism of air-ventilated NMP in a rat model of DCD liver by metabolomics, and to select biomarker to predict liver function recovery. MATERIALS AND METHODS: Peroxisome proliferator activator receptor-α (PPARα) agonist or antagonist was administered via the perfusion circuit in the air-ventilated NMP. Perfusate samples were taken for measurements of aminotransferases using standard biochemical methods, tumor necrosis factor-alpha and interleukin-6. Liver biopsies were allocated for detection of metabolomics, PPARα and cytochrome P450 1A2 (CYP1A2). RESULTS: Metabolomics analysis revealed the significant increased γ-linolenic acid and decreased adrenic acid during the air-ventilated NMP, indicating linoleic acid metabolism pathway was associated with a better DCD liver recovery; as a major enzyme involved in linolenic acid metabolism, CYP1A2 was found correlated with a less inflammation and better liver function with the air-ventilated NMP; PPARα agonist could increase CYP1A2 expression and activity, decrease inflammation response, and improve liver function with the air-ventilated NMP, while PPARα antagonist played the opposite. CONCLUSION: Air-ventilated NMP confers a better liver recovery from DCD rats through the activated linoleic acid metabolism and CYP1A2 upregulation; CYP1A2 expression and activity might function as biomarker to predict DCD liver function recovery with NMP.

Design, synthesis and biological evaluation of chalcone derivatives as potent and orally active hCYP3A4 inhibitors.

Human cytochrome P450 3A4 (hCYP3A4), one of the most important drug-metabolizing enzymes, catalyze the metabolic clearance of ∼50% therapeutic drugs. CYP3A4 inhibitors have been used for improving the in vivo efficacy of hCYP3A4-substrate drugs. However, most of existing hCYP3A4 inhibitors may trigger serious adverse effects or undesirable effects on endogenous metabolism. This study aimed to discover potent and orally active hCYP3A4 inhibitors from chalcone derivatives and to test their anti-hCYP3A4 effects both in vitro and in vivo. Following three rounds of screening and structural optimization, the isoquinoline chalcones were found with excellently anti-hCYP3A4 effects. SAR studies showed that introducing an isoquinoline ring on the A-ring significantly enhanced anti-CYP3A4 effect, generating A10 (IC50 = 102.10 nM) as a promising lead compound. The 2nd round of SAR studies showed that introducing a substituent group at the para position of the carbonyl group on B-ring strongly improved the anti-CYP3A4 effect. As a result, C6 was identified as the most potent hCYP3A4 inhibitor (IC50 = 43.93 nM) in human liver microsomes (HLMs). C6 also displayed potent anti-hCYP3A4 effect in living cells (IC50 = 153.00 nM), which was superior to the positive inhibitor ketoconazole (IC50 = 251.00 nM). Mechanistic studies revealed that C6 could potently inhibit CYP3A4-catalyzed N-ethyl-1,8-naphthalimide (NEN) hydroxylation in a competitive manner (Ki = 30.00 nM). Moreover, C6 exhibited suitable metabolic stability in HLMs and showed good safety profiles in mice. In vivo tests demonstrated that C6 (100 mg/kg, orally administration) significantly increased the AUC(0-inf) of midazolam by 3.63-fold, and strongly prolonged its half-life by 1.66-fold compared with the vehicle group in mice. Collectively, our findings revealed the SARs of chalcone derivatives as hCYP3A4 inhibitors and offered several potent chalcone-type hCYP3A4 inhibitors, while C6 could serve as a good lead compound for developing novel, orally active CYP3A4 inhibitors with improved druglikeness properties.