{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T01:58:32Z","timestamp":1780106312461,"version":"3.54.0"},"reference-count":50,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,4,14]],"date-time":"2025-04-14T00:00:00Z","timestamp":1744588800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key R&amp;D Program of China","award":["2019YFC1712303"],"award-info":[{"award-number":["2019YFC1712303"]}]},{"name":"National Key R&amp;D Program of China","award":["81872834"],"award-info":[{"award-number":["81872834"]}]},{"name":"the National Natural Science Foundation of China","award":["2019YFC1712303"],"award-info":[{"award-number":["2019YFC1712303"]}]},{"name":"the National Natural Science Foundation of China","award":["81872834"],"award-info":[{"award-number":["81872834"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Chinese herbal medicines have played a significant role in the development of new and effective drugs, but how to identify the active ingredients from complex extracts of traditional Chinese herbal medicines was a research difficulty. In recent years, few studies have focused on high-efficiency identification of small-molecule inhibitors of Programmed Death Ligand 1 with lower antigenicity and flexible structure tunability. In order to identify small molecule inhibitors of PD-L1 from complex Chinese herbal extracts, this study established a protein\u2013ligand trapping system based on high-performance liquid chromatography coupled with a photo-diode array detector, ion trap\/quadrupole time-of-flight tandem mass spectrometry, and a Programmed Death Ligand 1 affinity chromatography unit (ACPD-L1-HPLC-PDA-IT-TOF (Q-TOF)-MS) to rapidly screen and identify small-molecule inhibitors of Programmed Death Ligand 1 from Toddalia asiatica (L.) Lam. Fourteen components were then identified as PD-L1 binders, and surface plasmon resonance (SPR) validation results showed that six of them\u2014magnoflorine (6), nitidine (22), chelerythrine (24), jatrorrhizine (13), toddaculin (68), and toddanol (45)\u2014displayed PD-L1 binding activity. Laser scanning confocal microscopy results demonstrated that these compounds effectively inhibited the binding of PD-1 to PD-L1 in a dose-dependent manner. Additionally, flow cytometry analysis indicated they could promote human lung cancer cell line (A549) apoptosis when co-cultured with Peripheral Blood Mononuclear Cells (PBMCs). The system\u2019s innovation lies in its first integration of dynamic protein\u2013ligand trapping with multi-dimensional validation, coupled with high-throughput screening capacity for structurally diverse natural products. This workflow overcomes traditional phytochemical screening bottlenecks by preserving native protein conformations during affinity capture while maintaining chromatographic resolution, offering a transformative template for accelerating natural product-derived immunotherapeutics through the PD-1\/PD-L1 pathway.<\/jats:p>","DOI":"10.3390\/molecules30081754","type":"journal-article","created":{"date-parts":[[2025,4,14]],"date-time":"2025-04-14T09:06:51Z","timestamp":1744621611000},"page":"1754","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A New Protein\u2013Ligand Trapping System to Rapidly Screen and Discover Small-Molecule Inhibitors of PD-L1 from Natural Products"],"prefix":"10.3390","volume":"30","author":[{"given":"Yazhuo","family":"Huang","sequence":"first","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Senfeng","family":"Sun","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Runxin","family":"Yin","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6017-338X","authenticated-orcid":false,"given":"Zongtao","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daidong","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanwan","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangyu","family":"Fu","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8369-7453","authenticated-orcid":false,"given":"Jing","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyu","family":"Lei","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mimi","family":"Sun","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Shanxi University of Chinese Medicine, Xianyang 712046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shizhong","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100191, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,14]]},"reference":[{"key":"ref_1","first-page":"1432","article-title":"Breakthrough of the year 2013. Cancer immunotherapy","volume":"342","year":"2013","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1038\/nm1100","article-title":"Cancer immunotherapy: Moving beyond current vaccines","volume":"10","author":"Rosenberg","year":"2004","journal-title":"Nat. Med."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Rosenberg, S.A. (2012). Raising the bar: The curative potential of human cancer immunotherapy. Sci. Transl. Med., 4.","DOI":"10.1126\/scitranslmed.3003634"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e16","DOI":"10.1038\/cti.2014.7","article-title":"Clinical application of genetically modified T cells in cancer therapy","volume":"3","author":"Kershaw","year":"2014","journal-title":"Clin. Transl. Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1038\/nature10673","article-title":"Cancer immunotherapy comes of age","volume":"480","author":"Mellman","year":"2011","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1350","DOI":"10.1126\/science.aar4060","article-title":"Cancer immunotherapy using checkpoint blockade","volume":"359","author":"Ribas","year":"2018","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Zou, W., Wolchok, J.D., and Chen, L. (2016). PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci. Transl. Med., 8.","DOI":"10.1126\/scitranslmed.aad7118"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/515496a","article-title":"Cancer: Antitumour immunity gets a boost","volume":"515","author":"Wolchok","year":"2014","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/nm730","article-title":"Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion","volume":"8","author":"Dong","year":"2002","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5393","DOI":"10.1158\/0008-5472.CAN-11-0993","article-title":"IL-18 induces PD-1-dependent immunosuppression in cancer","volume":"71","author":"Terme","year":"2011","journal-title":"Cancer Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1093\/intimm\/8.5.765","article-title":"Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes","volume":"8","author":"Agata","year":"1996","journal-title":"Int. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1038\/nri1349","article-title":"Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity","volume":"4","author":"Chen","year":"2004","journal-title":"Nat. Rev. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5538","DOI":"10.4049\/jimmunol.169.10.5538","article-title":"Expression of programmed death 1 ligands by murine T cells and APC","volume":"169","author":"Yamazaki","year":"2002","journal-title":"J. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1038\/85330","article-title":"PD-L2 is a second ligand for PD-1 and inhibits T cell activation","volume":"2","author":"Latchman","year":"2001","journal-title":"Nat. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1038\/nrc3239","article-title":"The blockade of immune checkpoints in cancer immunotherapy","volume":"12","author":"Pardoll","year":"2012","journal-title":"Nat. Rev. Cancer"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1163","DOI":"10.1016\/j.str.2017.06.011","article-title":"Structural biology of the immune checkpoint receptor PD-1 and its ligands PD-L1\/PD-L2","volume":"25","author":"Zak","year":"2017","journal-title":"Structure"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Alsaab, H.O., Sau, S., Alzhrani, R., Tatiparti, K., Bhise, K., Kashaw, S.K., and Iyer, A.K. (2017). PD-1 and PD-L1 Checkpoint signaling inhibition for cancer immunotherapy: Mechanism, combinations, and clinical outcome. Front. Pharmacol., 8.","DOI":"10.3389\/fphar.2017.00561"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e9","DOI":"10.1016\/S1470-2045(14)71129-5","article-title":"Nivolumab success in untreated metastatic melanoma","volume":"16","author":"Wilkinson","year":"2015","journal-title":"Lancet Oncol."},{"key":"ref_19","first-page":"605","article-title":"Clinical analysis of docetaxel combined with PD-1\/PD-L1 inhibitor in second-line treatment of advanced non-small cell lung cancer","volume":"24","author":"Chen","year":"2021","journal-title":"Chin. J. Lung Cancer"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3117","DOI":"10.1158\/1078-0432.CCR-04-2332","article-title":"Resistance of tumor interstitial pressure to the penetration of intraperitoneally delivered antibodies into metastatic ovarian tumors","volume":"11","author":"Flessner","year":"2005","journal-title":"Clin. Cancer Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1886","DOI":"10.1158\/1078-0432.CCR-16-1417","article-title":"Atezolizumab: A PD-L1-blocking antibody for bladder cancer","volume":"23","author":"Inman","year":"2017","journal-title":"Clin. Cancer Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1056\/NEJMoa1411087","article-title":"PD-1 blockade with nivolumab in relapsed or refractory Hodgkin\u2019s lymphoma","volume":"372","author":"Ansell","year":"2015","journal-title":"N. Engl. J. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2455","DOI":"10.1056\/NEJMoa1200694","article-title":"Safety and activity of anti-PD-L1 antibody in patients with advanced cancer","volume":"366","author":"Brahmer","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1080\/00325481.2019.1709344","article-title":"Immune checkpoint inhibitors and endocrine side effects, a narrative review","volume":"132","author":"Agrawal","year":"2020","journal-title":"Postgrad. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1210\/er.2018-00006","article-title":"Endocrine toxicity of cancer immunotherapy targeting immune checkpoints","volume":"40","author":"Chang","year":"2019","journal-title":"Endocr. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"13354","DOI":"10.1038\/ncomms13354","article-title":"Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy","volume":"7","author":"Lee","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"111876","DOI":"10.1016\/j.ejmech.2019.111876","article-title":"Progress in PD-1\/PD-L1 pathway inhibitors: From biomacromolecules to small molecules","volume":"186","author":"Lin","year":"2020","journal-title":"Eur. J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1407","DOI":"10.1111\/bph.13450","article-title":"Immunotherapy of cancer: From monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18","volume":"173","author":"Carvalho","year":"2016","journal-title":"Br. J. Pharmacol."},{"key":"ref_29","first-page":"9","article-title":"Treating with checkpoint inhibitors-figure $1 million per patient","volume":"8","author":"Andrews","year":"2015","journal-title":"Am. Health Drug Benefits"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Guzik, K., Tomala, M., Muszak, D., Konieczny, M., Hec, A., Baszkiewicz, U., Pustua, M., Butera, R., Dmling, A., and Holak, T.A. (2019). Development of the inhibitors that target the PD-1\/PD-L1 interaction\u2014A brief Look at progress on small molecules, peptides and macrocycles. Molecules, 24.","DOI":"10.3390\/molecules24112071"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Li, T.T., Jiang, J.W., Qie, C.X., Xuan, C.X., Hu, X.L., Liu, W.M., Chen, W.T., and Liu, J. (2021). Identification of active small-molecule modulators targeting the novel immune checkpoint VISTA. BMC Immunol., 22.","DOI":"10.1186\/s12865-021-00446-4"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhuo, G., Fu, X., Wang, P.P., Tan, C.P., and Chen, C. (2025). Ultrasonic collaborative pulse extraction of sugarcane polyphenol with good antiaging and \u03b1-glucosidase inhibitory activity. Int. J. Biol. Macromol., 297.","DOI":"10.1016\/j.ijbiomac.2025.139930"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Yang, N., Yue, R., Ma, J., Li, W., Zhao, Z., Li, H., Shen, Y., Hu, Z., Lv, C., and Xu, X. (2019). Nitidine chloride exerts anti-inflammatory action by targeting Topoisomerase I and enhancing IL-10 production. Pharmacol. Res., 148.","DOI":"10.1016\/j.phrs.2019.104368"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1976","DOI":"10.1096\/fj.02-0402fje","article-title":"Ca2+ channel remodeling in perfused heart: Effects of mechanical work and interventions affecting Ca2+ cycling on sarcolemmal and sarcoplasmic reticulum Ca2+ channels","volume":"16","author":"Zucchi","year":"2002","journal-title":"FASEB J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1007\/s00436-012-3251-9","article-title":"Essential oil composition and larvicidal activity of Toddalia asiatica roots against the mosquito Aedes albopictus (Diptera: Culicidae)","volume":"112","author":"Xin","year":"2013","journal-title":"Parasitol. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1377","DOI":"10.1016\/S0031-9422(97)00678-X","article-title":"Anti-platelet aggregation constituents from Formosan Toddalia asiatica","volume":"48","author":"Tsai","year":"1998","journal-title":"Phytochemistry"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1016\/j.foodchem.2012.12.058","article-title":"Alkaloids from Toddalia asiatica and their cytotoxic, antimicrobial and antifungal activities","volume":"148","author":"Hu","year":"2014","journal-title":"Food Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5146","DOI":"10.1021\/acs.orglett.6b02572","article-title":"Antiviral spirotriscoumarins A and B: Two pairs of oligomeric coumarin enantiomers with a spirodienone-sesquiterpene skeleton from Toddalia asiatica","volume":"18","author":"Tang","year":"2016","journal-title":"Org. Lett."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Li, Y., Sun, S.W., Zhang, X.Y., Liu, Y., Liu, X.H., Zhang, S., Wang, W., Wang, J., and Wang, W. (2020). Separation of new coumarin glycosides from Toddalia asiatica using offline two-dimensional high-performance liquid chromatography. Plants, 9.","DOI":"10.3390\/plants9040428"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1016\/j.jsps.2021.05.003","article-title":"A systematic review on traditional medicine Toddalia asiatica (L.) Lam.: Chemistry and medicinal potential","volume":"29","author":"Zeng","year":"2021","journal-title":"Saudi Pharm. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"450","DOI":"10.3736\/jcim20040615","article-title":"A study on anti-inflammatory and analgesic effects of alkaloids of Toddalia asiatica","volume":"2","author":"Hao","year":"2004","journal-title":"J. Chin. Integr. Med."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1016\/j.jep.2012.07.006","article-title":"Antidiabetic and antioxidant activities of Toddalia asiatica (L.) Lam.: Leaves in streptozotocin induced diabetic rats","volume":"143","author":"Sunil","year":"2012","journal-title":"J. Ethnopharmacol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.jep.2012.01.005","article-title":"Antimicrobial activity of Ulopterol isolated from Toddalia asiatica (L.) Lam.: A traditional medicinal plant","volume":"140","author":"Balachandran","year":"2012","journal-title":"J. Ethnopharmacol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ke, M., Zhang, Z., Xu, B., Zhao, S., Ding, Y., Wu, X., Wu, R., Lv, Y., and Dong, J. (2019). Baicalein and baicalin promote antitumor immunity by suppressing PD-L1 expression in hepatocellular carcinoma cells. Int. Immunopharmacol., 75.","DOI":"10.1016\/j.intimp.2019.105824"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Tian, W.A., Xie, X.B., and Cao, P.C. (2020). Magnoflorine improves sensitivity to doxorubicin (DOX) of breast cancer cells via inducing apoptosis and autophagy through AKT\/mTOR and p38 signaling pathways. Biomed. Pharmacother., 121.","DOI":"10.1016\/j.biopha.2019.109139"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Sun, X.L., Zhang, X.W., Zhai, H.J., Zhang, D., and Ma, S.Y. (2020). Magnoflorine inhibits human gastric cancer progression by inducing autophagy, apoptosis and cell cycle arrest by JNK activation regulated by ROS. Biomed. Pharmacother., 125.","DOI":"10.1016\/j.biopha.2019.109118"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1038\/s41420-025-02396-x","article-title":"The natural alkaloid nitidine chloride targets RNA polymerase I to inhibit ribosome biogenesis and repress cancer cell growth","volume":"11","author":"Voukeng","year":"2025","journal-title":"Cell Death Discov."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1007\/s12272-011-0513-5","article-title":"Induction of apoptosis by chelerythrine chloride through mitochondrial pathway and Bcl-2 family proteins in human hepatoma SMMC-7721 cell","volume":"34","author":"Zhang","year":"2011","journal-title":"Arch. Pharm. Res."},{"key":"ref_49","first-page":"8","article-title":"Toddaculin, a natural coumarin from Toddalia asiatica, induces differentiation and apoptosis in U-937 leukemic cells","volume":"15","author":"Riveiro","year":"2012","journal-title":"Phytomedicine"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"3113","DOI":"10.1016\/j.apsb.2022.03.016","article-title":"Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor","volume":"12","author":"Cao","year":"2022","journal-title":"Acta Pharm. Sin. 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