Linlin Xie1, David Yue-Wei Lee2, Ying Shang1, Xiaotong Cao1, Siqi Wang1, Jun Liao1, Teng Zhang3, Ronghua Dai4. 1. School of Pharmacy, Department of Pharmaceutical Analysis, Shenyang Pharmaceutical University, Shenyang 110016, China. 2. Mailman Research Center, McLean Hospital, Harvard Medical School, Boston, MA, United States. Electronic address: dlee@mclean.harvard.edu. 3. School of Pharmacy, Department of Pharmaceutical Analysis, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address: zhangteng1200@sina.com. 4. School of Pharmacy, Department of Pharmaceutical Analysis, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address: ronghuadai@sina.com.
Abstract
BACKGROUND: Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR). METHODS: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies. RESULTS: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 μM. CONCLUSION: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.
BACKGROUND: Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR). METHODS: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies. RESULTS: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 μM. CONCLUSION: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.