title: High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida.
abstract: Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Yousof
authors.initials: Y
authors.lastname: Tahtah
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Sileshi G
authors.initials: SG
authors.lastname: Wubshet
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Kongstad
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Allison Maree
authors.initials: AM
authors.lastname: Heskes
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Irini
authors.initials: I
authors.lastname: Pateraki
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Jäger
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Electronic address: ds@sund.ku.dk.
authors.forename: Dan
authors.initials: D
authors.lastname: Staerk
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003924:Diabetes Mellitus, Type 2; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010936:Plant Extracts / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000037:antagonists & inhibitors*; D028083:Scrophulariaceae / Q000737:chemistry*; D052616:Solid Phase Extraction
chemical_list: D007004:Hypoglycemic Agents; D010936:Plant Extracts; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1
keywords: 3,6-dimethoxyapigenin (PubChem CID: 5352032); Caffeine (PubChem CID: 2519); Cirsimaritin (PubChem CID: 188323); Dinatin (PubChem CID: 5281628); Eremophila lucida; Gallic acid (PubChem CID: 370); Gramine (PubChem CID: 6890); HPLC–HRMS–SPE–NMR; High-resolution bioassay; Jaceidin (PubChem CID: 5464461); Luteolin (PubChem CID: 5280445); PTP1B; Quercetin (PubChem CID: 5280343); RK682 (PubChem CID: 54678922); Shikonin (PubChem CID: 479503); Tricin (PubChem CID: 5281702); Type 2 diabetes