title: Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screening.
abstract: Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL(-1) SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM, and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Meng
authors.initials: M
authors.lastname: Meng
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Zhihuan
authors.initials: Z
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Pei
authors.initials: P
authors.lastname: Song
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Faculty of Chemistry, M.V. Lomonosov Moscow State University , Moscow 119991, Russia.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Eremin
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University , Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
authors.forename: Rimo
authors.initials: R
authors.lastname: Xi
mesh_terms: D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007118:Immunoassay / Q000379:methods*; D049449:Luminescence; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015394:Molecular Structure; D012435:S-Adenosylhomocysteine / Q000032:analysis* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases