Molecular networking-based metabolite profiling of REV-ERB agonist SR9009

Abstract

SR9009 는 합성 약물로서 REV-ERB 를 약리학적으로 표적화 하는 대사 조절제이다. SR9009 의 잠재적인 효과로는 골격근의 미토콘드리아 수를 증가시켜 운동 능력을 향상시키는 것으로 알려져 있다. 따라서 세계반도핑기구(WADA)는 SR9009 를 호르몬 및 대사 조절제(S4)와 함께 금지 물질로 분류하고 있다. 본 연구에서는 금지물질 SR9009 의 도핑 검출 판단 자료 확보를 위해 GNPS(Global Natural Products Social Molecular Networking) 플랫폼의 기존 분자네트워킹 및 정보 기반 분자네트워킹 (FBMN)을 사용하여 사람과 말에서의 SR9009 의 대사 연구를 수행하였다. 연구 결과로 얻어진 SR9009 대사의 분자네트워킹 지도는 SR9009 검출을 위한 잠재적 타켓을 파악하기 위하여 정리되었다. 사람의 간 마이크로좀에서 FBMN 분석 결과 총 13 개의 대사산물(M1-M9) 노드 (M1, 하이드록실화; M2a, 하이드록실화 및 -H2O; M3, N탈알킬화(C3H5O2); M4, N-탈알킬화(C3H5O2) 및 하이드록실화 및 탈수소화; M5, N-탈알킬화(C8H14NO2); M6, N-탈알킬화(C5H4NO2S)), M7a-d, N탈알킬화(C5H4NO2S) 및 히드록실화, M8a-b, N-탈알킬화(C5H4NO2S) 및 히드록실화 및 -H2O, M9, N-탈알킬화(C5H4NO2S) 및 히드록실화 및 탈수소화)가 확인되었다. 이는 이전 다른 연구들에서 보고되지 않은 3 개의 대사체(M2, M8a-b)들이 추가로 확인된 결과이다. 또한 SR9009 의 FBMN 지도에서의 분자네트워킹 노드는 하이드록실화(M1-2), 탈수소화(M4, M9) 및 3 개의 N-탈알킬화(M3-4, M5, M6-9)로 그룹화할 수 있었다. 그중 M3 는 스펙트럼상 가장 높게 검출된 대사체였으며 M8b 가 두 번째로 높은 검출 감도를 나타내어 두 대사체는 SR9009 검출을 위한 잠재적 표적 대사체로써 제안될 수 있었다|SR9009 is a metabolic modulator targeted pharmacologically at REV-ERBs as a synthetic agonist. The potential performance-enhancing effects of SR9009 are known to improve exercise capacity by increasing mitochondrial counts in skeletal muscle; therefore, the World Anti-Doping Agency (WADA) classified SR9009 with hormone and metabolic modulators (S4) as a prohibited substance. In the present study, we conducted metabolism study of SR9009 in human and horse using classical molecular networking (MN) and feature-based molecular networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) platform. A MN map of SR9009 metabolism was established to identify potential targets for the detection of SR9009. A total of 13 metabolite (M1-M9) nodes in FBMN were structurally connected and elucidated based on MS1, MS2 (M1, hydroxylation; M2a, hydroxylation and -H2O; M3, N-dealkylation (C3H5O2); M4, N-dealkylation (C3H5O2) and hydroxylation and dehydrogenation; M5, N-dealkylation (C8H14NO2); M6, N-dealkylation (C5H4NO2S); M7a-d, N-dealkylation (C5H4NO2S) and hydroxylation; M8a-b, N-dealkylation (C5H4NO2S) and hydroxylation and -H2O; and M9, N-dealkylation (C5H4NO2S) and hydroxylation and dehydrogenation) in human liver microsomes assay. This resulted in the additional identification of three metabolites (M2, M8a-b) that had not been reported previously. In the FBMN map of SR9009, nodes were grouped by hydroxylation (M1-2), dehydrogenation (M4, M9), and three N-dealkylations (M3-4, M5, M6-9). M3 was the metabolite with the highest relative intensity of the spectrum and M8b exhibited the second highest relative intensity, therefore identifying both as potential targets for the detection of SR9009. A total of 13 metabolites (M1-M8c) were elucidated (M1, hydroxylation; M2b, dehydrogenation; M7b-d, N-dealkylation (C5H4NO2S) and hydroxylation; and M8a-c, N-dealkylation (C5H4NO2S) and hydroxylation and -H2O) in horse liver microsomes assay. This was result to additionally identify seven metabolites (M2a-b, M4, M7, M8a-c) which had not been reported previously. In FBMN map of SR9009 in vitro, nodes were grouped by hydroxylation (M1-2a), dehydrogenation (M2b, M4) and three N-dealkylation (M3-4; M5; M6-8c). According to in vivo assay, M6 and M7c were major metabolite as potential targets for detection of SR9009. Additionally, a quantitative method was developed for the determination of SR9009 in equine plasma. For quantitative detection of SR9009, plasma samples were pretreated by protein precipitation with methanol and were loaded onto an ACQUITY ultra performance liquid chromatography high-strength silica C18 column (2.1 × 150 mm, 1.8 µm) for chromatographic separation. The mobile phase consisted of 5 mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in acetonitrile, and a gradient elution was used at a flow rate of 0.25 mL⁄min. the selected reaction monitoring mode was used with transitions of 438.2→124.9 for SR9009, for the internal standard (testosterone-d3) in the positive ionization mode. For developed method, the linearity, lower limit of quantification, intra- and inter-day precision, accuracy, matrix effect, recovery, and stability were evaluated. The method was found to be accurate and reproducible for the quantitation of SR9009. The developed method was successfully applied to plasma samples of thoroughbreds injected intramuscularly with SR9009. These results suggest that the application method and performance of the MN technology, which was mainly used in the study of natural products, was confirmed in forensic science and metabolism research, and the obtained MN map will be used as basic scientific data for forensic interpretation of the illegal abuse of SR9009 in human sports and horse racing industry.