Bobcat 339 is a novel cytosine-based TET enzyme inhibitor with good selectivity, exhibiting IC50 values of 33μM for TET1 and 73μM for TET2 without inhibiting the DNA methyltransferase DNMT3a[1]. TET enzymes are key epigenetic modifiers that facilitate DNA demethylation and regulate cell differentiation and development[2]. By competitively inhibiting the TET-mediated oxidation of 5-methylcytosine (5mC), Bobcat 339 is particularly suitable for investigating TET functions in epigenetic studies and elucidating mechanisms in relevant disease models[3,4].
In vitro, treatment of mouse hippocampal neuronal HT-22 cells with 10μM Bobcat 339 (in 1%DMSO) for 24h significantly reduced global levels of 5-hydroxymethylcytosine (5hmC)[1]. Incubation of mouse hypothalamic GT1-7 neuronal cells with 10μM Bobcat 339 for 6h decreased TET3 protein expression without affecting its mRNA abundance[5]. Furthermore, 72h treatment of human breast cancer MDA-MB-231 cells with 25μM Bobcat 339 downregulated FANCA gene expression and significantly inhibited cell proliferation via TET inhibition[6].
In vivo, intraperitoneal injection of Bobcat 339 (2.5mg/kg) in mice with GFP-specific expression in agouti-related petide (AgRP) neurons of the hypothalamus resulted in a notable reduction in TET3-positive AgRP neurons within 2 days, accompanied by increased neuronal activation (indicated by elevated FOS protein expression)[5]. In C57BL/6 mice with transient middle cerebral artery occlusion (tMCAO) and a lipopolysaccharide (LPS)-induced (10mg/kg; i.p.) hyperinflammatory state, intravenous administration of Bobcat 339 (10mg/kg/day) for 3 consecutive days significantly reduced cerebral infarct volume and improved neurological functional recovery[7].
References:
[1] CHUA G N L, WASSARMAN K L, SUN H, et al. Cytosine-based TET enzyme inhibitors[J]. ACS Medicinal Chemistry Letters, 2019, 10(2): 180-185.
[2] WEIRATH N A, HURBEN A K, CHAO C, et al. Small molecule inhibitors of TET dioxygenases: Bobcat339 activity is mediated by contaminating copper (II)[J]. ACS Medicinal Chemistry Letters, 2022, 13(5): 792-798.
[3] FENG J, SHAO N, SZULWACH K E, et al. Role of Tet1 and 5-hydroxymethylcytosine in cocaine action[J]. Nature Neuroscience, 2015, 18(4): 536-544.
[4] XU Y, YANG Y, WANG Z, et al. ZNF397 deficiency triggers TET2-driven lineage plasticity and AR-targeted therapy resistance in prostate cancer[J]. Cancer Discovery, 2024, 14(8): 1496-1521.
[5] LV H, CATARINO J, LI D, et al. A small-molecule degrader of TET3 as treatment for anorexia nervosa in an animal model[J]. Proceedings of the National Academy of Sciences, 2023, 120(16): e2300015120.
[6] LUO L, YUAN F, PALOVCAK A, et al. Oncogenic properties of wild-type DNA repair gene FANCA in breast cancer[J]. Cell Reports, 2025, 44(4): 115480.
[7] WANG Y, ZHANG L, LYU T, et al. Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state[J]. Neural Regeneration Research, 2024, 19(10): 2229-2239.
Bobcat 339是一种具有良好选择性的新型胞嘧啶类TET酶抑制剂，对TET1和TET2的IC50值分别为33μM和73μM，但不抑制DNA甲基转移酶DNMT3a[1]。TET酶是关键的表观遗传修饰因子，可以促进DNA去甲基化并调控细胞的分化和发育[2]。Bocat 339可竞争性地抑制TET介导的5-methylcytosine（5mC）氧化过程,故适用于表观遗传学研究中TET酶功能探索和相关疾病模型的机制解析[3,4]。
在体外，Bobcat 339（10μM）在1%DMSO中处理小鼠海马神经元HT-22细胞系24h，可显著降低细胞整体的5-hydroxymethylcytosine（5hmC）水平[1]。Bobcat 339（10μM）与小鼠下丘脑GT1-7神经元细胞共同孵育6h，降低了TET3蛋白水平，而不影响mRNA丰度[5]。Bobcat 339（25μM）处理人乳腺病变MDA-MB-231细胞系72h，TET的抑制会下调FANCA基因的表达，并显著抑制细胞的增殖[6]。
在体内，Bobcat 339（2.5mg/kg）通过腹腔注射治疗下丘脑agouti-related peptide（AgRP）神经元中特异性表达GFP的小鼠，2天后TET3阳性的AgRP神经元数量明显减少，同时这些神经元被激活（FOS蛋白表达增加）[5]。Bobcat 339（10mg/kg/day）通过尾静脉注射给药经脂多糖LPS（10mg/kg; i.p.）诱导的高炎症脑缺血小鼠C57BL/6，连续给药3天后显著减少了梗死体积，改善了神经功能缺损[7]。