Exportin-1 (also named as CRM1) plays a prominent role in autoimmune disorders and has emerged as a potential therapeutic target for colitis. Here we report on the rational structure-based discovery of a small-molecule antagonist of exportin-1, LFS-829, with low-range nanomolar activities. The co-crystallographic structure, surface plasmon resonance binding assay, and cell-based phenotypic nuclear export functional assay validated that exportin-1 is a key target of LFS-829. Moreover, we demonstrated that the C528S mutation or the knockdown on exportin-1 can abolish the cellular activities of LFS-829. Strikingly, oral administration of LFS-829 can significantly reverse the pathological features of colitis model mice. We revealed that LFS-829 can attenuate dual NF-κB signaling and the Nrf2 cytoprotection pathway via targeting exportin-1 in colitis mice. Moreover, LFS-829 has a very low risk of cardiotoxicity and acute toxicity. Therefore, LFS-829 holds great promise for the treatment of colitis and may warrant translation for use in clinical trials.

利用计算机辅助药物设计,筛选新型染色体区域维持因子(CRM1)共价靶向抑制剂,并探究其对结外NK/T细胞淋巴瘤(ENKTL)细胞增殖的影响.利用基于片段的药物设计方法在LFS-01母核结构的基础上设计新型CRM1抑制剂并利用ADME/T、共价对接等手段进行药物筛选得到小分子化合物LFS-829.MALDI-TOF质谱分析表明,LFS-829对CRM1具有靶向作用.用CCK-8法检测LFS-829对ENKTL细胞系SNK6及HANK-1增殖活性的影响,活细胞工作站观察药物作用下细胞形态的变化.利用免疫荧光技术分析LFS-829对CRM1核输出功能的影响.通过蛋白质免疫印迹实验、双荧光素酶报告基因实验以及酶联免疫吸附技术分析不同浓度的LFS-829作用下NF-κB信号通路的变化.借助流式细胞仪分析检测细胞凋亡,并通过蛋白质免疫印迹实验检测凋亡通路相关蛋白的表达.根据外周血单核淋巴细胞(PBMC)毒性测试、血小板毒性测试以及小鼠急性毒性实验对LFS-829进行安全性评价.结果表明,LFS-829能够与CRM1蛋白疏水活性口袋的半胱氨酸残基共价靶向结合,并选择性杀伤SNK6及HANK-1细胞,72 h的IC50分别为366和158 nmol/L.800 nmol/L LFS-829能够显著抑制CRM1的细胞核输出功能,促使IκB-α的细胞核聚集,下调NF-κB信号通路的转录活性,并显著上调凋亡通路蛋白p53、剪切型Caspase 3和剪切型Caspase 9的表达,诱导细胞凋亡.LFS-829对PBMC以及血小板没有明显的杀伤效果.在300 mg/kg的大剂量作用下,LFS-829未对小鼠造成实质性的组织损伤,安全性良好,具有良好的应用前景.

The rapid development of fluorescent probes for monitoring target enzymes is still a great challenge owing to the lack of efficient ways to optimize a specific fluorophore. Herein, a practical two-dimensional strategy was designed for the development of an isoform-specific probe for CYP3A4, a key cytochrome P450 isoform responsible for the oxidation of most clinical drugs. In first dimension of the design strategy, a potential two-photon fluorescent substrate (NN) for CYP3A4 was effectively selected using ensemble-based virtual screening. In the second dimension, various substituent groups were introduced into NN to optimize the isoform-selectivity and reactivity. Finally, with ideal selectivity and sensitivity, NEN was successfully applied to the real-time detection of CYP3A4 in living cells and zebrafish. These findings suggested that our strategy is practical for developing an isoform-specific probe for a target enzyme.

alpha-Acetolactate decarboxylase (ALDC) catalyses a-acetolactate into acetoin (3-hydroxy-2-butanone, AC) and is considered to be the rate-limiting enzyme in the synthesis of 2,3-butanediol. In this work, the enzymatic activity of ALDC from Enterobacter aerogenes ALDC (E.a.-ALDC) was fully characterized with enzyme kinetics, indicating a K-m of 14.83 +/- 0.87 mM and a k(cat) of 0.81 +/- 0.09 s(-1). However, compared with the activities of ALDCs reported from other bacteria, the activity of E.a.-ALDC was determined to present a relatively lower value of 849.08 +/- 35.21 U/mg. The enzyme showed maximum activity at pH 5.5. In addition, the activity of Ea-ALDC was promoted by Mg2+. The crystal structure of E.a.-ALDC firstly solved by X-ray crystallography at resolution of 2.4 angstrom revealed a chelated zinc ion with conserved His199, His201, His212, G1u70 and G1u259. In the active center, the conservative Arg150 was particularly proven to deviate from the zinc ion of the active centre, by adopting a flexible conformational change, resulting in a weak interaction network of the enzyme and the substrate. Further in silico docking of E.a.-ALDC with two enantiomers, (R)-acetolactate and (S)-acetolactate, unaltered the interaction network of E.a.-ALDC from the apo structure, which confirmed the weakened role of Arg150 in the catalytic properties of E.a.-ALDC. Our results reveal a unique structure-function relationship of acetolactate decarboxylase and provide a fundamental basis for the enzymatic synthesis of acetoin.

β-Glucuronidase (GLU) as a vital factor in enterohepatic circulation and drug-inducing enteropathy has been given more and more attention in recent years. In this study, an off-on near-infrared (NIR) fluorescent probe (DDAO-glu) for selectively and sensitively sensing GLU was developed on the basis of its substrate preference. DDAO-glu can rapidly and selectively respond to bacterial GLU under physiological conditions for detecting the real-time intestinal GLU bioactivity of complex biological systems such as human feces in clinic. Meantime, DDAO-glu has been successfully applied for visualization of endogenous GLU in bacterial biofilm, thallus, and even in vivo. Using this NIR probe, we successfully visualized the real distribution of intestinal GLU in the enterohepatic circulation. Furthermore, a high-throughput screening method was successfully established by our probe, and a potent natural inhibitor of GLU was identified as (-)-epicatechin-3-gallate (ECG) for effectively preventing NSAIDs-inducing enteropathy in vivo. DDAO-glu could serve as a powerful tool for exploring real physical functions of intestinal GLU in enterohepatic circulation, under physiological and pathological contexts, and developing the novel inhibitors of GLU to therapy acute drug-inducing enteropathy in clinic.

Cutaneous hyperpigmentation from excess melanogenesis causes serious pigmentary disorders and even melasma. Short peptides (SPs) are garnering attention lately owing to their therapeutic potential in dermatological diseases and low systemic side effects. Here, we show an octapeptide, ansin2, designed de novo from antioxidant SPs we previously reported, significantly inhibiting melanogenesis in B16 cells by decreasing tyrosinase production via regulating the MITF pathway. Ansin2 could also inhibit tyrosinase function by covering its catalytic pocket, which was simulated in docking and LIGPLOT studies. Topical application of ansin2 exhibited evident protection in UVB-induced pigmentation in guinea pig models both in terms of prophylaxis and treatment. Interestingly, unlike other hydrophilic and peptidic drugs that need delivery systems, ansin2 can be efficiently delivered topically to the epidermis and dermis per se without an affiliated moiety. Given that ansin2 lacks unwanted toxicities and immunogenicity, it holds great potential in treating hyperpigmentation in the cosmetics and pharmaceutical industries.