Target identification of covalently binding drugs by activity-based protein profiling (ABPP)

The characterization of the target proteins of drug molecules has become an important goal in understanding its mode of action and origin of side effects due to off-target binding. This is especially important for covalently binding drugs usually containing electrophilic moieties, which potentially can react with nucleophilic residues found in many proteins. This review gives a comprehensive overview of the use of activity-based protein profiling (ABPP) as an efficient tool for the target identification of covalently binding drugs.     

耐药细菌蛋白质的分泌——新抗攻击的靶点

几十年来,由于抗生素的大量使用,导致了细菌对很多药物的抗药性.为了克服细菌的抗药性问题,需要用新的思路去发掘新的抗生素,包括发掘细菌细胞中存在的抗生素作用的新靶点.蛋白质的分泌过程对于细菌是生死攸关的,它可能成为新药物的适合靶点.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.     

Exploiting azide–alkyne click chemistry in the synthesis,tracking and targeting of platinum anticancer complexes

Click chemistry is fundamentally important to medicinal chemistry and chemical biology. It represents a powerful and versatile tool, which can be exploited to develop novel Pt-based anticancer drugs and to better understand the biological effects of Pt-based anticancer drugs at a cellular level. Innovative azide–alkyne cycloaddition–based approaches are being used to functionalise Pt-based complexes with biomolecules to enhance tumour targeting. Valuable information in relation to the mechanisms of action and resistance of Pt-based drugs is also being revealed through click-based detection, isolation and tracking of Pt drug surrogates in biological and cellular environments. Although less well-explored, inorganic Pt-click reactions enable synthesis of novel (potentially multimetallic) Pt complexes and provide plausible routes to introduce functional groups and monitoring Pt-azido drug localisation.     

MicroRNA-203在结直肠癌研究中的进展

结直肠癌是我国常见的消化道恶性肿瘤。目前认为结直肠癌的形成是一个多因素、多步骤的过程,其具体发病机制尚不清楚。microRNA是一类非编码小分子RNA,能在转录后水平调控靶基因的表达,参与肿瘤的增殖、侵袭和转移,甚至调节肿瘤化疗敏感性。学者普遍认为mircroRNA-203(mir-203)是抗癌小分子RNA,但与以往不同的是mir-203在结直肠癌肿的表达水平仍存在争议。本文将概述mir-203在结直肠癌的发生、发展、诊断、预后以及药物抗性中发挥的各类生物学作用。阐述mir-203在结直肠癌不同信号通路中的作用,探索其在结直肠癌研究中的全部潜能。     

NLS-RARα contributes to differentiation block and increased leukemogenic potential in vivo

The fusion oncogene, promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα), is crucial for acute promyelocytic leukemia (APL) pathogenesis. Previous studies have reported that PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. This study was designed to evaluate the value of NLS-RARα in the early diagnosis of APL. To investigate the potential functional role of NLS-RARα in leukemogenesis, HL-60 and U937 cell lines were transfected with NLS-RARα lentivirus and negative control (LVNC). The results showed that the induced expression of NLS-RARα down-regulated expressions of CD11b, CD11c, and CD14 compared to the LVNC group induced by 1α, 25-dihydroxyvitamin D₃(1,25(OH)₂D₃). This suggested that NLS-RARα overexpression inhibited granulocytic and monocytic differentiation of myeloid leukemia cells. In addition, Wright-Giemsa staining, flow cytometry, respiratory burst assay, and NBT reduction assay all confirmed the importance of NLS-RARα in differentiation. The mechanistic investigations revealed that induced NLS-RARα expression inhibited 1,25(OH)₂D₃-induced granulocytic differentiation by regulating the cell cycle regulators p19^INK4D, p21^WAF1/CIP1, cyclinD1, cyclin E1, and pRB. Furthermore, the cleaved protein NLS-RARα enhanced the oncogenicity of U937 cells in NOD/SCID mice. These findings collectively demonstrated that NLS-RARα blocked granulocytic and monocytic differentiation of myeloid leukemia cells by inhibiting the downstream targets of the RARα signal pathway and the cell cycle. This may provide a promising new target and method for diagnosing and treating APL.     

MicroRNA-29b过表达慢病毒载体的构建及其生物学特性的研究

目的:构建针对小鼠microRNA-29b过表达的慢病毒载体,研究其在小鼠神经元GT1-7细胞系中的生物学特性。方法:化学合成两条寡聚核苷酸单链,通过搭桥互补延伸成DNA双链,形成miR-29b的前体结构,将酶切后的慢病毒载体FUGW通过同源重组的方法与miR-29b的前体结构进行连接,构建相应microRNA-29b过表达慢病毒载体,并包装成病毒颗粒后转染小鼠神经元细胞系GT1-7,通过博来霉素药物筛选获得稳转株,RT-PCR检测相关基因在mRNA转录水平上表达量情况。结果:测序图谱证实重组慢病毒表达质粒f-F-miR-29b构建成功,GT1-7细胞稳转株中,miR-29b的表达量与对照组相比提高了约28倍,其靶基因DCX,Vdac1,Pten的表达量有所抑制,性发育相关基因LH-β,kiss-1,Inshulin,IGF-I,GPR54,GnRH,leptin-R没有明显变化。结论:利用慢病毒筛选的方法,成功在小鼠神经元GT1-7细胞中获得microRNA-29b过表达稳转株,为以后microRNA-29b的生物学特性的研究奠定了基础。     

Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus

The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.