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Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-13 00:00:00 , DOI: 10.1021/acs.biochem.7b00706 Sarah Benabdi 1 , François Peurois 1 , Agata Nawrotek 1 , Jahnavi Chikireddy 1 , Tatiana Cañeque 2, 3, 4 , Takao Yamori 5 , Isamu Shiina 6 , Yoshimi Ohashi 5 , Shingo Dan 5 , Raphaël Rodriguez 2, 3, 4 , Jacqueline Cherfils 1 , Mahel Zeghouf 1
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-13 00:00:00 , DOI: 10.1021/acs.biochem.7b00706 Sarah Benabdi 1 , François Peurois 1 , Agata Nawrotek 1 , Jahnavi Chikireddy 1 , Tatiana Cañeque 2, 3, 4 , Takao Yamori 5 , Isamu Shiina 6 , Yoshimi Ohashi 5 , Shingo Dan 5 , Raphaël Rodriguez 2, 3, 4 , Jacqueline Cherfils 1 , Mahel Zeghouf 1
Affiliation
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Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.
中文翻译：
家庭范围内的小分子抑制Arf鸟嘌呤核苷酸交换因子的分析：独特的抑制谱的证据。
Arf GTPases及其鸟嘌呤核苷酸交换因子（ArfGEFs）是细胞膜运输和细胞器结构的主要调节剂。它们与多种疾病有关，因此是被小分子抑制的有吸引力的治疗靶标。已经发现了几种不相关化学结构的抑制剂，这些抑制剂显示出它们在解剖分子途径和阻断疾病相关功能方面的潜力。但是，它们在ArfGEF家族中的特异性仍然难以捉摸。重要的是，膜的抑制反应是Arf和ArfGEF细胞功能的关键决定因素，目前尚未进行研究。在这里，我们比较了四种结构上不同的ArfGEF抑制剂，布雷菲德菌素A，SecinH3，M-COPA和NAV-2729对六个ArfGEF（人ARNO，军团菌和立克次体（ RalF）。使用纯蛋白质通过荧光动力学评估抑制作用，并在脂质体存在下用脂质化的GTPases确定膜对膜的调节作用。我们的分析表明，尽管与ArfGEF家族相似，但每种抑制剂都有特定的抑制特性。值得注意的是，M-COPA是有效的pan-ArfGEF抑制剂，而NAV-2729抑制所有GEF，最强的作用是对抗BRAG2和Arf1。此外，军团菌中存在膜结合结构域RalF揭示了BFA的强大抑制作用，而仅在其GEF结构域上无法测量。这项研究证明了掺入膜的家庭范围测定的价值，并且它应该能够通过这些抑制剂准确地分离Arf途径，从而最好地指导其作为治疗剂的使用和发展。
更新日期：2017-09-14
中文翻译：
家庭范围内的小分子抑制Arf鸟嘌呤核苷酸交换因子的分析：独特的抑制谱的证据。
Arf GTPases及其鸟嘌呤核苷酸交换因子（ArfGEFs）是细胞膜运输和细胞器结构的主要调节剂。它们与多种疾病有关，因此是被小分子抑制的有吸引力的治疗靶标。已经发现了几种不相关化学结构的抑制剂，这些抑制剂显示出它们在解剖分子途径和阻断疾病相关功能方面的潜力。但是，它们在ArfGEF家族中的特异性仍然难以捉摸。重要的是，膜的抑制反应是Arf和ArfGEF细胞功能的关键决定因素，目前尚未进行研究。在这里，我们比较了四种结构上不同的ArfGEF抑制剂，布雷菲德菌素A，SecinH3，M-COPA和NAV-2729对六个ArfGEF（人ARNO，军团菌和立克次体（ RalF）。使用纯蛋白质通过荧光动力学评估抑制作用，并在脂质体存在下用脂质化的GTPases确定膜对膜的调节作用。我们的分析表明，尽管与ArfGEF家族相似，但每种抑制剂都有特定的抑制特性。值得注意的是，M-COPA是有效的pan-ArfGEF抑制剂，而NAV-2729抑制所有GEF，最强的作用是对抗BRAG2和Arf1。此外，军团菌中存在膜结合结构域RalF揭示了BFA的强大抑制作用，而仅在其GEF结构域上无法测量。这项研究证明了掺入膜的家庭范围测定的价值，并且它应该能够通过这些抑制剂准确地分离Arf途径，从而最好地指导其作为治疗剂的使用和发展。
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