Arf6 Inhibition as Novel Treatment for Multidrug Resistance Gram Negative Infections

 描述(由申请人提供)：由革兰氏阴性杆菌(GNB)引起的多重耐药(MDR)生物已成为世界各地医疗保健系统的地方病。非常令人关注的是最近极端耐药(XDR)-A的频率显著上升。鲍曼氏(AB)感染在过去十年中增加了17倍。此外，AB的感染率正在超过铜绿假单胞菌(PA)引起的感染，PA是另一种耐多药GNB，其发病率在美国各地保持稳定。由XDR AB引起的血液感染有60%的死亡率。显然，需要新的方法来预防和治疗AB和其他耐多药GNB。败血症是AB和其他GNB感染的常见表现，是内毒素通过与TLR4结合而引起的。TLR4识别LPs通过MyD88/NF-kB级联反应触发有效的保护性炎性细胞因子免疫反应。然而，这种免疫反应往往会导致与过度血管渗漏相关的有害和狂热的过程，从而导致组织水肿、器官衰竭、休克和死亡。我们发现TLR4缺陷小鼠对AB感染具有抵抗力。与这些发现一致的是，AB(带有影响脂质A生物合成的LpxC抑制剂)内毒素合成的中断使细菌在小鼠体内完全无毒，尽管这些抑制剂在体外缺乏对细菌的活性。Navigen的首席财务官Dean Li博士最近的研究表明，在内毒素引发的败血症中，过量的血管渗漏是由小GTP酶Arf6引起的，该酶落在MyD88/Arno/Arf6途径中，独立于MyD88/NF-kB级联反应发挥作用。ARF6通过VE-钙粘附素的细胞内内化导致血管内皮细胞渗漏，导致细胞间连接中断。我们推测，AB菌血症(和其他GNB感染)经常导致抗生素治疗失败，因为内毒素导致过多的血管渗漏，导致浮肿和器官衰竭。此外，Arf6的药理抑制提供了一个机会，在不增加有害血管泄漏的情况下，保留由TLR4识别脂多糖而触发的有益炎症免疫反应。事实上，我们的初步结果显示，在AB感染的小鼠模型中，Arf6抑制剂之一具有良好的活性。我们建议基于这些令人兴奋的数据来描述MyD88/Arno/Arf6通过siRNA基因抑制、肽类似物和敲除小鼠在AB感染的发病机制中的作用。此外，我们将确定第二代改进的Arf6抑制剂在治疗小鼠AB感染方面的活性。实现这两个目标(R21目标)将确定两种主要的Arf6抑制剂，用于详细的PK/PD和毒理学研究。最后，我们将确定这两种抑制剂对小鼠其他GNB感染的活性范围，包括耐多药PA和肺炎克雷伯菌碳青霉烯酶(KPC)(R33AIMS)。我们的研究结论将为进一步开发Arf6抑制剂作为可行的新型抗生素治疗GNB感染奠定基础，包括GMP、GLP临床前毒性和IND申报。

 DESCRIPTION (provided by applicant): Multi-drug resistant (MDR) organisms due to Gram negative bacilli (GNB) have become endemic in healthcare systems throughout the world. Of great concern is the recent, remarkable rise in the frequency of extremely drug resistant (XDR)-A. baumannii (AB) infections which increased >17 fold in the last decade. Further, infections with AB are surpassing those caused by Pseudomonas aeruginosa (PA), another MDR GNB, the incidence of which has remained stable across the US. Bloodstream infections caused by XDR AB have >60% mortality rates. Clearly new methods to prevent and treat AB and other MDR GNB are needed. Septicemia is a common manifestation of AB and other GNB infections which is caused by the endotoxic bacterial lipopolysaccharide (LPS) through binding to TLR4. LPS recognition by TLR4 triggers a potent and protective inflammatory cytokine immune response via the MyD88/NF-kB cascade. However, this immune response can often lead to a harmful and frenetic process associated with excess vascular leak that leads to tissue edema, organ failure, shock, and death. We found TLR4 deficient mice to be resistant to AB infection. Consistent with these findings, disruption of LPS synthesis in AB (with LpxC inhibitors affecting lipid A biosynthesis) renders the bacterium totally avirulent in mice despite the lack of in vitro activity of these inhibitors against the bacterium. Recent research by Dr. Dean Li (CSO of Navigen) demonstrated that the excess vascular leak seen in LPS-triggered septicemia is caused by the small GTPase Arf6 which falls in the MyD88/ARNO/Arf6 pathway and functions independently from the MyD88/NF-kB cascade. Arf6 induces vascular endothelium leak via intracellular internalization of VE-cadherin which leads to cell-to-cell junction disruption. We hypothesize that AB bacteremia (and other GNB infections) often fail antibiotic therapy because LPS induces excess vascular leak that results in edema and organ failure. Also, the pharmacological inhibition of Arf6 provides an opportunity to retain the beneficial inflammatory immune response, trigged by TLR4 recognizing LPS, without the added harmful vascular leak. Indeed our preliminary results showed promising activity of one of the Arf6 inhibitors in the mouse model of AB infection. We propose to build on these exciting data to delineate the role of MyD88/ARNO/Arf6 in the pathogenesis of AB infections via siRNA gene inhibition, peptide analogues and knockout mice. Further, we will determine the activity of 2nd generation improved Arf6 inhibitors in treating murine AB infections. Achieving these two goals (R21 aims) will identify 2 lead Arf6 inhibitors for detailed PK/PD and toxicology studies. Finally, we will determine the breadth of activity of these 2 inhibitors against other GNB infections including MDR PA and Klebsiella pneumonia carbapenemase (KPC) in mice (R33 aims). Conclusion of our studies will lay the foundation for further development of Arf6 inhibitors as viable new class of antibiotics versus GNB infections including GMP, GLP preclinical toxicity, and IND filing.