Small molecules that imperfectly replicate protein function of missing proteins restore physiology in yeast and human epithelia

Ion channels;Natural products

Cioffi, Alexander Geoffrey

关键词: Ion channels;    Natural products;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/97529/CIOFFI-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Human diseases caused by an excess of protein function can often be treated with small molecules that bind and inhibit the corresponding proteins. In contrast, there are many currently incurable human diseases that are caused by missing protein ion transporters, including cystic fibrosis and microcytic anemia. Like many other human diseases caused by missing proteins, these diseases are difficult to treat, and new approaches are needed. Some small molecules can perform ion transporter-like functions, suggesting the possibility of replacing missing protein ion transporters with small molecule mimics. Closely replicating the functions of ion selective and tightly regulated protein transporters with small molecules is challenging. However, robust protein networks comprised of pumps and channels drive targeted ions in targeted directions throughout the spectrum of living systems. We hypothesized that relatively unselective and unregulated small molecule mimics of missing protein transporters might be capable of collaborating with the corresponding protein ion pumps to restore physiology.The ion channel-forming natural product amphotericin B (AmB) was first identified as a small molecule that could enable testing of this hypothesis. AmB is the prototypical small molecule capable of ion channel formation and can permeabilize both yeast and human cells to potassium and other ions. We observed vigorous and sustainable restoration of yeast cell growth by replacing potassium ion transporters with AmB. We next tested whether AmB could restore physiology in cystic fibrosis (CF) human lung epithelia. We observed restoration of key makers of physiology in CF epithelia upon treatment with AmB. Chemical inhibition experiments demonstrated evidence for collaboration between AmB and protein ion pumps in both yeast and human epithelia. To fully harness the ion channel property of AmB in the setting of protein deficiencies, a detailed understanding of how AmB interacts with membranes is required.For over 50 years, AmB has remained the powerful but toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. For decades, the dominant theory has been that AmB primarily exists in the form of small ion channel aggregates that are inserted into lipid bilayers and thereby permeabilize and kill yeast cells. Using solid-state NMR (SSNMR), we determined that AmB exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings suggest a roadmap for separating its toxic and ion channel activities. In this vein, the AmB channel is proposed to be funnel shaped, with the narrowest region near the C3 hydroxyl. Based on this model, we hypothesized that removal of the C3 hydroxyl would impact ion channel activity. The C3 alcohol was removed via synthesis in only 9 steps from AmB, resulting in C3deOAmB. Single ion channels of C3deoxyAmB in planar lipid bilayers revealed that C3deoxyAmB is still capable of ion channel formation, but its conductance is significantly reduced relative to AmB. This is consistent with models that place the C3 hydroxyl at a critical point for ion conductance and provide a potential site to tune ion selectivity.Encouraged by the results in yeast and CF lung epithelia, we hypothesized that a small molecule might be able to restore physiology in iron transporter deficiencies. We used iron-deficient yeast to screen for small molecules that could restore growth. We identified the small molecule hinokitiol (Hino) as capable of restoring growth in yeast lacking iron transporters. We then tested Hino in human colorectal epithelia lacking an iron transporter and observed Hino-mediated restoration of transepithelial iron transport. Together, these results illuminate a mechanistic framework for pursuing small molecule replacements for deficient protein ion transporters that underlie a range of challenging human diseases.