Numerous human diseases result from the failure of mutant proteins to exit the endoplasmic reticulum (ER), their site of synthesis. 11AT is a serine proteinase inhibitor (serpin) secreted by the liver into the plasma. 11AT functions to inhibit proteases in serum and, through delivery to the interstitium of the lung, maintain the proper balance of neutrophil lactase in the lung environment in order to prevent degradation of the extracellular matrix in the alveolar wall. Patients deficient in circulating 11AT have a high risk of developing lung disease. We address herein a critical need for novel HTS strategies to identify small molecules that will benefit the disease early-onset emphysema including childhood emphysema, a disease related to deficiency in alpha-1-antitrypsin (11AT) in the lung. We propose to develop a high throughput screening (HTS) to identify the small molecules that will reverse disease phenotype and promote delivery of 11AT to the extracellular space of the lung to prevent the pathophysiology of this protein misfolding disease. The objective of this proposal is to discover small molecules that promote the export of variant 11AT for transport through the secretory pathway. The discovery of such molecules will allow us to understand how variant 11AT transport from the ER can be sustained by adjusting the protein homeostasis or 'proteostasis'environment that is controlled by numerous signaling pathways that modulate the levels of folding chaperones and trafficking components that facilitate transfer of 11AT to the cell surface. This proposal is driven by the general hypothesis that small molecules identified in the HTS campaign can modulate proteostasis pathways to correct the folding problem and elicit a substantial ER exit response. We will explore two Aims to accomplish this goal using innovative and state-of-the-art HTS technologies.
Aim 1 will focus on development of our preliminary assay using an IB3 lung cell line expressing Z-variant 11AT (IB3-Z) as an HTS small molecule screen.
Aim 2 will build upon our success to date in the IB3-Z lung cell line to develop an assay in the mouse AML12 liver cell line, likely to be highly relevant to the human liver condition. This proposal addresses an important need to identify small molecules in an HTS campaign posed by Program Announcement PAR-08-024 'Assay Development for High throughput Molecular Screening'that can be used to modify the ER environment to promote secretion of 11AT reducing the risk of lung disease.
11AT functions to inhibit proteases in the interstitium of the lung to prevent degradation of the extracellular matrix in the alveolar wall. We address herein a critical need for high throughput screening (HTS) strategies that can be used to develop novel therapeutics to benefit the disease 11AT deficiency/early-onset emphysema including childhood emphysema that leads to premature lung failure in patients. We propose to use our HTS technologies to screen large chemical libraries provided by the MLCPN to identify biologically active compounds that will prevent the disease phenotype.
| Bouchecareilh, Marion; Balch, William E (2011) Proteostasis: a new therapeutic paradigm for pulmonary disease. Proc Am Thorac Soc 8:189-95 |
| Roth, Daniela M; Balch, William E (2011) Modeling general proteostasis: proteome balance in health and disease. Curr Opin Cell Biol 23:126-34 |
| Bouchecareilh, Marion; Conkright, Juliana J; Balch, William E (2010) Proteostasis strategies for restoring alpha1-antitrypsin deficiency. Proc Am Thorac Soc 7:415-22 |
| Routledge, Katy E; Gupta, Vijay; Balch, William E (2010) Emergent properties of proteostasis-COPII coupled systems in human health and disease. Mol Membr Biol 27:385-97 |
