RESUMEN
Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.
Asunto(s)
Calcio/química , Proteínas Recombinantes de Fusión/genética , Proteína Tumoral Controlada Traslacionalmente 1/genética , Proteasas Virales 3C/química , Sitios de Unión , Calcio/metabolismo , Clonación Molecular , Endopeptidasas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Modelos Moleculares , Oligopéptidos/genética , Oligopéptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Proteína Tumoral Controlada Traslacionalmente 1/química , Proteína Tumoral Controlada Traslacionalmente 1/metabolismoRESUMEN
Drug development is a complicated, slow and expensive process with high failure rates. One strategy to mitigate these factors is to recycle existing drugs with viable safety profiles and have gained Food and Drug Administration approval following extensive clinical trials. Cardiovascular and neurodegenerative diseases are difficult to treat, and there exist few effective therapeutics, necessitating the development of new, more efficacious drugs. Recent scientific studies have led to a mechanistic understanding of heart and brain disease progression, which has led researchers to assess myriad drugs for their potential as pharmacological treatments for these ailments. The focus of this review is to survey strategies for the selection of drug repurposing candidates and provide representative case studies where drug repurposing strategies were used to discover therapeutics for cardiovascular and neurodegenerative diseases, with a focus on anti-inflammatory processes where new drug alternatives are needed.