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    X-Ray Crystal Structure of Human 20s Proteasome in Complex with Carfilzomib

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    Date
    2015-02-09
    Author
    Harshbarger, Wayne
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    Abstract
    20S proteasomes are large, multicatalytic N- terminal threonine proteases which are tasked with maintaining intracellular homeostasis by the breaking down of mis-folded, oxidized, or tagged proteins into small peptides. Proteasomes are essential for cell division and differentiation, and proteasome inhibition represents a successful therapeutic strategy against cancers. There are two main isoforms of the proteasome, the constitutive proteasome and the immunoproteasome. While the constitutive proteasome is emerging as a medical rationale for treatment of solid tumors, the immunoproteasome shows promise in the treatment of autoimmune diseases such as rheumatoid arthritis. Inhibition of both proteasome forms is necessary for the treatment of multiple myeloma, and is accomplished by the FDA approved drugs, bortezomib and carfilzomib. While bortezomib equally targets each isoform, carfilzomib has nearly six-fold selectivity for the constitutive proteasome. Despite the importance of the proteasome in cancer treatment as well as carfilzomibs use in the clinic, there remains no crystal structure of the human 20S proteasome, and no crystal structure of a proteasome:carfilzomib complex. This hampers drug development efforts charged with designing the next generation proteasome inhibitors. The present dissertation describes the structural and functional characterization of the constitutive proteasome from human, which will facilitate structure-based development of novel drugs. Using X-ray crystallography, the 3D structures of the human proteasome in an unbound and carfilzomib bound state have been determined to high resolution. The ligand bound structure provides explanations for the chymotrypsin-like selectivity of the drug, as well as to its preferential binding to constitutive proteasomes over immunoproteasome. The caspase-like sites have closed off S3 binding pockets which sterically hamper binding of carfilzomibs P3 phenyl group. Tryspsin-like sites have very similar specificity pocket characteristics as chymotrypsin-like sites, however the S1 pockets are spacious in comparison, and do not provide as many interactions to carfilzomibs P1 leucyl group, which may be reflected in the decreased selectivity. Chymotrypsin-like subunits have hydrophobic specificity pockets which form van der Waals interactions with carfilzomib that favor ligand binding. In particular, the P3 and P4 positions are found to be important for overcoming the structural rearrangements of Met45, α-helix H1, and β-sheets S5 and S6 upon carfilzomib binding. Additionally in this dissertation, high throughput screening techniques were used to identify novel proteasome inhibitors. A natural product polyphenol, PPH-1, was identified from microbial extracts, and a second natural product, α -mangostin, was identified from the National Cancer Institute’s library of clinically active compounds. The binding of each to the proteasome’s catalytic subunits was confirmed using Western Blotting techniques. Finally, the kinetic characterization of a series of compounds with dual proteasome and fatty acid synthase are described. The inhibition of multiple enzymes by a single drug may decrease the likelihood that diseases such as cancer will develop resistance, due to the need to become resistant along multiple pathways. The structural and functional characterization of the human proteasome described herein, now facilitates the design of novel drugs with therapeutic potential in cancer.
    URI
    http://hdl.handle.net/1969.1/155004
    Subject
    20S Proteasome
    carfilzomib
    multiple myeloma
    cellular regulation
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    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Harshbarger, Wayne (2015). X-Ray Crystal Structure of Human 20s Proteasome in Complex with Carfilzomib. Doctoral dissertation, Texas A & M University. Available electronically from http : / /hdl .handle .net /1969 .1 /155004.

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