Date of Award

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Mamoun A. Alhamadsheh

First Committee Member

William K. Chan

Second Committee Member

Miki S. Park

Third Committee Member

David Thomas

Abstract

Transthyretin (TTR) amyloidosis is a progressive, fatal disease in which deposition of amyloid derived from either mutant or wild-type TTR causes severe organ damage and dysfunction. TTR cardiomyopathy is an infiltrative, restrictive cardiomyopathy characterized by progressive left and right heart failure. Familial amyloid cardiomyopathy (FAC) is driven by pathogenic point mutations in the TTR gene that destabilize the TTR tetramer, prompting its dissociation into dimers and monomers, with subsequent misfolding, aggregation and deposition of toxic TTR amyloid aggregates in the myocardium. The most prevalent mutation that causes FAC is the V122I variant, carried by 3.4% of African Americans, that increases the risk of cardiomyopathic events several-fold in this population. AG10, a potent TTR kinetic stabilizer, prevents dissociation of V122I-TTR in serum samples obtained from patients with FAC. Further, we have described structural, biochemical, and animal studies of AG10 which reveal mechanistic and structural insights on the ability of AG10 to mimic the disease suppressing T119M variant in stabilizing TTR.

The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life (t1/2) of therapeutic peptides. Native peptides typically display short in vivo t1/2, however conjugation of peptides to macromolecules causes steric hindrance which often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Utilizing Gonadotropin Releasing Hormone (GnRH) as a model peptide, we show that t1/2 may be extended without compromising potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Our strategy was effective in enhancing the t1/2 of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

The third and final part of the thesis describes our effort on developing a fluorescent probe to quantify TTR in human serum using fluorescence polarization. TTR is used as a marker for nutritional and inflammatory status in critical patients. This assay development has the potential to minimize lab cost, effort, and time with regards to determination of TTR concentration in patients.

Pages

147

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