Chemical Synthesis and Biological Mechanism of Intracellular Delivery of Engineered Biomolecules [electronic resource].

9780438973435

Ann Arbor : ProQuest Dissertations & Theses, 2018.

1 online resource (377 p.)

Access is available to the Yale community.

Peptide and protein therapeutics—biologics—embody an important and growing branch of the modern pharmaceutical industry. Biologics have been developed to treat a variety of diseases, such as cancer, diabetes, and autoimmune inflammatory disorders. Despite the immense potential of peptides and proteins as therapeutics, all currently available FDA-approved biologics stimulate, inhibit, or replace proteins that are localized on the plasma membrane, in the blood stream, or within endolysosomes. Not a single one acts within the cytosol of cells. The effort to develop intracellular protein therapeutics has been obstructed by the presence of biological lipid bilayers; the plasma membrane has evolved over millions of years to be a highly selective barrier only allowing a select few classes of molecules to enter the cytosol. This acts as a natural check-and-balance against the passage of toxic proteins and organisms, preventing them from entering cells and preserving the sensitive equilibrium necessary for cellular life. A few exceptions to this rule are known. The 13-residue Tat peptide derived from the trans-activator of transcription protein from human immunodeficiency virus (HIV) and the 16-residue penetratin peptide from antennapedia homeoprotein embody the first two reported examples of canonical cell-penetrating peptides (CPPs). CPPs are able to translocate biological membranes and elicit a biological response in cells. They enter cells via endocytosis and escape from endosomes into the cytosol. The problem is that, even though some fraction of the CPP molecules present in endosomes are able to reach the cytosol (and nucleus) of cells, the majority of the endocytosed CPPs remain trapped in endolysosomes and are eventually degraded in lysosomes. To fully harness the potential of CPPs for protein delivery, better CPPs and a better understanding of their mechanism of endosomal escape are desperately needed. Here, we present four projects that broadly relate to either the chemical synthesis or the intracellular delivery, and in some cases to both, of rationally engineered cell-permeant (semi-) synthetic lipids, peptides, or proteins. First, we outline our work with cell-permeant miniature proteins (CPMPs), a group of chemically synthesized, folded polypeptides carrying a pentaarginine (5.3) motif endowing them with the ability to circumnavigate biological membranes and reach the cytosol of cells. This work includes the implementation of assays to measure cytosolic and nuclear CPMP concentrations with precision and accuracy using fluorescence correlation spectroscopy and our efforts towards elucidating the mechanism of CPMP/CPP endosomal release using a broad spectrum of techniques. Furthermore, we describe a collaborative project with the Miller laboratory, which entailed the development of an aqueous phenolic O-glycosylation reaction for completely unprotected peptides. Glycosylation of peptides is another exciting strategy with the potential to improve the bioavailability of peptides within cells. We also recount our unsuccessful efforts towards engineering a bio-orthogonal compartment in live cells using fluorinated biomolecules. Finally, we include two projects exploiting β3-amino acids. These unnatural amino acids contain an additional methylene unit in the amino acid backbone. Peptides and proteins consisting solely of (33-amino acids have the ability to form protein-like structures and exhibit exceptional resistance to proteases. The first of these two projects describes our negative results attempting to design a cell-permeant α/β-peptide of the Puma-BH3 domain carrying a penta-arginine motif. The second describes a collaborative effort with the Pentelute laboratory, which entailed the preparation of the difficult-to-synthesize β-peptide bundle monomer Z28 using continuous flow. Together, these projects illustrate the diverse chemical space available for the manipulation of the cell permeability properties of naturally occurring lipids, peptides, and proteins. We anticipate that our results will aid in the future development of peptide- and protein-based therapeutics.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

August 21, 2019

Thesis (Ph.D.)--Yale University, 2018.

Yale University.