Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Physics

Department

Physics

First Advisor

Yana Reshetnyak

Abstract

The pH-dependent interactions of pHLIPs® (pH (Low) Insertion Peptides) with lipid bilayer of membrane provides an opportunity to study and address fundamental questions of protein folding/insertion into membrane and unfolding/exit, as well as develop novel approach to target acidic diseased tissue such as cancer, ischemic myocardium, infection and others.

The main goal of the work presented here is to answer the following questions:

- What is the molecular mechanism of spontaneous insertion and folding of a peptide in a lipid bilayer of membrane;

- What is the molecular mechanism of unfolding and exit of a peptide from a lipid bilayer of membrane;

- How polar cargo attached to a peptide's inserting end might affect the process of insertion into a lipid bilayer of membrane;

How sequence variation will affect a peptide's interactions with a lipid bilayer of membrane (partitioning into bilayer at neutral and low pH; apparent pK of insertion) with the main goal to identify the best pHLIP variants for imaging and therapy of pathological states such as cancer and others.

It has been demonstrated that pHLIP insertion into a membrane is associated with the protonation of Asp/Glu residues, which leads to an increase of hydrophobicity that triggers the folding and insertion of the peptide across a lipid bilayer. The insertion of the pHLIP is unidirectional and it is accompanied by the release of energy. Therefore, the energy of membrane associated-folding can be used to favor the movement of cell-impermeable polar cargo molecules across the hydrophobic membrane bilayer when they are attached to the inserting end of pHLIP. Both pH-targeting behavior and molecular translocation have been demonstrated in cultured cells and in vivo. Thus, there is an opportunity to develop a novel concept in drug delivery, which is based on the use of a monomeric, pH-sensitive peptide molecular transporter, to deliver agents that are significantly more polar than conventional drugs. Understanding the molecular events that occur when a peptide inserts across a membrane, folds, or exits from it and unfolds provides crucial information for the development of new drug delivery agents, as well as improving our understanding of the first step of membrane-associate protein folding.

The promise of exploiting tumor acidosis as a cancer biomarker has not been fully realized in clinical practice, even though the acidity has been a known property since the work of Otto Warburg nearly a century ago. The problem has been to find a practical way to target acidity. pHLIP reversibly folds and inserts across membranes in response to pH changes, and this discovery has led to a novel way to target acidic tissue. Steady state biophysical studies have revealed the molecular mechanism of pHLIP action, which is based on the increase of hydrophobicity of carboxyl groups when they become protonated under mildly acidic conditions, leading to peptide insertion into a membrane. It has been shown that pHLIP can target acidic tissue and selectively translocate polar, cell-impermeable molecules across membranes in response to low extracellular pH. As noted in the Molecular Imaging and Contrast Agent Database (MICAD) at NCBI, a pHLIP labeled with a fluorescent dye, or a PET- and SPECT- agents (64Cu-DOTA, 18F, 99Tc) is a marker for in vivo acidity,.

All prior studies in vivo were carried out with the WT-pHLIP sequence and showed that a good contrast and tumor to blood ratio can be achieved only more than 24 hours after pHLIP injection, when it has accumulated in the tumor and largely cleared from the blood. However, for the use of pHLIP-based radioactive imaging agents in the clinic, a more rapid background signal reduction is absolutely essential. We have conducted research in order to address this important need, to tune tumor targeting properties, and to broaden our understanding of the molecular mechanism of pHLIP action. A family of 16 pHLIP variants has been designed based on chemical and physical principles and comprehensive biophysical studies were performed with non-labeled peptides. We have successfully established a set of design criteria and identified the pHLIP candidates for imaging and therapeutic applications, including lead compounds for PET/SPECT and fluorescence/MR imaging.

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