DISCOVERY OF ANTI-APOPTOTIC MACROMOLECULAR CANCER THERAPEUTICS AND DEVELOPMENT OF A BIOCOMPATIBLE NANOPARTICLE SYSTEM FOR TARGETED DRUG DELIVERY

Abstract

Two of the biggest challenges facing medicine today are 1) the discovery of new molecular entities that can serve as drug molecules and 2) the efficient delivery of drug molecules to their desired target in the body. This thesis addresses both of these challenges. For the purpose of drug discovery, tools of directed evolution and protein engineering have been used to develop peptide inhibitors of the Bcl-2 family of proteins. Pro-survival Bcl-2 proteins are found at elevated levels in many cancers and can cause resistance of a cancer to current chemotherapy treatments. An interactome was measured between all anti-apoptotic Bcl-2 proteins and the highly conserved BH3 domain of 17 pro-apoptotic peptides. Bak and Bim were determined to be the two peptides with strongest affinity toward all 5 anti-apoptotic proteins. Through affinity maturation, several Bak and Bim variants were identified to possess improved affinity and improved cytotoxicity when they were delivered to different types of cancer cells either through fusion with a cell penetrating peptide or techniques of peptide stapling. The peptide inhibitors described in this thesis hold promise as new cancer drugs since they compare favorably to small molecules currently being used or tested as cancer therapeutics in clinical trials.

My work on drug delivery was focused on the development of new block copolymers that can form nanoparticles using a technique pioneered by the Prud'homme lab, termed Flash NanoPrecipitation (FNP). Previous work in the Prud'homme lab has demonstrated that FNP can result in nanoparticles composed of block copolymers with very high drug loading. The focus of this thesis was to develop nanoparticles that can be readily targeted to different locations in the body. This targeting requires the specific attachment of different molecules to the surface of the nanoparticles that direct the particles to specific cells in the body. A robust synthesis of a biocompatible block copolymer that can be easily functionalized using click chemistry techniques was developed. It was subsequently shown that both small molecules and proteins can be attached to the surface of the block-copolymer nanoparticles with high conversion. This approach will allow us to attach essentially any molecule to nanoparticles made by FNP and is expected to have applications in both drug delivery and non-invasive imaging of diseased tissue.

To demonstrate the versatility of a nanoparticle system, the crosslinking of a polymeric nanoparticle core to increase its thermostability as well as the decoration of a nanoparticle corona with oligonucleotides and amplification via polymerase chain reaction (PCR) are also included in this thesis.