Defense Date

2016

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutical Sciences

First Advisor

UMESH R DESAI

Abstract

Influenza A virus (IAV) is a seasonal infectious agent that could cause a major worldwide catastrophe. Due to its genetic properties, IAV generates new viral particles that resist the body’s immune defense and antiviral drug therapy. This occurs when a host cell has been co-infected by different IAV strains leading to the generation of hybrid viruses. This process is called reassortment. The majority of these new IAVs contain genetically altered hemagglutinin (HA) and neuraminidase (NA). Unfortunately, all current IAV drug therapies target the highly mutated proteins, HA and NA, which is not very useful. Influenza matrix protein 1 (M1) is a structural protein that accounts for a number of critical viral events. It displays a highly conserved sequence compared to other proteins, HA and NA. It is the most abundant structural viral protein. M1 has a key role in viral replication and viral assembly. During all viral steps of cellular invasion, not a single step appears to occur without the contribution of M1 in one way or another. M1 protein forms a layer underneath the lipid bilayer membrane, which contributes to vital integrity and provides an intact viral entity. Upon cellular viral entry, the M1 layer dissociates to release an RNA genome that migrates to the nucleus to utilize the host’s cellular machinery for synthesizing viral proteins. More interestingly, M1 protein exhibits different structural conformations that correlate with its physiological activity. These conformational changes come with a variety of M1-M1 interactions. Crystallographic structures have revealed a tremendous amount of information regarding the M1 mechanism in self-oligomerization and depolymerization. xi Various crystal structures of M1 are available. Our collaborator at the FDA identified an M1 mutant with G88E substitution, which is unable to form an intact M1 layer as wt-M1. In order to understand the role of a single mutated residue, M1 protein (G88E-M1) has been crystallized and its crystal structure was resolved by the groups of Desai and Safo. This crystal forms three monomers in an asymmetric unit. G88E-M1 concentration was 15 mg/mL in a buffer of 55 mM KH2PO4/K2HPO4/H3PO4, 0.2 M NaCl, pH 3.4. The condition of the reservoir was 0.1 M Tris, pH 8.5, 8% PEG (8K). The estimated pH of the crystallization drop was 6.2. In combination with the literature, significant structural manifestations were observed in different pH conditions. Under acidic conditions, this M1 mutant forms a face-to-face dimer, which is stabilized by hydrophobic interactions as well as hydrogen bond interactions. Although the monomers have less hydrophobic interactions at the monomer-monomer interface due to mutation of Gly88 into a polar amino acid, Glu88, it forms a stable dimer. That is because Glu88 generates at the interface a number of hydrogen bond interactions with Tyr100, Lys104 and Arg134. M1 is an attractive a therapeutic target protein. Recently, the Desai’s group has identified through computer-based drug design a promising anti-IAV drug candidate, called PHE that interferes with M1 layer formation leading to defects in cellular production of new viral particles. However, PHE binding affinity to M1 was unknown. Experiment of PHE-M1 binding affinity was performed using surface plasmon resonance with NeutrAvidin gold chip on which biotinylated M1 was immobilized under neutral pH. An affinity constant (Kd) of ~ 1 µM was determined. Likewise, PHE-M1 affinity was studied using microscale thermophoresis (MST), which yielded an affinity constant (Kd) of ~ 1.5 µM. Another project undertaken in this study is to evaluate the affinity of small-molecule inhibitors that bind to signaling proteins. Small-molecules that could interfere with signaling pathways are highly valued in cancer therapy. G2.2, which is a highly sulfated molecule, has previously shown anticancer activity. It seems to be safe, potent, and selective toward colorectal cancer. The mechanism of action of G2.2 mainly triggers multiple important signaling pathways of cancer stem cells including fibroblast growth factor, epidermal growth factor (EGF), bone morphogenetic protein 4, wingless-int, and transforming growth factor-β (TGFβ). EGF and TGFβ were labeled with reactive dye NT-647 on the free thiol group of cysteine residues. MST experiments were performed using phosphate buffer (pH 7.4) and serial dilution of G2.2 (1 mM as the highest concentration). MST binding studies have revealed Kd of 80 µM and 54 µM for EGF and TGFβ, respectively. xii In conclusion, this project has elucidated the crystal structure of G88E-M1 protein with valuable structural manifestations. PHE has high affinity for M1, which was confirmed using two different biophysical techniques. Moreover, G2.2 seems to be a promising drug therapy that targets cancer stem cells through inhibition of growth factors and cytokines associated with their survival and activity. However, G2.2 has low affinity for EGF and TGFβ.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

10-5-2016

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