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Discovery of Novel Inhibitors for the Human Papillomavirus E6 Protein

thesis
posted on 14.01.2021, 18:25 by Dino P. Petrov
The human papillomavirus (HPV) has been a “companion” of humanity for as long as humanity has existed. The migration of peoples around the globe has given rise to more than 170 different types of the virus, which cause a variety of conditions. All five genera of HPV infect epithelial cells in the body, but only the Alphapapillomaviruses infect the genital mucosa. Most infections are benign and typically regress to subclinical within two years, but persistent infections can cause precancerous lesions. HPV types 16 and 18 are among the highest risk and account for the majority of cervical cancer, and more than 90% of all other HPV-related cancers. While the two vaccines, Gardasil and Cervarix, have been successfully implemented in the US market and some European and Asian countries, complete world penetrance has been burdened by multiple factors, including financial constraints and social norms. Treatments for established papillomas are invasive (cryosurgery, conization, etc.) and advanced malignant HPV-related tumors have been targeted with chemo- and radiotherapy with varied success. The high morbidity and long-term effects of current treatment options make clear the need for easy-to-administer, low-cost therapies, which can specifically treat both early and advanced HPV-associated cancers.

The hallmark of HPV tumors is the inactivation of p53, an evasion strategy key to the progression of HPV- derived cancers. Through an interaction between the viral protein E6 and the E3 ubiquitin ligase E6AP, p53 is polyubiquitinated and targeted for proteasomal degradation, allowing infected cells to bypass their own defense mechanisms. This work explores interruption of the association between E6 and E6AP as an opportunity to combat the infection and resulting malignancies.

In the first part of this project, disruption of the E6-E6AP interactions is pursued through the development of helical stabilized peptidomimetics of the LxxLL motif, which E6AP uses for E6 recognition and binding. Several reports have indicated that targeting the E6 binding groove is a viable means for disrupting the interaction. However, reported peptides were not cell permeable or optimized for α-helicity and proteolytic resistance (for reference, the LxxLL motif is an α-helix when bound). To address this challenge a peptide stabilization strategy was applied, which uses an all-hydrocarbon chain to connect two non-adjacent residues and enforce α-helicity. Results from in silico simulations and biochemical assay with these new stapled peptides showed that affinity for E6, α-helicity, and cell permeability can all be improved with the installment of the proper staple.

The second question examined by this work is whether fragment-based drug design can be successfully employed to derive new small-molecule inhibitors of the formation of the E6-E6AP complex. From a design perspective, the significant challenge was to define discreet binding hot-spots capable of accommodating fragments with reasonable affinity, which can then be linked together into a complete ligand. Using existing structural knowledge of the E6 protein and computational hot-spot searching tools, three previously-unidentified regions (sub-pockets) on E6 were discovered, which are near but not directly engaged by either the E6AP motif or p53. Using high-throughput in silico and biochemical screening, three sets of sub-pocket specific fragments were defined and elaborated into larger molecules with two different scaffolds. As a result, the work herein presents a stepwise approach to targeting the E6-E6AP protein-protein interaction – the discovery of new binding hot spots, the identification of site-specific fragments, and the design of complete molecules with versatile scaffold.

History

Degree Type

Doctor of Philosophy

Department

Medicinal Chemistry and Molecular Pharmacology

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Vincent Jo Davisson

Additional Committee Member 2

Mark Cushman

Additional Committee Member 3

Markus Lill

Additional Committee Member 4

Mark Lipton

Additional Committee Member 5

Chittaranjan Das

Licence

Exports