Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Ernst Schönbrunn, Ph.D.

Co-Major Professor

Yu Chen, Ph.D.

Committee Member

Vince Luca, Ph.D.

Committee Member

Michael Teng, Ph.D.

Committee Member

Vladimir Uversky, Ph.D.

Committee Member

Laura Blair, Ph.D.

Committee Member

Xingmin Sun, Ph.D.

Keywords

BET proteins, ZMYND8, EML4-ALK, EP300/CBP, X-ray crystallography

Abstract

Cancer is a significant global health burden, as ~40% of the human population is diagnosed with cancer over a lifetime, half of which succumb to the disease. To develop novel compounds as potential cancer therapeutics, an in-depth structural and biochemical understanding of proteins involved in oncogenesis is key. This dissertation centers around the comprehensive structural and biochemical characterization of two classes of proteins critical for cancer cell survival: bromodomain-containing proteins (BRDs) and fusion tyrosine kinases (FTKs).In 2010, small molecule inhibitors targeting bromodomains (epigenetic reader proteins) became heavily popularized due to the druggability and potent anticancer effects of BET inhibitors. Despite strong preclinical evidence of therapeutic efficacy, a challenge with these inhibitors incurred in clinical trials caused by toxicity, and the dosage had to be reduced to below the efficacious dosage. Chapter 1 addresses alternative means to target BET BRDs using dual BRD-kinase inhibitors with increased intra-BET selectivity and simultaneous inhibition of certain kinases.The frontier of epigenetic bromodomain inhibition shifted from BET to non-BET proteins, specifically with EP300 and CBP being considered as promising oncology targets. To address this, I studied EP300, CBP, ZMYND8, and TAF1 in detail. Chapter 2 details the use of structure-based drug design to develop selective EP300/CBP inhibitors. Highlights include the first allosteric compound to bind to bromodomains, the first PROTAC that selectively targets EP300 over CBP, and studies of the first clinical trial inhibitor to target EP300/CBP. This work culminated in new inhibitors as promising cancer therapeutics for patients with neuroblastoma or hematological malignancies containing c-Myc amplifications in addition to other cancers. Chapter 3 discusses the two other bromodomain-containing proteins, ZMYND8 and TAF1. For ZMYND8, there are no known inhibitors so the therapeutic utility of targeting ZMYND8 with small molecule inhibitors has yet to be established. Herein we show several confirmed micromolar inhibitors that may not only lead to understanding the ZMYND8 role in cancer pathogenesis but lay the foundation for new inhibitors targeting cancers established to be ZMYND8-dependent. Secondly, no TAF1 inhibitor has reached the clinic. Recent studies suggested that chemical inhibition of TAF1 is insufficient to prevent cancer cell growth. Therefore, we focused on the discovery of small molecules capable of simultaneously inhibiting TAF1 and a kinase with the potential to produce synergistic or synthetic lethal effects. The second class of protein under investigation, EML4-ALK, is a fusion tyrosine kinase (FTK). Fusion tyrosine kinases (FTKs) have been identified as an oncogene in 5–10% of the overall population in non-small cell lung cancer (NSCLC), which is a major cause of cancer-related mortalities. FTKs result from chromosomal translocation of a tyrosine kinase that becomes hyper-activated by gene fusion to an adjacent oligomerization domain of a different protein. Structural information on any FTK is unknown, without which a rational drug design approach towards the development of more selective and efficacious inhibitors is less feasible. Chapter 4 details the establishment of procedures for the overexpression and purification of homogeneous EML4-ALK and biochemical assays with clinically used ATP-site directed inhibitors, in addition to preliminary cryo-EM studies.Lastly, an additional COVID-19 project is discussed. During the COVID-19 pandemic, the precise extent of SARS-CoV-2 infection in the human population was needed to conduct epidemiologic studies to ascertain virus transmission and spread. Chapter 5 details the development of a SARS-CoV-2 antibody test using enzyme-linked immunosorbent assay (ELISA) to determine the SARS-CoV-2 seroprevalence of two populations: Hillsborough County residents and cancer patients (which may have compromised immune systems). Combined, these structural and biochemical studies on epigenetic and fusion tyrosine kinase targets may aid the future development of novel cancer therapeutics.

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