Material Type  

 학위논문

 

0017165102

Date and Time of Latest Transaction  

20250211153124

ISBN  

9798346852223

DDC  

574

Author  

Castellon, Jose Omar.

Title/Author  

High-Throughput Screening and Chemoproteomic Approaches to Identify Procaspase Inhibitors.

Publish Info  

[S.l.] : University of California, Los Angeles., 2024

Publish Info  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Material Info  

344 p.

General Note  

Source: Dissertations Abstracts International, Volume: 86-06, Section: B.

General Note  

Advisor: Backus, Keriann Marie.

학위논문주기  

Thesis (Ph.D.)--University of California, Los Angeles, 2024.

Abstracts/Etc  

요약Caspases are a highly conserved family of cysteine-aspartyl endoproteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Aside from the canonical roles in apoptosis, their functions in diverse cell functions and diseases, including neurodegenerative disease, autoimmune disorders, and cancers, remain poorly defined. Studying caspases is very difficult as they have complex activation mechanisms. Most caspases exist as inactive proenzymes. There are multiple caspase isoforms, some of which have redundant functions. Additionally, they behave differently in living cells and tissues, rendering in vitro assays ineffective, and are involved in crosstalk with other cellular processes such as autophagy and immune responses. Due to these challenges, new approaches can elucidate the biological function of specific caspases. Complementary to genetic approaches, small molecule inhibitors have emerged as useful tools for modulating caspase activity. However, achieving high selectivity remains a central challenge for caspase-directed inhibitor development efforts due to all twelve human caspases' high sequence and structure homology. Here, using a chemoproteomics and high-throughput screening (HTS) approach, I identified lead compounds that selectively label and inhibit procaspase-2 and identified new pan-caspase reactive inhibitors. First, using a chemical-proteomic platform termed isoTOP-ABPP, I identified a highly reactive non-catalytic cysteine residue, C370, located near the active site of caspase-2. I assayed a panel of cysteine reactive electrophiles using an engineered TEV-cleavable caspase-2 construct to validate the hits against pro-caspase-2 activity. I found a selective pro-caspase-2 inhibitor that targets the non-catalytic cysteine residue and binds the monomeric form of the enzyme. I also confirmed target engagement using cellular thermal shift assays (CETSA). Next, I identified a group of caspase inhibitors using a high-throughput screening assay. From a screen of approximately 120,000 compounds, I found pifithrin-µ (PFTµ), a known p53 inhibitor, as a caspase reactive covalent inhibitor and interesting scaffold molecule. From that same group of compounds, I found that the decomposed product of compound SO265 was driving caspase inhibition in my initial screen. Target engagement was also confirmed for both compounds using CETSA. I found that PFTµ and the other pan-caspase reactive electrophiles could protect Jurkat cells from Fas ligand and staurosporine-mediated apoptosis. This study demonstrates the potential of chemoproteomics and high-throughput approaches to help identify selective caspase inhibitors.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Cellular biology.

Subject Added Entry-Topical Term  

Molecular biology.

Subject Added Entry-Topical Term  

Genetics.

Index Term-Uncontrolled  

Caspases

Index Term-Uncontrolled  

High-throughput screening

Index Term-Uncontrolled  

Proteomics

Index Term-Uncontrolled  

Chemoproteomics

Index Term-Uncontrolled  

Cellular thermal shift assays

Added Entry-Corporate Name  

University of California, Los Angeles Biochemistry Molecular and Structural Biology 0090

Host Item Entry  

Dissertations Abstracts International. 86-06B.

Electronic Location and Access  

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Control Number  

joongbu:657737