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High-Throughput Screening and Chemoproteomic Approaches to Identify Procaspase Inhibitors
High-Throughput Screening and Chemoproteomic Approaches to Identify Procaspase Inhibitors
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211153124
- ISBN
- 9798346852223
- DDC
- 574
- 서명/저자
- High-Throughput Screening and Chemoproteomic Approaches to Identify Procaspase Inhibitors
- 발행사항
- [Sl] : University of California, Los Angeles, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 344 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 86-06, Section: B.
- 주기사항
- Advisor: Backus, Keriann Marie.
- 학위논문주기
- Thesis (Ph.D.)--University of California, Los Angeles, 2024.
- 초록/해제
- 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.
- 일반주제명
- Biochemistry
- 일반주제명
- Cellular biology
- 일반주제명
- Molecular biology
- 일반주제명
- Genetics
- 키워드
- Caspases
- 키워드
- Proteomics
- 키워드
- Chemoproteomics
- 기타저자
- University of California, Los Angeles Biochemistry Molecular and Structural Biology 0090
- 기본자료저록
- Dissertations Abstracts International. 86-06B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520250211153124
■006m o d
■007cr#unu||||||||
■020 ▼a9798346852223
■035 ▼a(MiAaPQ)AAI31764442
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574
■1001 ▼aCastellon, Jose Omar.
■24510▼aHigh-Throughput Screening and Chemoproteomic Approaches to Identify Procaspase Inhibitors
■260 ▼a[Sl]▼bUniversity of California, Los Angeles▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a344 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 86-06, Section: B.
■500 ▼aAdvisor: Backus, Keriann Marie.
■5021 ▼aThesis (Ph.D.)--University of California, Los Angeles, 2024.
■520 ▼aCaspases 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.
■590 ▼aSchool code: 0031.
■650 4▼aBiochemistry
■650 4▼aCellular biology
■650 4▼aMolecular biology
■650 4▼aGenetics
■653 ▼aCaspases
■653 ▼aHigh-throughput screening
■653 ▼aProteomics
■653 ▼aChemoproteomics
■653 ▼aCellular thermal shift assays
■690 ▼a0487
■690 ▼a0379
■690 ▼a0369
■690 ▼a0307
■71020▼aUniversity of California, Los Angeles▼bBiochemistry, Molecular and Structural Biology 0090.
■7730 ▼tDissertations Abstracts International▼g86-06B.
■790 ▼a0031
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17165102▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
