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Engineering Small Protein Based Inhibitors and Biodegraders for Cytosolic Delivery and Targeting of the Undruggable Proteome
Engineering Small Protein Based Inhibitors and Biodegraders for Cytosolic Delivery and Targeting of the Undruggable Proteome
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211150954
- ISBN
- 9798382829876
- DDC
- 610
- 저자명
- Chan, Alexander.
- 서명/저자
- Engineering Small Protein Based Inhibitors and Biodegraders for Cytosolic Delivery and Targeting of the Undruggable Proteome
- 발행사항
- [Sl] : University of Pennsylvania, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 278 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-12, Section: B.
- 주기사항
- Advisor: Tsourkas, Andrew.
- 학위논문주기
- Thesis (Ph.D.)--University of Pennsylvania, 2024.
- 초록/해제
- 요약The so-called "undruggable proteome" encompasses all intracellular proteins lacking small-molecule binding sites for pharmacological modulation. It is estimated that 80% of proteins fall under this category. While protein-based drugs can bind to nearly any target, thereby hitting undruggables, their large size and hydrophilicity restricts their passage across cell membranes. Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases. Herein, we develop protein-based inhibitors and protein-based degraders that can be shuttled across the plasma membrane with charged lipids to gain access to the cytosol. To accomplish this, we first engineer small protein scaffolds with an anionic polypeptide (ApP), conferring cargo with a net negative charge. Then, we use off-the-shelf cationic lipids or optimized lipid nanoparticle (LNP) formulations to encapsulate these charged proteins for intracellular delivery. Due to the ApP grafted onto proteins, an electrostatic interaction is enforced between cargo and lipids, akin to nucleic acid transfection. In Chapter 2, we deliver small-protein inhibitors of two oncogenes, Myc and Ras, with this strategy. We further develop LNP formulations for the delivery a potent Ras-inhibiting binder, DARPinK27. This study culminates in the validation of intracellular delivery in a mouse model of hepatocellular carcinoma (HCC). In Chapter 3, we convert protein inhibitors into targeted degraders by fusing E3 ligase or E3-like domains onto existing binders. We characterize optimal degraders, identify a suitable LNP formulation for delivery, and validate their activity in vitro. Finally, Ras degraders are delivered in a pancreatic ductal adenocarcinoma (PDAC) cell line. We demonstrate potent and rapid target depletion and induce anti-proliferative effects in this therapeutic model. In the final chapter, I propose strategies to further improve bioPROTAC design, nanoparticle formulations, as well as live-animal cytosolic delivery assays. A preliminary mathematical framework is provided to better intuit bioPROTAC parameters for enhancing targeted protein degradation.In summary, this work expands the ability to target clinically-relevant proteins that elude inhibition by conventional drug modalities. We establish therapeutic utility with an emphasis on targeted therapy in cancers. However, the technology developed here can be applied to many disease areas beyond oncology.
- 일반주제명
- Bioengineering
- 일반주제명
- Cellular biology
- 일반주제명
- Oncology
- 일반주제명
- Biochemistry
- 키워드
- Biodegrader
- 키워드
- bioPROTAC
- 키워드
- Drug delivery
- 기타저자
- University of Pennsylvania Bioengineering
- 기본자료저록
- Dissertations Abstracts International. 85-12B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■020 ▼a9798382829876
■035 ▼a(MiAaPQ)AAI30993487
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a610
■1001 ▼aChan, Alexander.
■24510▼aEngineering Small Protein Based Inhibitors and Biodegraders for Cytosolic Delivery and Targeting of the Undruggable Proteome
■260 ▼a[Sl]▼bUniversity of Pennsylvania▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a278 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-12, Section: B.
■500 ▼aAdvisor: Tsourkas, Andrew.
■5021 ▼aThesis (Ph.D.)--University of Pennsylvania, 2024.
■520 ▼aThe so-called "undruggable proteome" encompasses all intracellular proteins lacking small-molecule binding sites for pharmacological modulation. It is estimated that 80% of proteins fall under this category. While protein-based drugs can bind to nearly any target, thereby hitting undruggables, their large size and hydrophilicity restricts their passage across cell membranes. Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases. Herein, we develop protein-based inhibitors and protein-based degraders that can be shuttled across the plasma membrane with charged lipids to gain access to the cytosol. To accomplish this, we first engineer small protein scaffolds with an anionic polypeptide (ApP), conferring cargo with a net negative charge. Then, we use off-the-shelf cationic lipids or optimized lipid nanoparticle (LNP) formulations to encapsulate these charged proteins for intracellular delivery. Due to the ApP grafted onto proteins, an electrostatic interaction is enforced between cargo and lipids, akin to nucleic acid transfection. In Chapter 2, we deliver small-protein inhibitors of two oncogenes, Myc and Ras, with this strategy. We further develop LNP formulations for the delivery a potent Ras-inhibiting binder, DARPinK27. This study culminates in the validation of intracellular delivery in a mouse model of hepatocellular carcinoma (HCC). In Chapter 3, we convert protein inhibitors into targeted degraders by fusing E3 ligase or E3-like domains onto existing binders. We characterize optimal degraders, identify a suitable LNP formulation for delivery, and validate their activity in vitro. Finally, Ras degraders are delivered in a pancreatic ductal adenocarcinoma (PDAC) cell line. We demonstrate potent and rapid target depletion and induce anti-proliferative effects in this therapeutic model. In the final chapter, I propose strategies to further improve bioPROTAC design, nanoparticle formulations, as well as live-animal cytosolic delivery assays. A preliminary mathematical framework is provided to better intuit bioPROTAC parameters for enhancing targeted protein degradation.In summary, this work expands the ability to target clinically-relevant proteins that elude inhibition by conventional drug modalities. We establish therapeutic utility with an emphasis on targeted therapy in cancers. However, the technology developed here can be applied to many disease areas beyond oncology.
■590 ▼aSchool code: 0175.
■650 4▼aBioengineering
■650 4▼aCellular biology
■650 4▼aOncology
■650 4▼aBiochemistry
■653 ▼aBiodegrader
■653 ▼abioPROTAC
■653 ▼aCytosolic protein delivery
■653 ▼aDrug delivery
■653 ▼aProtein engineering
■653 ▼aTargeted protein degradation
■690 ▼a0202
■690 ▼a0379
■690 ▼a0992
■690 ▼a0487
■71020▼aUniversity of Pennsylvania▼bBioengineering.
■7730 ▼tDissertations Abstracts International▼g85-12B.
■790 ▼a0175
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17160310▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
