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Purification and Ligand-Binding Mech...

Cai, Yingying.

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Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics./
作者:	Cai, Yingying.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2017,
面頁冊數:	196 p.
附註:	Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.
Contained By:	Dissertation Abstracts International79-05B(E).
標題:	Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10767217
ISBN:	9780355681314

Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics.
Cai, Yingying.

Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics. - Ann Arbor : ProQuest Dissertations & Theses, 2017 - 196 p.

Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.

Thesis (Ph.D.)--Yale University, 2017.

Family B G protein-coupled receptors (GPCRs) have attracted increasing attention as targets for the treatment of metabolic diseases, including type 2 diabetes and osteoporosis. The 15 members of family B GPCRs all bind to peptide hormones as their native ligands, making the discovery of small-molecule drugs extremely challenging. There is a lack of structural and functional information necessary to depict druggable binding sites, largely due to the immense difficulty in purifying sufficient quantities of functional receptors. It is therefore imperative to establish new purification methods and explore the molecular mechanisms of ligand-induced activation that may facilitate the next generation of drug development. This dissertation describes our efforts made toward a better understanding of how family B GPCRs operate as well as furthering advances in drug development, using parathyroid hormone 1 receptor (PTH 1 R) and glucagon-like peptide-1 receptor (GLP 1 R) as model systems.

ISBN: 9780355681314Subjects--Topical Terms:

518028
Biochemistry.

Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics.
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100 1 $a Cai, Yingying. $3 3352411
245 1 0 $a Purification and Ligand-Binding Mechanism of Family B G Protein-Coupled Receptors: Guiding the Rational Design of Peptide-Based Therapeutics.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2017
300 $a 196 p.
500 $a Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.
500 $a Adviser: Elsa C. Y. Yan.
502 $a Thesis (Ph.D.)--Yale University, 2017.
520 $a Family B G protein-coupled receptors (GPCRs) have attracted increasing attention as targets for the treatment of metabolic diseases, including type 2 diabetes and osteoporosis. The 15 members of family B GPCRs all bind to peptide hormones as their native ligands, making the discovery of small-molecule drugs extremely challenging. There is a lack of structural and functional information necessary to depict druggable binding sites, largely due to the immense difficulty in purifying sufficient quantities of functional receptors. It is therefore imperative to establish new purification methods and explore the molecular mechanisms of ligand-induced activation that may facilitate the next generation of drug development. This dissertation describes our efforts made toward a better understanding of how family B GPCRs operate as well as furthering advances in drug development, using parathyroid hormone 1 receptor (PTH 1 R) and glucagon-like peptide-1 receptor (GLP 1 R) as model systems.
520 $a With the increasing acceptance of peptide therapeutics, cognate peptide ligands of family B GPCRs and their derivatives have been developed as promising drug candidates. For example, truncated parathyroid hormone PTH(1-34), acting on PTH1R, is the only FDA-approved anabolic medication for the treatment of osteoporosis. However, peptide drugs generally suffer from poor in vivo stability and short half-lives, requiring high doses and frequent injections. To overcome these disadvantages, we proposed an affinity-enhancing design of triblock peptide-linker-lipid construct, where a therapeutic peptide is covalently bound to a lipid molecule through a linker group. We tested the feasibility of the triblock constructs using the short N-terminus of PTH, PTH(1-14), as the therapeutic peptide to target PTH1R. We showed that PTH(1-14)-based triblock constructs form micelle-like structures for improved biostability and insert into lipid membranes to enhance bioactivity up to 100-fold. Subsequently, we explored the fine-tuning feature of the triblock design enabled by the flexible linker region. Using a combined experimental and computational " approach, we identified the "Goldilocks" linker length of PTH(1-14)-based triblock molecules for optimal construct-PTH1R interaction. Our findings showed that shorter linkers have limited flexibility, impairing the favorable peptide-receptor interaction, while longer linkers exhibit linker-receptor interactions that compete with essential peptide-receptor interactions. These results will likely guide the future design of similar triblock constructs that could target PTH1 R for osteoporosis treatment. The tunable character of the triblock design in the ligand, linker, and lipid moieties implies the broad applicability to all family B GPCRs and as well as other transmembrane targets with extracellular ligand binding sites.
520 $a Furthermore, the triblock design allows for the independent studies of ligand binding to the juxtamembrane and N-terminal domains that are common to the family B GPCR two-domain ligand binding model. Specifically, the ligand C-terminal portion binds the receptor N-terminal domain, creating an affinity trap and bringing the N-terminus of the peptide in contact with the receptor juxtamembrane domain to activate signaling. The application of the triblock design can overcome the extremely low affinity of the peptide N-terminus thus, allowing for the examination of receptor signaling processes without the binding effect of the peptide C-terminal portion. Preliminary studies suggest that beyond the well-accepted two-domain model, potential allosteric modulations exist between the C- and N-terminal fragments of the peptide ligand upon receptor binding and between the receptor's extracellular and transmembrane domains via interactions with the peptide ligands. Since the future development of peptide and/or non-peptide therapeutics is ultimately determined by our knowledge of receptor binding mechanisms, completion of this study would establish a new paradigm of allostery in family B GPCR signaling through hormone - interaction, suggesting further avenues for the development of allosteric drugs.
520 $a Lastly, since the difficulty in purifying large quantities of the functional receptor has limited the mechanistic studies of ligand binding and receptor activation, we have made extensive efforts to develop a new purification method for family B GPCRs using nanodiscs. The method incorporates the receptor into the native-like lipid environment of nanodiscs immediately after solubilizing the membrane followed by chromatographic purification, minimizing detergent contact to reduce denaturation and prolonging receptor stabilization in lipid bilayers. We initially applied this method to purify PTH1R, and in this work, the protocol was optimized and used toward GLP1R, which is one of the best-validated drug targets for type 2 diabetes. We successfully obtained purified GLP1R in nanodiscs that maintain ligand-binding and G protein activation abilities and demonstrated the nanodisc purification method as a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
520 $a Throughout these experiments, the importance of purification and ligand-binding mechanism of family B GPCRs were emphasized and demonstrated for guiding future rational drug design. We provided valuable insights into the complex ligand-receptor interaction and offered novel platforms for receptor purification and therapeutic development.
590 $a School code: 0265.
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