Mechanistic and Structural Features of PROTAC Ternary Complexes

Ryan Casement; Adam Bond; Conner Craigon; Alessio Ciulli

doi:10.1007/978-1-0716-1665-9_5

Mechanistic and Structural Features of PROTAC Ternary Complexes

Ryan Casement, Adam Bond, Conner Craigon, Alessio Ciulli (Lead / Corresponding author)

Biological Chemistry and Drug Discovery

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

33 Citations (Scopus)

798 Downloads (Pure)

Abstract

The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.

Original language	English
Title of host publication	Targeted Protein Degradation
Subtitle of host publication	Methods and Protocols
Editors	Angela M. Cacace, Christopher M. Hickey, Miklós Békés
Place of Publication	New York
Publisher	Humana Press
Pages	79-113
Number of pages	35
Edition	1
ISBN (Electronic)	9781071616659
ISBN (Print)	9781071616642 (hbk), 9781071616673 (pbk)
DOIs	https://doi.org/10.1007/978-1-0716-1665-9_5
Publication status	Published - 2021

Publication series

Name	Methods in Molecular Biology
Volume	2365
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Biophysical methods, crystal structures
Cooperativity
E3 ubiquitin ligase
Hook effect
Protein ubiquitination
Target engagement
Targeted protein degradation (TPD)
Ternary complex
proteolysis-targeting chimeras (PROTACs)

ASJC Scopus subject areas

Molecular Biology
Genetics

Access to Document

10.1007/978-1-0716-1665-9_5

Author Accepted ManuscriptAccepted author manuscript, 1.05 MB

Cite this

@inbook{ffa8b560399e48979774ae26a441efd7,

title = "Mechanistic and Structural Features of PROTAC Ternary Complexes",

abstract = "The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.",

keywords = "Biophysical methods, crystal structures, Cooperativity, E3 ubiquitin ligase, Hook effect, Protein ubiquitination, Target engagement, Targeted protein degradation (TPD), Ternary complex, proteolysis-targeting chimeras (PROTACs)",

author = "Ryan Casement and Adam Bond and Conner Craigon and Alessio Ciulli",

note = "Funding Information: We would like to thank many members of the Ciulli group for helpful discussions. We apologise to the authors of many studies in the field which could not be mentioned due to space restriction. The Ciulli laboratory's work on PROTACs has received funding from the European Research Council (ERC) under the European Union?s Seventh Framework Programme (FP7/2007-2013) as a Starting Grant to A.C. (grant agreement No. ERC-2012-StG-311460 DrugE3CRLs). R.C. is funded by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio doctoral training programme (BB/M010996/1). A.B. is funded by a PhD studentship from the Medical Research Scotland (MRS) (1170-2017). C.C. is funded by a PhD studentship from the UK Medical Research Council (MRC) under the doctoral training programme in Quantitative and Interdisciplinary approaches to biomedical science (QI Biomed) (MR/N0123735/1). The Ciulli laboratory receives or has received sponsored research support from Boehringer Ingelheim, Eisai, Nurix, Ono Pharmaceuticals and Amphista Therapeutics. Publisher Copyright: {\textcopyright} 2021, Springer Science+Business Media, LLC, part of Springer Nature.",

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language = "English",

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pages = "79--113",

editor = "Cacace, {Angela M.} and Hickey, {Christopher M.} and Mikl{\'o}s B{\'e}k{\'e}s",

booktitle = "Targeted Protein Degradation",

address = "United States",

edition = "1",

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Mechanistic and Structural Features of PROTAC Ternary Complexes. / Casement, Ryan; Bond, Adam; Craigon, Conner et al.
Targeted Protein Degradation: Methods and Protocols. ed. / Angela M. Cacace; Christopher M. Hickey; Miklós Békés. 1. ed. New York: Humana Press, 2021. p. 79-113 (Methods in Molecular Biology; Vol. 2365).

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

TY - CHAP

T1 - Mechanistic and Structural Features of PROTAC Ternary Complexes

AU - Casement, Ryan

AU - Bond, Adam

AU - Craigon, Conner

AU - Ciulli, Alessio

N1 - Funding Information: We would like to thank many members of the Ciulli group for helpful discussions. We apologise to the authors of many studies in the field which could not be mentioned due to space restriction. The Ciulli laboratory's work on PROTACs has received funding from the European Research Council (ERC) under the European Union?s Seventh Framework Programme (FP7/2007-2013) as a Starting Grant to A.C. (grant agreement No. ERC-2012-StG-311460 DrugE3CRLs). R.C. is funded by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio doctoral training programme (BB/M010996/1). A.B. is funded by a PhD studentship from the Medical Research Scotland (MRS) (1170-2017). C.C. is funded by a PhD studentship from the UK Medical Research Council (MRC) under the doctoral training programme in Quantitative and Interdisciplinary approaches to biomedical science (QI Biomed) (MR/N0123735/1). The Ciulli laboratory receives or has received sponsored research support from Boehringer Ingelheim, Eisai, Nurix, Ono Pharmaceuticals and Amphista Therapeutics. Publisher Copyright: © 2021, Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2021

Y1 - 2021

N2 - The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.

AB - The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.

KW - Biophysical methods, crystal structures

KW - Cooperativity

KW - E3 ubiquitin ligase

KW - Hook effect

KW - Protein ubiquitination

KW - Target engagement

KW - Targeted protein degradation (TPD)

KW - Ternary complex

KW - proteolysis-targeting chimeras (PROTACs)

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U2 - 10.1007/978-1-0716-1665-9_5

DO - 10.1007/978-1-0716-1665-9_5

M3 - Chapter (peer-reviewed)

C2 - 34432240

SN - 9781071616642 (hbk)

SN - 9781071616673 (pbk)

T3 - Methods in Molecular Biology

SP - 79

EP - 113

BT - Targeted Protein Degradation

A2 - Cacace, Angela M.

A2 - Hickey, Christopher M.

A2 - Békés, Miklós

PB - Humana Press

CY - New York

ER -

Mechanistic and Structural Features of PROTAC Ternary Complexes

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Novel PROTAC-based Chemical Biology Approaches to Induced Protein Knockdown Across the Proteome (PhD Studentship - with Boehringer Ingelheim)

DrugE3CRL's: Probing Druggability of Multisubunit Complexes: E3 Cullin RING Ligases (ERC Starting Grant)

Development and application of BromoTag : A “Bump-&-Hole”- PROTAC degron system for rapid, potent, and selective degradation of tagged target proteins

Development of Next-Generation Protein Degradation Systems: BromoTag and Other PROTACs

Cite this

Mechanistic and Structural Features of PROTAC Ternary Complexes

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Projects

Novel PROTAC-based Chemical Biology Approaches to Induced Protein Knockdown Across the Proteome (PhD Studentship - with Boehringer Ingelheim)

DrugE3CRL's: Probing Druggability of Multisubunit Complexes: E3 Cullin RING Ligases (ERC Starting Grant)

Student theses

Development and application of BromoTag : A “Bump-&-Hole”- PROTAC degron system for rapid, potent, and selective degradation of tagged target proteins

Development of Next-Generation Protein Degradation Systems: BromoTag and Other PROTACs

Cite this