Structure-Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Here, we report the design and synthesis of a first macrocyclic PROTAC by adding a second cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second Brd4 bromodomain validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.

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Structure-Based Design of a Macrocyclic PROTAC

10.26434/chemrxiv.8967941.v1 ◽

2019 ◽

Author(s):

Andrea Testa ◽

Scott J. Hughes ◽

Xavier Lucas ◽

Jane E. Wright ◽

Alessio Ciulli

Keyword(s):

Crystal Structure ◽

Rational Design ◽

Enzyme Inhibitors ◽

Protein Complexes ◽

Linear Molecule ◽

Chemical Probes ◽

Bifunctional Molecules ◽

Design And Synthesis ◽

Biophysical Studies ◽

New Interactions

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to design enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Here, we report the design and synthesis of a first macrocyclic PROTAC based on co-crystal structure of a parent linear molecule, by adding a cyclizing linker. A co-crystal structure of the macrocyclic PROTAC bound in a ternary complex with VHL and Brd4 validated the rational design and identified new interactions formed by the new linker. Biophysical studies revealed that the macrocycle selectively discriminated the second against the first bromodomains of BET proteins, and is a potent Brd4 degrader. Macrocyclization provides a viable strategy to induce protein-protein complexes and protein degradation inside cells.

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Rational Design and Synthesis of Enzyme Inhibitors: Significance in Probing the Function of Enzymes

ChemInform ◽

10.1002/chin.200315288 ◽

2003 ◽

Vol 34 (15) ◽

Author(s):

Jun Hiratake

Keyword(s):

Rational Design ◽

Enzyme Inhibitors ◽

Design And Synthesis

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Design and synthesis of new enzymes based on the lactate dehydrogenase framework

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1991.0047 ◽

1991 ◽

Vol 332 (1263) ◽

pp. 177-184 ◽

Cited By ~ 98

Keyword(s):

Crystal Structure ◽

Lactate Dehydrogenase ◽

Malate Dehydrogenase ◽

Functional Groups ◽

Maximum Rate ◽

Chemical Probes ◽

Protein Surface ◽

Design And Synthesis ◽

Tryptophan Residues ◽

Lactate Dehydrogenases

Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.

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In Silico Modeling and Scoring of PROTAC-Mediated Ternary Complex Poses

10.26434/chemrxiv-2021-ldzzz ◽

2021 ◽

Author(s):

Junzhuo Liao ◽

Xueqing Nie ◽

Ilona Unarta ◽

Spencer Ericksen ◽

Weiping Tang

Keyword(s):

Residence Time ◽

Rational Design ◽

Ternary Complex ◽

Mechanical Energy ◽

Protein Structures ◽

Target Protein ◽

Change Analysis ◽

Bifunctional Molecules ◽

Protein Ligands ◽

Threshold Score

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that can induce the ubiquitination of targeted proteins via the formation of ternary complexes between an E3 ubiquitin ligase and a target protein. The poly-ubiquitinated target protein will be escorted to the proteasome for degradation. Rational design of PROTACs require knowledge of an accurate configuration of the PROTAC induced ternary complex. This study demonstrates that native ternary poses can be distinguished by scoring candidate poses based on the pose residence time. The scoring is essentially heat-and-dissociate trials of candidate poses sampled by MD and pre-ranked by the classic MM/GBSA method. It is practical, simple to use and self-intuitive, relying on the observation that the assumed more stable native crystal ternary poses maintained a longer residence time than non-native ones at both room and higher temperatures. A time score and temperature score were generated from multiple replicate trajectories. These scores were able to correctly identify the native pose from non-native ones in all the systems examined. The absolute numbers were comparable across different systems in all currently available VHL and CRBN-containing ternary crystal structures. Therefore, it is also possible to provide an empirical criteria for unresolved ternary structures that under the conditions of this study. If a ternary pose is stable up to over a certain threshold score, it is likely a native pose. The success of the method is in part attributed to the dynamic nature of the pose change analysis which naturally involves entropic effects, one that is intrinsically unavailable with faster static scoring methods that consider molecular mechanical energy only. Protein-protein binding entropy is much more significant than in protein-ligands binding. The success is also attributed to the fact that the protein structures themselves were all stable in the short heating trials.

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In Silico Modeling and Scoring of PROTAC-Mediated Ternary Complex Poses

10.26434/chemrxiv-2021-ldzzz-v2 ◽

2021 ◽

Author(s):

Junzhuo Liao ◽

Xueqing Nie ◽

Ilona Unarta ◽

Spencer Ericksen ◽

Weiping Tang

Keyword(s):

Residence Time ◽

Rational Design ◽

Ternary Complex ◽

Mechanical Energy ◽

Protein Structures ◽

Target Protein ◽

Change Analysis ◽

Bifunctional Molecules ◽

Protein Ligands ◽

Threshold Score

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that can induce the ubiquitination of targeted proteins via the formation of ternary complexes between an E3 ubiquitin ligase and a target protein. The poly-ubiquitinated target protein will be escorted to the proteasome for degradation. Rational design of PROTACs require knowledge of an accurate configuration of the PROTAC induced ternary complex. This study demonstrates that native ternary poses can be distinguished by scoring candidate poses based on the pose residence time. The scoring is essentially heat-and-dissociate trials of candidate poses sampled by MD and pre-ranked by the classic MM/GBSA method. It is practical, simple to use and self-intuitive, relying on the observation that the assumed more stable native crystal ternary poses maintained a longer residence time than non-native ones at both room and higher temperatures. A time score and temperature score were generated from multiple replicate trajectories. These scores were able to correctly identify the native pose from non-native ones in all the systems examined. The absolute numbers were comparable across different systems in all currently available VHL and CRBN-containing ternary crystal structures. Therefore, it is also possible to provide an empirical criteria for unresolved ternary structures that under the conditions of this study. If a ternary pose is stable up to over a certain threshold score, it is likely a native pose. The success of the method is in part attributed to the dynamic nature of the pose change analysis which naturally involves entropic effects, one that is intrinsically unavailable with faster static scoring methods that consider molecular mechanical energy only. Protein-protein binding entropy is much more significant than in protein-ligands binding. The success is also attributed to the fact that the protein structures themselves were all stable in the short heating trials.

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Rational Design and Synthesis of Enzyme Inhibitors: Significance in Probing the Function of Enzymes.

Journal of Synthetic Organic Chemistry Japan ◽

10.5059/yukigoseikyokaishi.60.1095 ◽

2002 ◽

Vol 60 (11) ◽

pp. 1095-1103 ◽

Cited By ~ 2

Author(s):

Jun Hiratake

Keyword(s):

Rational Design ◽

Enzyme Inhibitors ◽

Design And Synthesis

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In Silico Modeling and Scoring of PROTAC-Mediated Ternary Complex Poses

10.26434/chemrxiv-2021-ldzzz-v3 ◽

2021 ◽

Author(s):

Junzhuo Liao ◽

Xueqing Nie ◽

Ilona Unarta ◽

Spencer Ericksen ◽

Weiping Tang

Keyword(s):

Protein Interactions ◽

Rational Design ◽

Ternary Complex ◽

Protein Protein Interactions ◽

Dynamic Nature ◽

Bifunctional Molecules ◽

In Silico Modeling ◽

Subsequent Degradation ◽

Heating Scheme ◽

Entropic Effects

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce ubiquitination and subsequent degradation of proteins via formation of ternary complexes between an E3 ubiquitin ligase and a target protein. Rational design of PROTACs requires accurate knowledge of the native configuration of the PROTAC induced ternary complex. This study demonstrates that native and non-native ternary complex poses can be distinguished based on pose occupancy time in MD, where native poses exhibit longer occupancy times than non-native ones at both room and higher temperatures. Candidate poses are generated by MD sampling and pre-ranked by the classic MM/GBSA method. A specific heating scheme is then applied to induce ternary pose departure, generating an occupancy score and temperature score reflecting pose occupancy time and fraction. The scoring approach enables identification of the native pose in all the test systems. Beyond providing a relative rank of hypothetical poses of a given ternary system, the method could also provide empirical guidance to whether a given ternary pose is likely a native one or not. The success of the method is in part attributed to the dynamic nature of the pose departure analysis which accounts for solute entropic effects, typically neglected in the faster static pose scoring methods, while solute entropic contributions play a greater role in protein-protein interactions than in protein-ligand systems.

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