The Vital Role of Proteomics in Characterizing Novel Protein Degraders

Zhang, Andrew X.; Cassidy, Katelyn; Dahl, Göran; Moreau, Kévin; Pachl, Fiona; Zuhl, Andrea M.

doi:10.1177/2472555220985776

Cited by 14 publications

(10 citation statements)

References 40 publications

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“…One of the most critical steps in determining the suitability of a target POI for TPD, is understanding its intrinsic degradation and synthesis within the relevant cell type and disease state. As reviewed previously, 36 the protein turnover rate (PTR) is a parameter to predict dosing schedules for effective PROTAC responses. After a PROTAC degrades a target POI and is cleared from the animal, in vivo functional activity may return quickly if the synthesis rate of the target POI is fast.…”

Section: D Protein Turnover and Synthesis Considerationsmentioning

confidence: 99%

“…SILAC (stable isotope labelling by amino acids in cell culture) mass spectrometry provides one of the best approaches at probing the turnover and synthesis of proteins in cell culture versus immunoassays or protein synthesis inhibitors like cycloheximide. 36 Pulsed SILAC approaches can be used to determine PTRs in non-dividing cells or to evaluate whole tissues in vivo , 37 giving insights into the turnover of long lived proteins. Additionally, investigators should keep in mind that in theory synthesis rates after PROTAC-mediated knockdown may not match synthesis rates under basal homeostatic conditions due to compensatory cellular responses.…”

Section: Target Selectivitymentioning

confidence: 99%

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Target and tissue selectivity of PROTAC degraders

et al. 2022

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Section: D Protein Turnover and Synthesis Considerationsmentioning

confidence: 99%

Section: Target Selectivitymentioning

confidence: 99%

Target and tissue selectivity of PROTAC degraders

et al. 2022

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“…To date, proteomic assays have been widely used to assess the degradation selectivity of first-generation degraders, to investigate mechanisms of action, to assess binding affinity for the target, and even to determine the endogenous turnover rate of the target, informing dose predictive PK/PD models. 85 In fact, these assays have been readily organized into a preferred cascade, 86 making assay prioritization relatively straightforward. In this cascade, the earliest suggested activities focus on off-target profiling of initial lead drug candidates.…”

Section: Possibilities For Novel Tpd Modalitiesmentioning

confidence: 99%

Redefining the Scope of Targeted Protein Degradation: Translational Opportunities in Hijacking the Autophagy–Lysosome Pathway

Cassidy

Zhao

2021

Biochemistry

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The advent of multi-specific targeted protein degradation (TPD) therapies has made it possible to drug targets that have long been considered to be inaccessible. For this reason, the foremost TPD modalities - molecular glues and proteolysis targeting chimeras (PROTACs) -have been widely adopted and developed in therapeutic programs across the pharmaceutical and biotechnology industries. While there are many clear advantages to these two approaches, there are also blind spots. Specifically, PROTACs and molecular glues are inherently mechanistically analogous in that targets of both are degraded via the 26s proteasome; however, not all disease-relevant targets are suitable for ubiquitin proteasome system (UPS)-mediated degradation. The alternative mammalian protein degradation pathway, the autophagy–lysosome system (or ALS), is capable of degrading targets that elude the UPS such as long-lived proteins, insoluble protein aggregates, and even abnormal organelles. Emerging TPD strategies- such as ATTEC, AUTAC, and LYTAC- take advantage of the substrate diversity of the ALS to greatly expand the clinical utility of TPD. In this Perspective, we will discuss the array of current TPD modalities, with a focus on critical evaluation of these novel ALS-mediated degradation techniques.

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“…[23][24] Thus, there is an urgent need for robust, sensitive, and high-throughput methods that can determine off-target degradation in such PROTACs. 25 Currently, off-target degradation can be assessed by mass-spectrometry-based methods [26][27][28][29] that detect protein levels, but these techniques lack sensitivity for low abundant proteins. 30 In addition to expense, the implementation of mass spectrometry is technically challenging when analyses include profiling the off-target degradation affected by specific PROTACs across multiple tissue types for tissue-specific expression of lineage-specific proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Currently, off-target degradation can be assessed by mass–spectrometry-based methods 26–29 that detect protein levels, but these techniques lack sensitivity for low abundant proteins. 30 In addition to the expense, the implementation of mass spectrometry is technically challenging and requires examination PROTACs across multiple tissue types owing to lineage-specific protein expression.…”

Section: Introductionmentioning

confidence: 99%

Proteolysis Targeting Chimeras With Reduced Off-targets

Nguyen

Deb

Kokkonda

et al. 2021

Preprint

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Proteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce significant degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a rationalized library of pomalidomide analogs with distinct exit vector modifications on the C4 and C5 positions of the phthalimide ring and profiled their propensities for ZF protein degradation. We found that modifications on the C5 position with nucleophilic aromatic substitution (SNAr) reduce off-target ZF degradation. We applied our newfound design principles on a previously developed ALK oncoprotein-targeting PROTAC and generated PROTACs with enhanced potency and minimal off-target degradation. We envision the reported off-target profiling platform and pomalidomide analogs will find utility in design of specific PROTACs.

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The Vital Role of Proteomics in Characterizing Novel Protein Degraders

Cited by 14 publications

References 40 publications

Target and tissue selectivity of PROTAC degraders

Target and tissue selectivity of PROTAC degraders

Redefining the Scope of Targeted Protein Degradation: Translational Opportunities in Hijacking the Autophagy–Lysosome Pathway

Proteolysis Targeting Chimeras With Reduced Off-targets

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