ABSTRACT
The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.
Subject(s)
Fluorescence Polarization , High-Throughput Screening Assays , Microtubule-Associated Proteins , Protein Binding , Humans , Microtubule-Associated Proteins/metabolism , High-Throughput Screening Assays/methods , Fluorescence Polarization/methods , Apoptosis Regulatory Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Autophagy/drug effects , Peptides/metabolism , Peptides/chemistry , Ligands , Autophagy-Related Protein 8 Family/metabolismABSTRACT
Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Calorimetry , Microtubule-Associated Proteins , Protein Binding , Thermodynamics , Calorimetry/methods , Humans , Ligands , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/chemistry , Apoptosis Regulatory Proteins/metabolism , Apoptosis Regulatory Proteins/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Escherichia coli/metabolism , Peptides/chemistry , Peptides/metabolismABSTRACT
Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/ .
Subject(s)
Fluorescence Resonance Energy Transfer , Fluorescence Resonance Energy Transfer/methods , Crowdsourcing , Humans , Fluorescent Dyes/chemistry , Drug Discovery/methods , Ligands , Databases, Factual , Bioluminescence Resonance Energy Transfer Techniques/methods , Fluorescence Polarization/methodsABSTRACT
Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/ß-catenin pathway, which promotes the degradation of ß-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/ß-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of ß-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/ß-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate ß-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/ß KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-ß-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/ß-catenin pathway at the level of ß-catenin and Axin.
Subject(s)
Casein Kinase Ialpha , Wnt Signaling Pathway , beta Catenin , Humans , Alternative Splicing , beta Catenin/metabolism , beta Catenin/genetics , Casein Kinase Ialpha/metabolism , Casein Kinase Ialpha/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/genetics , HEK293 Cells , Phosphorylation , Wnt3A Protein/metabolism , Wnt3A Protein/geneticsABSTRACT
The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.
ABSTRACT
The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR-Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days.
Subject(s)
Luciferases , Proteolysis , Humans , Luciferases/metabolism , Luciferases/genetics , Genes, Reporter , Small Molecule Libraries/pharmacology , HEK293 Cells , Ubiquitin-Protein Ligases/metabolismABSTRACT
Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.
Subject(s)
Proteolysis , Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Humans , Ligands , Proteolysis/drug effects , Drug Design , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/metabolism , Molecular StructureABSTRACT
Bromodomain and extra-terminal domain (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors, such as pancreatic cancer. Here, we developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100 nM range in cellular NanoBRET target engagement assays. For one of our lead molecules, we could also show the selective inhibition of HDAC1/2 over all other zinc-dependent HDACs. Importantly, this on-target activity translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate the oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.
Subject(s)
Antineoplastic Agents , Carcinoma , Pancreatic Neoplasms , Humans , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/chemistry , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Pharmacophore , Pancreatic Neoplasms/drug therapy , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , RNA-Binding Proteins , Bromodomain Containing Proteins , Cell Cycle Proteins/metabolismABSTRACT
The multi-step degradation process of PROteolysis TArgeting Chimeras (PROTACs) poses a challenge for their rational development, as the rate-limiting steps that determine PROTACs efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here, we developed cell-based assays using the NanoLuciferase and HaloTag that allow measuring PROTAC-induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for the degradation of WD40 repeat domain protein 5 (WDR5), the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC-induced degradation pathway.
Subject(s)
Proteins , Ubiquitin-Protein Ligases , Proteolysis , Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Induction of Atg8-family protein (LC3/GABARAP proteins in human) interactions with target proteins of interest by proximity-inducing small molecules offers the possibility for novel targeted protein degradation approaches. However, despite intensive screening campaigns during the last 5 years, no potent ligands for LC3/GABARAPs have been developed, rendering this approach largely unexplored and unsuitable for therapeutic exploitation. In this Viewpoint, we analyze the reported attempts identifying LC3/GABARAP inhibitors and provide our own point of view why no potent inhibitors have been found. Additionally, we designate reasonable directions for the identification of potent and probably selective LC3/GABARAP inhibitors for alternative therapeutic applications.
ABSTRACT
Small molecule degraders such as PROTACs (PROteolysis TArgeting Chimeras) have emerged as new promising pharmacological modalities and the first PROTAC drug candidates are now studied clinically. The catalytic properties of PROTACs, acting as chemical degraders of a protein of interest (POI), represent an attractive new strategy for drug development. The development and characterization of PROTACs requires an array of additional assay systems that track the degradation pathway leading ultimately to degradation of the POI, identifying critical steps for PROTAC optimization. In addition to their exciting translational potential, PROTACs represent versatile chemical tools that considerably expanded our chemical biology toolbox and significantly enlarged the proteome that can be modulated by small molecules. Similar to conventional chemical probes, PROTACs used as chemical probes in target validation require comprehensive characterization. As a consequence, PROTAC-specific quality criteria should be defined by the chemical biology community. These criteria need to comprise additional or alternative parameters compared to those for conventional occupancy-driven chemical probes, such as the maximum level of target degradation (Dmax), confirmation of a proteasome dependent degradation mechanism and, importantly, also kinetic parameters of POI degradation. The kinetic aspects are particularly relevant for PROTACs that harbor covalent binding moieties. Here, we review recent progress in the development of assay systems for PROTAC characterization and suggest a set of criteria for PROTACs as high quality chemical probes.
Subject(s)
Proteasome Endopeptidase Complex , Proteome , Proteasome Endopeptidase Complex/metabolism , ProteolysisABSTRACT
E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.
ABSTRACT
Lysine acetylation creates docking sites for epigenetic reader domains of BET bromodomain proteins that have emerged as principal regulators of linage specific gene transcription. The development of potent and highly selective inhibitors, that have been soon widely available, enabled mechanistic studies in a diversity of disease models leading to a rapid translation into the clinic. Initial studies on pan-BET inhibitors lead now to second generation inhibitors with improved domain selectivity, but also to highly potent bifunctional and dual inhibitors extending the toolbox for basic research on acetylation dependent transcription, BET associated diseases and further translational efforts targeting this interesting family of epigenetic reader domains.
Subject(s)
Antineoplastic Agents , Transcription Factors , Acetylation , Lysine/metabolism , Protein Domains , Transcription Factors/metabolismABSTRACT
Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor, RMS-07, targeting a poorly conserved cysteine in the kinase's hinge region. RMS-07 shows potent MPS1 inhibitory activity and selectivity against all protein kinases with an equivalent cysteine but also in a broader kinase panel. We demonstrate potent cellular target engagement and pronounced activity against various cancer cell lines. The covalent binding mode was validated by mass spectrometry and an X-ray crystal structure. This proof of MPS1 covalent ligandability may open new avenues for the design of MPS1-specific chemical probes or drugs.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Cell Line, Tumor , Crystallography, X-Ray , Drug Design , Drug Screening Assays, Antitumor , Female , Humans , In Vitro Techniques , Male , Mass Spectrometry , Mice , Microsomes, Liver , Models, Molecular , Triple Negative Breast Neoplasms/drug therapyABSTRACT
The ribosomal protein S6 kinase beta 2 (S6K2) is thought to play an important role in malignant cell proliferation, but is understudied compared to its closely related homolog S6 kinase beta 1 (S6K1). To better understand the biological function of S6K2, chemical probes are needed, but the high similarity between S6K2 and S6K1 makes it challenging to selectively address S6K2 with small molecules. We were able to design the first potent and highly isoform-specific S6K2 inhibitor from a known S6K1-selective inhibitor, which was merged with a covalent inhibitor engaging a cysteine located in the hinge region in the fibroblast growth factor receptor kinase (FGFR) 4 via a nucleophilic aromatic substitution (SNAr) reaction. The title compound shows a high selectivity over kinases with an equivalently positioned cysteine, as well as in a larger kinase panel. A good stability towards glutathione and Nα-acetyl lysine indicates a non-promiscuous reactivity pattern. Thus, the title compound represents an important step towards a high-quality chemical probe to study S6K2-specific signaling.
ABSTRACT
Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.