ABSTRACT
[This corrects the article DOI: 10.1016/j.isci.2023.107059.].
ABSTRACT
Small molecules that induce protein degradation hold the potential to overcome several limitations of the currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activityâhow can we convert an inhibitor into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, we demonstrate that an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are tolerated, including both aromatic and nonaromatic groups. We used these findings to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and confirm the mechanism of degradation. These results suggest that general design principles can be generated for the development and optimization of monovalent degraders.
Subject(s)
Cyclins , Proteolysis , Cell Cycle Checkpoints , Cyclins/metabolismABSTRACT
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , ProteolysisABSTRACT
To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.
ABSTRACT
Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.
Subject(s)
Colorectal Neoplasms , Proteomics , Humans , Proteome/analysis , Proteomics/methods , Reproducibility of Results , WorkflowABSTRACT
The use of phenotypic screening was central to the discovery and development of novel thalidomide analogs, the IMiDs (immunomodulatory drugs) agents. With the discovery that these agents bind the E3 ligase, CRL4CRBN, and alter its substrate specificity, there has been a great deal of endeavor to discover other small molecules that can modulate alternative E3 ligases. Furthermore, the chemical properties necessary for drug discovery and the rules by which neo-substrates are selected for degradation are being defined in the context of phenotypic alterations in specific cellular systems. This review gives a detailed summary of these recent advances and the methodologies being exploited to understand the mechanism of action of emerging protein degradation therapies.
Subject(s)
Drug Development , Immunologic Factors/pharmacology , Ubiquitin-Protein Ligases/metabolism , Drug Evaluation, Preclinical , Humans , Immunologic Factors/chemistry , PhenotypeABSTRACT
Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (γ-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression.
Subject(s)
Centrosome/metabolism , Microtubules/metabolism , Muscle Proteins/metabolism , Nuclear Proteins/metabolism , A Kinase Anchor Proteins/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Cell Cycle Proteins , Cell Proliferation , Cytoskeletal Proteins/metabolism , G1 Phase Cell Cycle Checkpoints , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Muscle Proteins/chemistry , Muscle Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.
Subject(s)
Cell Communication/genetics , Exosomes/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Oncogene Protein pp60(v-src)/genetics , Syntenins/genetics , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Amino Acid Motifs , Cell Movement , Cell Proliferation , Culture Media, Conditioned/pharmacology , Endocytosis , Endosomes/metabolism , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , MCF-7 Cells , Oncogene Protein pp60(v-src)/metabolism , Phospholipase D/genetics , Phospholipase D/metabolism , Phosphorylation , Signal Transduction , Syndecans/genetics , Syndecans/metabolism , Syntenins/metabolismABSTRACT
Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT-actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration.