Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.

Measuring Ligand-bound Protein Complexes with Proximity Labeling: A Practical Guide.

Identifying the drug-target interactome of small molecule therapeutics is essential for understanding the full pharmacological effects of a compound. These therapies often induce changes within the cellular proteome, leading to unexpected consequences such as changes in the targets complexation state or off-target interactions between the compound and additional proteins. Currently, unbiased target-ID approaches are being used to embark on this task. Here we provide an overview of the strengths and limitations of these methods, and a practical step-by-step protocol for using the BioTAC system to assist with drug target and interactome ID.

Discovery of a selective inhibitor of doublecortin like kinase 1.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1OE -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1PAR ). Quantitative proteome analysis of MKN1OE -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1PAR -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.

Synthesis and structure activity relationships of a series of 4-amino-1H-pyrazoles as covalent inhibitors of CDK14.

The TAIRE family of kinases are an understudied branch of the CDK kinase family, that have been implicated in a number of cancers. This manuscript describes the design, synthesis and SAR of covalent CDK14 inhibitors, culminating in identification of FMF-04-159-2, a potent, covalent CDK14 inhibitor with a TAIRE kinase biased selectivity profile.

Characterization of a highly selective inhibitor of the Aurora kinases.

Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases have proved popular cancer targets and many inhibitors have been developed. The majority of these clinical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants of ligand binding and selectivity.

Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.

Pan-RAF:MEK Molecular Glues Take Center Stage.

In this issue, Ryan and colleagues describe the preclinical development of a pan-RAF:MEK molecular glue with superior efficacy, brain penetrance, and tolerability in xenograft models of Ras/Raf/MAPK pathway-driven tumors. See related article by Ryan et al., p. 1190 (1).

Application of induced proximity for therapeutic discovery.

In this Voices piece, the Cell Chemical Biology editors ask researchers from a range of backgrounds: what are some exciting discoveries in the induced proximity field and the next frontier for therapeutic development?

ZBTB11 Depletion Targets Metabolic Vulnerabilities in K-Ras Inhibitor Resistant PDAC.

Over 95% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic mutations in K-Ras. Upon treatment with K-Ras inhibitors, PDAC cancer cells undergo metabolic reprogramming towards an oxidative phosphorylation-dependent, drug-resistant state. However, direct inhibition of complex I is poorly tolerated in patients due to on-target induction of peripheral neuropathy. In this work, we develop molecular glue degraders against ZBTB11, a C2H2 zinc finger transcription factor that regulates the nuclear transcription of components of the mitoribosome and electron transport chain. Our ZBTB11 degraders leverage the differences in demand for biogenesis of mitochondrial components between human neurons and rapidly-dividing pancreatic cancer cells, to selectively target the K-Ras inhibitor resistant state in PDAC. Combination treatment of both K-Ras inhibitor-resistant cell lines and multidrug resistant patient-derived organoids resulted in superior anti-cancer activity compared to single agent treatment, while sparing hiPSC-derived neurons. Proteomic and stable isotope tracing studies revealed mitoribosome depletion and impairment of the TCA cycle as key events that mediate this response. Together, this work validates ZBTB11 as a vulnerability in K-Ras inhibitor-resistant PDAC and provides a suite of molecular glue degrader tool compounds to investigate its function.

DCLK1 induces a pro-tumorigenic phenotype to drive gastric cancer progression.

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell-intrinsic and-extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1D511N). MKN1D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1DCLK1) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

A new avenue for molecular glues: Rapid discovery of a NFKB1 degrader.

Targeted protein degradation using molecular glues is a powerful method for targeting traditionally undruggable proteins. One challenge in molecular glue discovery is the absence of rational discovery methods. Here, King et al. leverage covalent library screening with chemoproteomics platforms to rapidly discover a molecular glue targeting NFKB1 via UBE2D recruitment.

A Biotin Targeting Chimera (BioTAC) System to Map Small Molecule Interactomes in situ.

Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.

A biotin targeting chimera (BioTAC) system to map small molecule interactomes in situ.

Understanding how small molecules bind to specific protein complexes in living cells is critical to understanding their mechanism-of-action. Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules would provide advantages over target-focused approaches, including the ability to detect previously unknown ligand targets and complexes. However, there are few current methods for unbiased profiling of small molecule interactomes. To address this, we envisioned a technology that would combine the sensitivity and live-cell compatibility of proximity labelling coupled to mass spectrometry, with the specificity and unbiased nature of chemoproteomics. In this manuscript, we describe the BioTAC system, a small-molecule guided proximity labelling platform that can rapidly identify both direct and complexed small molecule binding proteins. We benchmark the system against µMap, photoaffinity labelling, affinity purification coupled to mass spectrometry and proximity labelling coupled to mass spectrometry datasets. We also apply the BioTAC system to provide interactome maps of Trametinib and analogues. The BioTAC system overcomes a limitation of current approaches and supports identification of both inhibitor bound and molecular glue bound complexes.

Targeted Protein Degradation: Design Considerations for PROTAC Development.

Targeted protein degradation has recently gained widespread interest as both a novel therapeutic strategy and a useful tool in biomedical research. Targeted protein degraders are often sub-stoichiometric and do not require strong binding affinity for their targets, enabling access to previously inaccessible targets. Proteolysis-targeting chimeras (PROTACs) are one class of targeted protein degraders that promote degradation by recruiting a target protein to an E3-ligase complex via a heterobifunctional molecule. The modular nature of PROTACs allows for their rational design and systematic optimization. Here we suggest resources and methodologies for developing PROTAC degraders for researchers that may be new to the field. © 2022 Wiley Periodicals LLC.

Targeted protein degradation: Emerging concepts and protein state-specific targeting principles.

Targeted protein degraders are heterobifunctional small molecules that link a target ligand or bait to an E3-ligase binder via a chemical spacer. Upon entering the cell, these ligands trigger the formation of a ternary complex between the target protein, degrader and E3-ligase, which leads to target polyubiquitination and proteasomal degradation. In recent years, TPD has expanded rapidly as a field, becoming the modality of choice in drug discovery and chemical probe development. This has been driven by the unique pharmacology of these molecules, which allows for fast and reversible knockdown of the target protein. Recent studies have demonstrated that degraders with specificity for a defined subpopulation of a protein-of-interest can be developed, giving rise to the emerging concept of protein state-specific targeting. In this article, we review advances towards developing degraders that differentiate between target protein subpopulations based on their; activation state, oligomerization state, cellular localization state, and cell type.

Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy.

Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties.