Discovery of ASP6918, a KRAS G12C inhibitor: Synthesis and structure–activity relationships of 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivatives as covalent inhibitors with good potency and oral activity for the treatment of solid tumors

Tomoyoshi Imaizumi, Itsuro Shimada, Yoshiki Satake, Susumu Yamaki, Takanori Koike, Takahiro Nigawara, Osamu Kaneko, Yasushi Amano, Kenichi Mori, Yosuke Yamanaka, Ayako Nakayama, Yoshihiro Nishizono, Masashi Shimazaki, Takeyuki Nagashima, Kazuyuki Kuramoto,

Bioorganic & Medicinal Chemistry, 2023, 117581

https://doi.org/10.1016/j.bmc.2023.117581.

Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration.

Keywords: KRAS G12C mutation; non-small cell lung cancer; structure-based drug design; hydrophobic pocket; steric hindrance; oral activity

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors

Kristopher W. Hoyt, Daniel A. Urul, Blessing C. Ogboo, Florian Wittlinger, Stefan A. Laufer, Erik M. Schaefer, Earl W. May, and David E. Heppner

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01502

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure−kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

Using a Function-First “Scout Fragment”-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes

Jared R. Ramsey, Patrick M. M. Shelton, Tyler K. Heiss, Paul Dominic B. Olinares, Lauren E. Vostal, Heather Soileau, Michael Grasso, Sara W. Casebeer, Stephanie Adaniya, Michael Miller, Shan Sun, David J. Huggins, Robert W. Myers, Brian T. Chait, Ekaterina V. Vinogradova, and Tarun M. Kapoor

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c10581

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic “scout fragments”, which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells

Sobinoff, Alexander P.; Di Maro, Salvatore; Low, Ronnie R.J.; Benedetti, Rosaria; Tomassi, Stefano; D'Aniello, Antonia; Russo, Rosita; Baglivo, Ilaria; Chianese, Ugo; Pedone, Paolo V.; Chambery, Angela; Cesare, Anthony J.;  Altucci, Lucia; Pickett, Hilda A.,; Cosconati, Sandro

Cell Chemical Biology, 2023

 doi: 10.1016/j.chembiol.2023.11.008

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation

Hartmann, P., Bohdan, K., Hommrich, M. et al.Nat. Chem. 2023 

https://doi.org/10.1038/s41557-023-01388-7

Cysteine conjugation is an important tool in protein research and relies on fast, mild and chemoselective reactions. Cysteinyl thiols can either be modified with prefunctionalized electrophiles, or converted into electrophiles themselves for functionalization with selected nucleophiles in an independent step. Here we report a bioconjugation strategy that uses a vinyl thianthrenium salt to transform cysteine into a highly reactive electrophilic episulfonium intermediate in situ, to enable conjugation with a diverse set of bioorthogonal nucleophiles in a single step. The reactivity profile can connect several nucleophiles to biomolecules through a short and stable ethylene linker, ideal for introduction of infrared labels, post-translational modifications or NMR probes. In the absence of reactive exogenous nucleophiles, nucleophilic amino acids can react with the episulfonium intermediate for native peptide stapling and protein–protein ligation. Ready synthetic access to isotopologues of vinyl thianthrenium salts enables applications in quantitative proteomics. Such diverse applications demonstrate the utility of vinyl-thianthrenium-based bioconjugation as a fast, selective and broadly applicable tool for chemical biology.

Accelerating multiplexed profiling of protein-ligand interactions: High-throughput plate-based reactive cysteine profiling with minimal input

Ka Yang,Rebecca L. Whitehouse,Shane L. Dawson,Lu Zhang,Jeffrey G. Martin,Douglas S. Johnson,Joao A. Paulo,Steven P. Gygi,Qing Yu

Cell Chemical Biology 2023

DOI: https://doi.org/10.1016/j.chembiol.2023.11.015

Chemoproteomics has made significant progress in investigating small-molecule-protein interactions. However, the proteome-wide profiling of cysteine ligandability remains challenging to adapt for high-throughput applications, primarily due to a lack of platforms capable of achieving the desired depth using low input in 96- or 384-well plates. Here, we introduce a revamped, plate-based platform which enables routine interrogation of either ∼18,000 or ∼24,000 reactive cysteines based on starting amounts of 10 or 20 μg, respectively. This represents a 5–10X reduction in input and 2–3X improved coverage. We applied the platform to screen 192 electrophiles in the native HEK293T proteome, mapping the ligandability of 38,450 reactive cysteines from 8,274 human proteins. We further applied the platform to characterize new cellular targets of established drugs, uncovering that ARS-1620, a KRASG12C inhibitor, binds to and inhibits an off-target adenosine kinase ADK. The platform represents a major step forward to high-throughput proteome-wide evaluation of reactive cysteines.

Discovery of Orally Available and Brain Penetrant AEP Inhibitors

Daniela Krummenacher, Weiping He, Bernd Kuhn, Christian Schnider, Angélica Beurier, Virginie Brom, Thulase Sivasothy, Christine Marty, Andreas Tosstorff, David S. Hewings, Stefanie Mesch, Emmanuel Pinard, Mathis Brändlin, Remo Hochstrasser, Paul Westwood, Judith Rothe, Alexandra Kronenberger, Federica Morandi, Simon Gutbier, Angelika Schuler, Dominik Heer, Ludivine Esteves Gloria, Lisa Joedicke, Markus G. Rudolph, Lutz Müller, Fiona Grüninger, Karlheinz Baumann, Senthilvelrajan Kaniyappan, Nenad Manevski, and Björn Bartels

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c01804

Alzheimer’s Disease (AD) is the most widespread form of dementia, with one of the pathological hallmarks being the formation of neurofibrillary tangles (NFTs). These tangles consist of phosphorylated Tau fragments. Asparagine endopeptidase (AEP) is a key Tau cleaving enzyme that generates aggregation-prone Tau fragments. Inhibition of AEP to reduce the level of toxic Tau fragment formation could represent a promising therapeutic strategy. Here, we report the first orthosteric, selective, orally bioavailable, and brain penetrant inhibitors with an irreversible binding mode. We outline the development of the series starting from reversible molecules and demonstrate the link between inhibition of AEP and reduction of Tau N368 fragment both in vitro and in vivo.

Covalent Targeting of Splicing in T Cells

Kevin A. Scott, Hiroyuki Kojima, Nathalie Ropek, Charles D. Warren, Tiffany L. Zhang, Simon J. Hogg, Caroline Webster, Xiaoyu Zhang, Jahan Rahman, Bruno Melillo, Benjamin F. Cravatt, Jiankun Lyu, Omar Abdel-Wahab, Ekaterina V Vinogradova

bioRxiv 2023.12.18.572199; 

doi: https://doi.org/10.1101/2023.12.18.572199

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2 inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics

Tianyang Yan, Lisa M. Boatner, Liujuan Cui, Peter J. Tontonoz, and Keriann M. Backus

JACS Au 2023

DOI: 10.1021/jacsau.3c00707

The plasma membrane proteome is a rich resource of functionally important and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here, we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our “Cys-Surf” platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of low-density lipoprotein (LDL) particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders

Nathalie M. Grob, Clint Remarcik, Simon L. Rössler, Jeffrey Y. K. Wong, John C. K. Wang, Jason Tao, Corey L. Smith, Andrei Loas, Stephen L. Buchwald, Dan L. Eaton, Magdalena Preciado López, and Bradley L. Pentelute

ACS Chemical Biology 2023

DOI: 10.1021/acschembio.3c00538

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions with these topologies and advance the development of PPI-focused therapeutics, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand and demonstrate its application in a model PPI. Cysteine mutants of a known ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a model PPI with the 9-mer peptide antigen VL9 and major histocompatibility complex (MHC) class I protein HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 h. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94–NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential application of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

Qianni Peng, Eranthie Weerapana 

Cell Chemical Biology, 2023

DOI: https://doi.org/10.1016/j.chembiol.2023.11.010

The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy

Yasir S. Raouf, Abootaleb Sedighi, Mulu Geletu, Geordon A. Frere, Rebecca G. Allan, Nabanita Nawar, Elvin D. de Araujo, and Patrick T. Gunning

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c01236

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1–3, sub-μM activity in MV4–11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Covalent targeting of non-cysteine residues in PI4KIIIβ

Brett Cosgrove, Emma K. Grant, Sophie Bertrand, Kenneth D. Down, Don O. Somers, John P. Evans d, Nicholas C. O. Tomkinson and Michael D. Barker

RSC Chem. Biol., 2023, 4, 1111-1122

DOI: 10.1039/D3CB00142C 

The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIβ is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.

Covalent PROTAC design method based on a sulfonyl pyridone probe

Qinhong Luo, Yaqi Wang,   Zhanfeng Hou,  Huiting Liang,  Licheng Tu,  Yun Xing, Chuan Wan, Jianbo Liu,  Rui Wang, Lizhi Zhu, Wei Han,   Jianlong Wu,  Fei Lu,   Feng Yin   and  Zigang Li 

Chem. Commun., 2024

DOI: 10.1039/D3CC05127G

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel–Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 μM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.

A simple method for developing lysine targeted covalent protein reagents

Gabizon, Ronen; Tivon, Barr; Reddi, Rambabu N.; van den Oetelaar; Maxime C. M.; Amartely, Hadar; Cossar, Peter J.; Ottmann, Christian London, Nir

 Nat Commun 14, 7933 (2023).

https://doi.org/10.1038/s41467-023-42632-5

Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters—electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3σ via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration

Ahmed Fnaiche, Hwai-Chien Chan, Alexis Paquin, Narjara González Suárez, Victoria Vu, Fengling Li, Abdellah Allali-Hassani, Michelle Ada Cao, Magdalena M. Szewczyk, Albina Bolotokova, Frédéric Allemand, Muriel Gelin, Dalia Barsyte-Lovejoy, Vijayaratnam Santhakumar, Masoud Vedadi, Jean-François Guichou, Borhane Annabi, and Alexandre Gagnon

ACS Medicinal Chemistry Letters 2023

DOI: 10.1021/acsmedchemlett.3c00386

The transcription factor YAP–TEAD is the downstream effector of the Hippo pathway which controls cell proliferation, apoptosis, tissue repair, and organ growth. Dysregulation of the Hippo pathway has been correlated with carcinogenic processes. A co-crystal structure of TEAD with its endogenous ligand palmitic acid (PA) as well as with flufenamic acid (FA) has been disclosed. Here we report the development of HC-258, which derives from FA and possesses an oxopentyl chain that mimics a molecule of PA as well as an acrylamide that reacts covalently with TEAD’s cysteine. HC-258 reduces the CTGF, CYR61, AXL, and NF2 transcript levels and inhibits the migration of MDA-MB-231 breast cancer cells. Co-crystallization with hTEAD2 confirmed that HC-258 binds within TEAD’s PA pocket, where it forms a covalent bond with its cysteine.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active Site Inhibitor Cystargolide A

Illigmann, Astrid, Vielberg, Marie-Theres, Lakemeyer, Markus, Wolf, Felix, Dema, Taulant, Stange, Patrik, Kuttenlochner, Wolfgang, Liebhart, Elisa, Kulik, Andreas, Staudt, Nicole, Malik, Imran, Grond, Stephanie, Sieber, Stephan A., Kaysser, Leonard, Groll, Michael, Brötz-Oesterhelt, Heike, Angew. Chem. Int. Ed. 2023, e202314028.

https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202314028

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Here, we describe the potent cystargolides as the first natural β-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semi-synthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, as well as Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by β-lactones, the pioneering group of ClpP inhibitors.

Discovery of a Drug-like, Natural Product-Inspired DCAF11 Ligand Chemotype

Xue, G., Xie, J., Hinterndorfer, M. et al. Discovery of a Drug-like, Natural Product-Inspired DCAF11 Ligand Chemotype. Nat Commun 14, 7908 (2023). https://doi.org/10.1038/s41467-023-43657-6

Targeted proteasomal and autophagic protein degradation, often employing bifunctional modalities, is a new paradigm for modulation of protein function. In an attempt to explore protein degradation by means of autophagy we combine arylidene-indolinones reported to bind the autophagy-related LC3B-protein and ligands of the PDEδ lipoprotein chaperone, the BRD2/3/4-bromodomain containing proteins and the BTK- and BLK kinases. Unexpectedly, the resulting bifunctional degraders do not induce protein degradation by means of macroautophagy, but instead direct their targets to the ubiquitin-proteasome system. Target and mechanism identification reveal that the arylidene-indolinones covalently bind DCAF11, a substrate receptor in the CUL4A/B-RBX1-DDB1-DCAF11 E3 ligase. The tempered α, β-unsaturated indolinone electrophiles define a drug-like DCAF11-ligand class that enables exploration of this E3 ligase in chemical biology and medicinal chemistry programs. The arylidene-indolinone scaffold frequently occurs in natural products which raises the question whether E3 ligand classes can be found more widely among natural products and related compounds.