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.

 

Lysine-Reactive N-Acyl-N-aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant

Masaharu Kawano, Syunsuke Murakawa, Kenji Higashiguchi, Kenji Matsuda, Tomonori Tamura, and Itaru Hamachi

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c08740

The covalent inhibition of a target protein has gained widespread attention in the field of drug discovery. Most of the current covalent drugs utilize the high reactivity of cysteines toward modest electrophiles. However, there is a growing need for warheads that can target lysine residues to expand the range of covalently druggable proteins and to deal with emerging proteins with mutations resistant to cysteine-targeted covalent drugs. We have recently developed an N-acyl-N-alkyl sulfonamide (NASA) as a lysine-targeted electrophile. Despite its successful application, this NASA warhead suffered from instability in physiological environments, such as serum-containing medium, because of its high intrinsic reactivity. In this study, we sought to modify the structure of the NASA warhead and found that N-acyl-N-aryl sulfonamides (ArNASAs) are promising electrophiles for use in a lysine-targeted covalent inhibition strategy. We prepared a focused library of ArNASA derivatives with diverse structures and reactivity and identified several warhead candidates with suppressed hydrolysis-mediated inactivation and reduced nonspecific reactions with off-target proteins, without sacrificing the reactivity toward the target. These reaction properties enabled the improved covalent inhibition of intracellular heat shock protein 90 (HSP90) in the presence of serum and the development of the first irreversible inhibitor for ibrutinib-resistant Bruton’s tyrosine kinase (BTK) bearing the C481S mutation. This study clearly demonstrated the use of a set of ArNASA warheads to create highly potent covalent drugs and highlighted the importance of enriching the current arsenal of lysine-reactive warheads.

Real-time monitoring of the reaction of KRAS G12C mutant specific covalent inhibitor by in vitro and in-cell NMR spectroscopy

Qingci Zhao, Ryoka Haga, Satoko Tamura, Ichio Shimada & Noritaka Nishida 

Sci Rep 13, 19253 (2023). 

https://doi.org/10.1038/s41598-023-46623-w

KRAS mutations are major drivers of various cancers. Recently, allele-specific inhibitors of the KRAS G12C mutant were developed that covalently modify the thiol of Cys12, thereby trapping KRAS in an inactive GDP-bound state. To study the mechanism of action of the covalent inhibitors in both in vitro and intracellular environments, we used real-time NMR to simultaneously observe GTP hydrolysis and inhibitor binding. In vitro NMR experiments showed that the rate constant of ARS-853 modification is identical to that of GTP hydrolysis, indicating that GTP hydrolysis is the rate-limiting step for ARS-853 modification. In-cell NMR analysis revealed that the ARS-853 reaction proceeds significantly faster than that in vitro, reflecting acceleration of GTP hydrolysis by endogenous GTPase proteins. This study demonstrated that the KRAS covalent inhibitor is as effective in the cell as in vitro and that in-cell NMR is a valuable validation tool for assessing the pharmacological properties of the drug in the intracellular context.

Assessing Squarates as Amine-Reactive Probes

Katherine I. Taylor, Jordan S. Ho, Hallie O. Trial, Alan W. Carter, and Laura L. Kiessling

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.2c05691

Probes that covalently label protein targets facilitate the identification of ligand-binding sites. Lysine residues are prevalent in the proteome, making them attractive substrates for covalent probes. However, identifying electrophiles that undergo amine-specific, regioselective reactions with binding site lysine residues is challenging. Squarates can engage in two sequential conjugate addition–elimination reactions with amines. Nitrogen donation reduces the second reaction rate, making the mono squaramide a mild electrophile. We postulated that this mild electrophilicity would demand a longer residence time near the amine, affording higher selectivity for binding site lysines. Therefore, we compared the kinetics of squarate and monosquaramide amine substitution to alternative amine bioconjugation handles. The data revealed that N-hydroxy succinimidyl esters react 4 orders of magnitude faster, consistent with their labeling promiscuity. Squarate reactivity can be tuned by a substitution pattern. Electron-withdrawing groups on the vinylogous ester or amide increase reaction rates. Dithionosquarates react more rapidly than squarates, while vinylogous thioester analogs, dithiosquarates, react more slowly. We assessed squarate selectively using the UDP-sugar processing enzyme GlfT2 from Mycobacterium tuberculosis, which possesses 21 surface-exposed lysines. The reaction predominately modified one lysine proximal to a binding site to afford covalent inhibition. These findings demonstrate the selectivity of squaric esters and squaramides, which is a critical feature for affinity-based chemoproteomic probes.

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs

Wouter van Bergen, Kristina Žuna, Jan Fiala, Elena E. Pohl, Albert J.R. Heck, and Marc P. Baggelaar

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.3c00637

preprint: https://doi.org/10.1101/2023.10.19.562725

Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-targets, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy, PhosID-ABPP, on a timsTOF HT mass spectrometer. This refinement enables dual dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely PF-06672131 and PF-6422899, facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, while both ABPs showed comparable affinities for the EGFR, PF-06672131 had a broader off-target reactivity profile. In contrast, PF-6422899 exhibited higher affinity for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF-06672131 also effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nanomolar. Additionally, analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on– and off-target engagement in a dose– and site-specific manner.

Graph Neural Networks for Identifying Protein-Reactive Compounds

Cano Gil, V. H.; Rowley, C. N.

ChemRxiv 2023. 

https://doi.org/10.26434/chemrxiv-2023-d0dqp

The identification of protein-reactive electrophilic compounds is critical to the design of new covalent modifier drugs, screening for toxic compounds, and the exclusion of reactive compounds from high throughput screening. In this work, we employ traditional and graph machine learning algorithms to classify molecules being reactive towards proteins or nonreactive. For training data, we built a new dataset, ProteinReactiveDB, comprised primarily of covalent and noncovalent inhibitors from DrugBank, BindingDB, and CovalentInDB databases. To assess the transferability of the trained models, we created a custom set of covalent and noncovalent inhibitors, which was constructed from recent literature. Baseline models were developed using Morgan fingerprints as training inputs, but they performed poorly when applied to compounds outside the training set. We then trained various Graph Neural Networks (GNNs), with the best GNN model achieving an Area Under the Receiver Operator Characteristic (AUROC) curve of 0.84, precision of 0.92, and recall of 0.73. We also explore the interpretability of these GNNs using Gradient Activation Mapping (GradCAM), which shows regions of the molecules GNNs deem most relevant when making a prediction. These maps indicated that our trained models can identify electrophilic functional groups in a molecule and classify molecules as protein-reactive based on their presence.

Covalent 14-3-3 Molecular Glues and Heterobifunctional Molecules Against Nuclear Transcription Factors and Regulators

Qian Shao, Tuong Nghi Duong, Inji Park, Daniel K Nomura

bioRxiv 2023.11.06.565850; 

doi: https://doi.org/10.1101/2023.11.06.565850

14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates to sequester and inhibit their function has remained elusive. One of the 14-3-3 proteins, 14-3-3 or SFN, has a cysteine C38 at the substrate binding interface near sites where previous 14-3-3σ molecular glues have been found to bind. In this study, we screened a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance interaction of 14-3-3σ with not only druggable transcription factors such as estrogen receptor (ERα), but also challenging oncogenic transcription factors such as YAP and TAZ that are part of the Hippo transducer pathway. We identified a hit EN171 that covalently targets 14-3-3 to enhance 14-3-3 interactions with ERα, YAP, and TAZ leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions, but also could be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and also demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels

Léa N. C. Rochet,  Calise Bahou,  Jonathan P. Wojciechowski,   Ilias Koutsopetras,   Phyllida Britton,   Richard J. Spears, ORCID logo a   Ioanna A. Thanasi,   Baihao Shao,   Lisha Zhong,   Dejan-Krešimir Bučar,    Abil E. Aliev,   Michael J. Porter,   Molly M. Stevens,  James R. Baker, and  Vijay Chudasama

Chem. Sci. 2023

https://doi.org/10.1039/D3SC04976K

Reversible cysteine modification has been found to be a useful tool for a plethora of applications such as selective enzymatic inhibition, activity-based protein profiling and/or cargo release from a protein or a material. However, only a limited number of reagents display reliable dynamic/reversible thiol modification and, in most cases, many of these reagents suffer from issues of stability, a lack of modularity and/or poor rate tunability. In this work, we demonstrate the potential of pyridazinediones as novel reversible and tuneable covalent cysteine modifiers. We show that the electrophilicity of pyridazinediones correlates to the rates of the Michael addition and retro-Michael deconjugation reactions, demonstrating that pyridazinediones provide an enticing platform for readily tuneable and reversible thiol addition/release. We explore the regioselectivity of the novel reaction and unveil the reason for the fundamental increased reactivity of aryl bearing pyridazinediones by using DFT calculations and corroborating findings with SCXRD. We also applied this fundamental discovery to making more rapid disulfide rebridging agents in related work. We finally provide the groundwork for potential applications in various areas with exemplification using readily functionalised “clickable” pyridazinediones on clinically relevant cysteine and disulfide conjugated proteins, as well as on a hydrogel material.

Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction

 Benjamin P. M. Lake and Anthony F. Rullo

ACS Central Science 2023

DOI: 10.1021/acscentsci.3c01052

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate–protein binding interactions (Kd ≈ 10–3–10–6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional “proximity-inducing” molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large kinact/KI), driven by a favorably small KI for binding. We hypothesized the much less favorable KI associated with carbohydrate–protein binding interactions can be offset by a favorably large kinact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

Ashley R Julio, Flowreen Shikwana, Cindy Truong, Nikolas R Burton, Emil Dominguez, Alexandra Turmon, Jian Cao, Keriann Backus

bioRxiv 2023.10.30.564067; 

doi: https://doi.org/10.1101/2023.10.30.564067

Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of undruggable protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 non-structural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.

Covalent Degrader of the Oncogenic Transcription Factor β-Catenin

Flor A Gowans, Nafsika Forte, Justin Hatcher, Yangzhi Wang, Belen E Altamirano Poblano, Ingrid E Wertz, Daniel K Nomura

bioRxiv 2023.10.31.565018; 

doi: https://doi.org/10.1101/2023.10.31.565018

β-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drives the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging deeming this transcription factor as undruggable. Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify a monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin proteasome dependent manner. We show that EN83 directly and covalently targets CTNNB1 through targeting four distinct cysteines within the armadillo repeat domain (C439, C466, C520, and C619) leading to a destabilization of CTNNB1. Using covalent chemoproteomic approaches, we show that EN83 directly engages CTNNB1 in cells with a moderate degree of selectivity. We further demonstrate that direct covalent targeting of three of these four cysteines (C466, C520, and C619) in cells contributes to CTNNB1 degradation in cells. We also demonstrate that EN83 can be further optimized to yield more potent CTNNB1 binders and degraders. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through a destabilization-mediated degradation.

Covalent and noncovalent strategies for targeting Lys102 in HIV-1 reverse transcriptase

Giavana R. Prucha, Sean Henry, Klarissa Hollander, Zachary J. Carter, Krasimir A. Spasov, William L. Jorgensen, Karen S. Anderson,

European Journal of Medicinal Chemistry, 2023, 262, 115894, 

https://doi.org/10.1016/j.ejmech.2023.115894

Reverse transcriptase (RT) is one of three key proteins responsible for the replication cycle of HIV-1 in the host. Several classes of inhibitors have been developed to target the enzyme, with non-nucleoside reverse transcriptase inhibitors forming first-line treatment. Previously, covalent RT inhibitors have been identified and found to bind irreversibly to commonly mutated residues such as Y181C. In this work we aim to circumvent the issue of NNRTI resistance through targeting K102, which has not yet been identified to confer drug resistance. As reported here, 34 compounds were synthesized and characterized biochemically and structurally with wild-type (WT) HIV-1 RT. Two of these inhibitors demonstrate covalent inhibition as evidenced by protein crystallography, enzyme kinetics, mass spectrometry, and antiviral potency in HIV-1 infected human T-cell assays.