Exploring Targeted Degradation Strategy for Oncogenic KRASG12C

Mei Zeng, Yuan Xiong, Nozhat Safaee, Radoslaw P. Nowak, Katherine A. Donovan, Christine J. Yuan, Behnam Nabet, Thomas W. Gero, Frederic Feru, Lianbo Li, Sudershan Gondi, Lincoln J. Ombelets, Chunshan Quan, Pasi A. Jänne, Milka Kostic, David A. Scott, Kenneth D. Westover, Eric S. Fischer Nathanael S. Gray

Cell Chemical Biology, 2019

DOI: https://doi.org/10.1016/j.chembiol.2019.12.006.

KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, KRAS G12C can be targeted by small-molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Our lead degrader successfully engaged CRBN in cells, bound KRAS G12C in vitro, induced CRBN/KRAS G12C dimerization, and degraded GFP-KRAS G12C in reporter cells in a CRBN-dependent manner. However, it failed to degrade endogenous KRAS G12C in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively poly-ubiquitinate endogenous KRAS G12C underlies the lack of activity. We discuss challenges for achieving targeted KRAS G12C degradation and proposed several possible solutions which may lead to efficient degradation of endogenous KRAS G12C.

A chemical proteomic probe for the mitochondrial pyruvate carrier complex

Yamashita, Y., Vinogradova, E., Zhang, X., Suciu, R. and Cravatt, B.

Angew. Chem. Int. Ed.. 2019 
doi:10.1002/anie.201914391

Target engagement assays are crucial for establishing the mechanism‐of‐action of small molecules in living systems. Integral membrane transporters, due to their specialized biophysical properties and activity assays, can present a challenging protein class for assessing cellular engagement by small molecules. Here, we describe the chemical proteomic discovery of alpha‐chloroacetamide (aCA) compounds that covalently modify cysteine‐54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) complex. We leverage this finding to create an alkyne‐modified aCA, YY4‐yne, that serves as a versatile cellular target engagement probe for MPC2 in click chemistry‐enabled western blotting or global mass spectrometry‐based proteomic experiments. Using YY4‐yne, we demonstrate that UK‐5099, an alpha‐cyanocinnamate inhibitor of the MPC complex, first discovered more than 30 years ago, but still with a poorly defined mechanism‐of‐action, engages MPC2 with remarkable selectivity in human cells. These findings support a model where UK‐5099 inhibits the MPC complex by binding to C54 of MPC2 in a covalent reversible manner that can be quantified in cells using the YY4‐yne probe.

Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

Kathrin Bach Bert L. H. Beerkens Patrick R. A. Zanon Stephan M. Hacker
ChemRxiv, 2019
doi: 10.26434/chemrxiv.11352101.v1

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72

Jonathan Pettinger, Michael Carter, Keith Jones, and Matthew D. Cheeseman

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01709

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cysteine residues as nucleophiles but the low abundance of cysteine in the proteome has limited its application. We have recently reported our discovery that lysine-56 in the difficult-to-drug target HSP72 could form a covalent bond with a small-molecule inhibitor. We now disclose the optimization of these targeted covalent inhibitors using rational design. Essential to our optimization was the development of a new covalent fluorescence polarization assay, which allows for the direct measurement of the key kinetic parameter in covalent inhibitor design, kinact/KI, extrapolation of the underlying parameters, kinact and Ki, and direct comparison to reversible analogues. Using our approach, we demonstrate a >100-fold enhancement in covalent efficiency and key learnings in lysine-selective electrophile optimization.

Discovery of a covalent inhibitor of KRASG12C (AMG 510) for the treatment of solid tumors

Brian A Lanman, Jennifer R. Allen, John G. Allen, Albert K Amegadzie, Kate S. Ashton, Shon K. Booker, Jian Jeffrey Chen, Ning Chen, Michael J Frohn, Guy Goodman, David J Kopecky, Longbin Liu, Patricia Lopez, Jonathan D Low, Vu Ma, Ana Elena Minatti, Thomas T Nguyen, Nobuko Nishimura, Alexander J. Pickrell, Anthony B. Reed, Youngsook Shin, Aaron Siegmund, Nuria A. Tamayo, Christopher M Tegley, Mary C Walton, Hui-Ling Wang, Ryan P. Wurz, May Xue, Kevin C Yang, Pragathi Achanta, Michael D. Bartberger, Jude Canon, L Steven Hollis, John D McCarter, Christopher Mohr, Karen Rex, Anne Y Saiki, Tisha San Miguel, Laurie Volak, Kevin H Wang, Douglas A. Whittington, Stephan G Zech, J. Russell Lipford, and Victor J. Cee

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01180

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-“undruggable” target, however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in Phase I clinical trials (NCT03600883).

Global targeting of functional tyrosines using sulfur-triazole exchange chemistry

Heung Sik Hahm, Emmanuel K. Toroitich, Adam L. Borne, Jeffrey W. Brulet, Adam H. Libby, Kun Yuan, Timothy B. Ware, Rebecca L. McCloud, Anthony M. Ciancone & Ku-Lung Hsu

Nat Chem Biol (2019) doi:10.1038/s41589-019-0404-5

Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.

Use of Pyridazinediones as Extracellular Cleavable Linkers Through Reversible Cysteine Conjugation

Calise Bahou , Richard Spears , Abil Aliev , Antoine Maruani , Marcos Fernandez , Faiza Javaid , Peter Szijj , James Baker and Vijay Chudasama

Chem. Commun. 2019
DOI: 10.1039/C9CC08362F

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-target modification with blood thiols (e.g. glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker.

Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina X. Van Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Matthew Harger, Clint D. J. Tavares, Jihyun Park, Mohamed F. Radwan, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose & Kevin N. Dalby

Nat. Commun. 2019, 10, 5232

DOI: https://doi.org/10.1038/s41467-019-12996-8

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.

Structure–Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Theresa D. Manz, Sindhu C. Sivakumaren, Adam Yasgar, Matthew D. Hall, Mindy I. Davis, Hyuk-Soo Seo, Joseph D. Card, Scott B. Ficarro, Hyeseok Shim, Jarrod A. Marto, Sirano Dhe-Paganon, Atsuo T. Sasaki, Matthew B. Boxer, Anton Simeonov, Lewis C. Cantley, Min Shen, Tinghu Zhang, Fleur M. Ferguson, and Nathanael S. Gray

ACS Medicinal Chemistry Letters  2019, DOI: 10.1021/acsmedchemlett.9b00402

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors B-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Crosslinking with Michael Acceptor Motif-Containing Drugs [@TateScience]

Ward, J.; Pinto-Fernández, A.; Cornelissen, L.; Bonham, S.; Saez, L. D.; Riant, O.; Huber, K.; Kessler, B. M.; Feron, O.; Tate, E. W

ChemRxiv. 2019

doi: 10.26434/chemrxiv.10028444.v1

Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors with a shared α,β-unsaturated carbonyl substructure motif. Initially taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has since been put on full clinical hold due to dose limiting toxicity. Through a proteomic approach, here we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation through cross-linking, providing a molecular explanation for general cellular toxicity.

The KRASG12C Inhibitor, MRTX849, Provides Insight Toward Therapeutic Susceptibility of KRAS Mutant Cancers in Mouse Models and Patients

James G. Christensen, Jill Hallin, Lars D Engstrom, Lauren Hargis, Andrew Calinisan, Ruth Aranda, David M Briere, Niranjan Sudhakar, Vickie Bowcut, Brian R Baer, Joshua A Ballard, Michael R Burkard, Jay B Fell, John P Fischer, Guy P Vigers, Jenny Y Xue, Sole Gatto, Julio Fernandez-Banet, Adam Pavlicek, Karen Velastegui, Richard C Chao, Jeremy Barton, Mariaelena Pierobon, Elisa Baldelli, Emmanuel F Patricoin, Douglas P Cassidy, Matthew A Marx, Igor I Rybkin, Melissa L Johnson, Sai-Hong Ignatius Ou, Piro Lito, Kyriakos P. Papadopoulos, Pasi A Janne and Peter Olson

Cancer Discov.  2019 DOI: 10.1158/2159-8290.CD-19-1167
DOI: 10.1158/2159-8290.CD-19-1167

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) of KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types and objective responses have been observed in KRASG12C-positive lung and colon adenocarcinoma patients. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant non-clinical models identified mechanisms implicated in limiting anti-tumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of RTKs, bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models.

Recent Advances in Selective and Irreversible Covalent Ligand Development and Validation

Tinghu Zhang, John M. Hatcher, Mingxing Teng, Nathanael S. Gray, Milka Kostic

Cell Chemical Biology, 2019
doi: 10.1002/wcms.1446

Some of the most widely used drugs, such as aspirin and penicillin, are covalent drugs. Covalent binding can improve potency, selectivity, and duration of the effects, but the intrinsic reactivity represents a potential liability and may result in idiosyncratic toxicity. For decades, the cons were believed to outweigh the pros, and covalent targeting was deprioritized in drug discovery. Recently, several covalent inhibitors have been approved for cancer treatment, thus rebooting the field. In this review, we briefly reflect on the history of selective covalent targeting, and provide a comprehensive overview of emerging developments from a chemical biology stand-point. Our discussion will reflect on efforts to validate irreversible covalent ligands, expand the scope of targets, and discover new ligands and warheads. We conclude with a brief commentary of remaining limitations and emerging opportunities in selective covalent targeting.

Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity [@dannomura]

Yosuke Isobe, Mikiko Okumura, Ross White, Lynn M McGregor, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Thomas J Maimone, Daniel K Nomura

BioRxiv, 2019
doi: https://www.biorxiv.org/content/10.1101/814285v1

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Ekaterina Vinogradova, Daniel Lazar, Radu Suciu, Yujia Wang, Giulia Bianco, Yu Yamashita, Vincent Crowley, Dave Remillard, Kenneth Lum, Gabriel Simon, Esther Kemper, Michael Lazear, Sifei Yin, Megan Blewett, Melissa Dix, Nhan Nguyen, Maxim Shokhirev, Emily Chin, Luke Lairson, Stefano Forli, John Teijaro, Benjamin Cravatt
BioRxiv, 2019
doi: https://www.biorxiv.org/content/10.1101/808113v1

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Synthesis of peptides with cysteine sulfinic acid via the cysteine methoxybenzyl sulfone

Urmey, AR, Zondlo, NJ.

Pept Sci. 2019; e24137.  
doi: https://doi.org/10.1002/pep2.24137

Cysteine sulfinic acid is a protein posttranslational modification that is formed under oxidative conditions and is regulated both enzymatically and nonenzymatically. Cysteine oxidation to the sulfinic acid has been observed broadly throughout the proteome and can induce activation or inhibition of function in proteins. Recently, wide‐scale, reversible regulation of the sulfinic acid state of cysteine within proteins was identified, posing new questions in cysteine sulfinic acid biology. Existing methods to synthesize peptides with cysteine sulfinic acid can suffer from low yield, due to the formation of side products in the disulfide, sulfenic acid, and/or sulfonic acid oxidation states. Herein, a method for the synthesis of peptides with cysteine sulfinic acids was developed, via protection of cysteine sulfinic acid as the methoxybenzyl (Mob) sulfone. Cysteine Mob sulfone was synthesized as an Fmoc amino acid in one step from the commercially available Mob‐protected Fmoc‐cysteine (Fmoc‐Cys(Mob)‐OH). This amino acid was directly incorporated into peptides via solid‐phase peptide synthesis. Alternatively, peptides were synthesized using Fmoc‐Cys(Mob)‐OH, followed by subsequent oxidation within peptides of the thioether to the Mob sulfone via H2O2 and catalytic niobium carbide. Deprotection of peptides under strongly acidic conditions (50% triflic acid, 45% trifluoroacetic acid, 5% water) generated peptides with cysteine sulfinic acid. This approach was applied to the synthesis of peptides containing cysteine sulfinic acid within diverse peptide sequence contexts.

Covalent‐Allosteric Inhibitors to Achieve Akt Isoform‐Selectivity

Quambusch, L. , Landel, I. , Depta, L. , Weisner, J. , Uhlenbrock, N. , Müller, M. P., Glanemann, F. , Althoff, K. , Siveke, J. T. and Rauh, D.

Angew. Chem. Int. Ed., 2019

doi: 10.1002/anie.201909857

Isoforms of protein kinase Akt (Akt1/2/3) are involved in a myriad of essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Here, we present a structure‐guided approach for the design of structurally diverse and pharmacologically beneficial covalent‐allosteric modifiers which enabled an investigation of the isoform‐specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The first set of isoform‐selective covalent‐allosteric Akt inhibitors that emerged from this approach showed a conclusive structure‐activity relationship and broke ground for further structure‐guided development of selective probes to delineate the isoform‐specific functions of Akt kinases.

https://www.rauh-lab.de/

Development of a Gram-Scale Synthesis of PBRM, an Irreversible Inhibitor of 17beta-Hydroxysteroid Dehydrogenase Type 1

René Maltais and Donald Poirier
Organic Process Research & Development 2019
DOI: 10.1021/acs.oprd.8b00402

Efforts toward the development of a reliable gram scale synthesis of PBRM, a potent and selective steroidal covalent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), are described. Among the three synthetic routes (C-E) developed herein, route E is the most efficient one with only 6 chemical steps from commercially available estrone, and an overall yield of 13% leading to PBRM with a high HPLC grade purity (99.7%) after recrystallization. Important improvements have been achieved in this sequence from previous reported routes (A and B). Notably, we used a palladium catalyzed Suzuki-Miyaura cross-coupling reaction to rapidly install the requested C3 chain on estrone. Also, catalytic hydrogenation of the C16-enone was shortened by half using Pearlman’s catalyst. Finally, we used a selective bromination through deoxygenation of alcohol at the last step of the sequence to provide PBRM without dehydration of its carboxamide functionality, a persistent problem observed in other routes. Crystals of PBRM were also obtained from recrystallization in acetonitrile and submitted to x-ray analysis, which confirmed the PBRM structure. This work now makes it possible to start a proof-of-principle in a non-human primate model for the treatment of endometriosis, while supporting its future pharmacological development.

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Patrick R. A. Zanon, Lisa Lewald, Stephan M. Hacker
ChemRxiv, 2019
doi: 10.26434/chemrxiv.9853445.v1

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in Staphylococcus aureus and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-
action.

https://chemrxiv.org/articles/Isotopically_Labeled_Desthiobiotin_Azide_isoDTB_Tags_Enable_Global_Profiling_of_the_Bacterial_Cysteinome/9853445/1

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

Qinheng Zheng, Jordan L. Woehl, Seiya Kitamura, Diogo Santos-Martins, Christopher J. Smedley, Gencheng Li, Stefano Forli, John E. Moses, Dennis W. Wolan, and K. Barry Sharpless

PNAS,  2019 116 (38) 18808-18814
doi: 10.1073/pnas.1909972116

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Limitations of ligand-only approaches for predicting the reactivity of covalent inhibitors

Angus Voice, Gary Tresadern, Herman Van Vlijmen and Adrian J. Mulholland

J. Chem. Inf. Model. 2019
DOI:https://doi.org/10.1021/acs.jcim.9b00404

Abstract

Covalent inhibition has undergone a resurgence and is an important modern-day drug design and chemical biology approach. To avoid off-target interactions, and to fine tune reactivity, the ability to accurately predict reactivity is vitally important for the design and development of safer and more effective covalent drugs. Several ligand-only metrics have been proposed that promise quick and simple ways of determining covalent reactivity. In particular, we examine proton affinity and reaction energies calculated with the density functional B3LYP-D3/6-311+G**//B3LYP-D3/6-31G* method to assess the reactivity of a series of ,-unsaturated carbonyl compounds that form covalent adducts with cysteine. We demonstrate that, whilst these metrics correlate well with experiment for a diverse range of covalent fragments, these approaches fail for predicting the reactivity of drug-like compounds. We conclude that ligand-only metrics such as proton affinity and reaction energies do not capture determinants of reactivity in situ and fail to account for important factors such as conformation, solvation and intra-molecular interactions.

Quantum Chemical Methods for Modeling Covalent Modification of Biological Thiols

Ernest Awoonor‐Williams, William C. Isley III, Stephen G. Dale, Erin R. Johnson, Haibo Yu, Axel D. Becke, Benoît Roux, Christopher N. Rowley

J. Comput. Chem. 2019 doi: https://doi.org/10.1002/jcc.26064

Targeted covalent inhibitor drugs require computational methods that go beyond simple molecular‐mechanical force fields in order to model the chemical reactions that occur when they bind to their targets. Here, several semiempirical and density‐functional theory (DFT) methods are assessed for their ability to describe the potential energy surface and reaction energies of the covalent modification of a thiol by an electrophile. Functionals such as PBE and B3LYP fail to predict a stable enolate intermediate. This is largely due to delocalization error, which spuriously stabilizes the prereaction complex, in which excess electron density is transferred from the thiolate to the electrophile. Functionals with a high‐exact exchange component, range‐separated DFT functionals, and variationally optimized exact exchange (i.e., the LC‐B05minV functional) correct this issue to various degrees. The large gradient behavior of the exchange enhancement factor is also found to significantly affect the results, leading to the improved performance of PBE0. While ωB97X‐D and M06‐2X were reasonably accurate, no method provided quantitative accuracy for all three electrophiles, making this a very strenuous test of functional performance. Additionally, one drawback of M06‐2X was that molecular dynamics (MD) simulations using this functional were only stable if a fine integration grid was used. The low‐cost semiempirical methods, PM3, AM1, and PM7, provide a qualitatively correct description of the reaction mechanism, although the energetics is not quantitatively reliable. As a proof of concept, the potential of mean force for the addition of methylthiolate to methylvinyl ketone was calculated using quantum mechanical/molecular mechanical MD in an explicit polarizable aqueous solvent. 

Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine

Thomas Schlatzer, Julia Kriegesmann, Hilmar Schröder, Melanie Trobe, Christian Lembacher-Fadum, Simone Santner, Alexander V. Kravchuk, Christian F. W. Becker, and Rolf BreinbauerJ. Am. Chem. Soc. 2019

DOI: 10.1021/jacs.9b08279

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji–Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.

Discovery and Development of a Series of Pyrazolo[3,4-d]pyridazinone Compounds as the Novel Covalent Fibroblast Growth Factor Receptor Inhibitors by the Rational Drug Design

Yulan Wang, Yang Dai, Xiaowei Wu, Fei Li, Bo Liu, Chunpu Li, Qiufeng Liu, Yuanyang Zhou, Bao Wang, Mingrui Zhu, Rongrong Cui, Xiaoqin Tan, Zhaoping Xiong, Jia Liu, Minjia Tan, Yechun Xu, Meiyu Geng, Hualiang Jiang, Hong Liu, Jing Ai, and Mingyue Zheng

J. Med. Chem. 2019, 62 (16), 7473-7488

DOI: 10.1021/acs.jmedchem.9b00510

Alterations of fibroblast growth factor receptors (FGFRs) play key roles in numerous cancer progression and development, which makes FGFRs attractive targets in the cancer therapy. In the present study, based on a newly devised FGFR target-specific scoring function, a novel FGFR inhibitor hit was identified through virtual screening. Hit-to-lead optimization was then performed by integrating molecular docking and site-of-metabolism predictions with an array of in vitro evaluations and X-ray cocrystal structure determination, leading to a covalent FGFR inhibitor 15, which showed a highly selective and potent FGFR inhibition profile. Pharmacokinetic assessment, protein kinase profiling, and hERG inhibition evaluation were also conducted, and they confirmed the value of 15 as a lead for further investigation. Overall, this study exemplifies the importance of the integrative use of computational methods and experimental techniques in drug discovery.

Pd-catalyzed site-selective C(sp2)–H radical acylation of phenylalanine containing peptides with aldehydes

Marcos San Segundo  and  Arkaitz Correa

Chem. Sci., 2019
doi: 10.1039/C9SC03425K

The site-selective functionalization of C–H bonds within a peptide framework remains a challenging task of prime synthetic importance. Herein, the first Pd-catalyzed δ-C(sp2)–H acylation of Phe containing peptides with aldehydes is described. This oxidative coupling is distinguished by its site-specificity, tolerance of sensitive functional groups, scalability, and enantiospecificity and exhibits entire chemoselectivity for Phe motifs over other amino acid units. The compatibility of this dehydrogenative acylation platform with a number of oligopeptides of high structural complexity illustrates its ample opportunities for the late-stage peptide modification and bioconjugation.

Spontaneous Isomerization of Long-Lived Proteins Provides a Molecular Mechanism for the Lysosomal Failure Observed in Alzheimer’s Disease

Tyler R. Lambeth, Dylan L. Riggs, Lance E. Talbert, Jin Tang, Emily Coburn, Amrik S. Kang, Jessica Noll, Catherine Augello, Byron D. Ford, Ryan R. Julian

ACS Central Science, 2019
DOI: 10.1021/acscentsci.9b00369

Proteinaceous aggregation is a well-known observable in Alzheimer’s disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l-isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

Discovery of Evobrutinib: An Oral, Potent and Highly Selective, Covalent Bruton’s Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases

Richard Caldwell, Hui Qiu, Ben C Askew, Andrew T Bender, Nadia Brugger, Montserrat Camps, Mohanraj Dhanabal, Vikram Dutt, Thomas Eichhorn, Anna S Gardberg, Andreas Goutopoulos, Roland Grenningloh, Jared Head, Brian Healey, Brian L Hodous, Bayard R Huck, Theresa L Johnson, Christopher Jones, Reinaldo C Jones, Igor Mochalkin, Federica Morandi, Ngan Nguyen, Michael Meyring, Justin R Potnick, Dusica Cvetinovic Santos, Ralf Schmidt, Brian Sherer, Adam Shutes, Klaus Urbahns, Ariele Viacava Follis, Ansgar A Wegener, Simone C. Zimmerli, and Lesley Liu-Bujalski

J. Med. Chem., 2019
DOI: 10.1021/acs.jmedchem.9b00794 

Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity) which preclude their evaluation in non-oncology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations

Mélissanne de Wispelaere, Guangyan Du, Katherine A. Donovan, Tinghu Zhang, Nicholas A. Eleuteri, Jingting C. Yuan, Joann Kalabathula, Radosław P. Nowak, Eric S. Fischer, Nathanael S. Gray & Priscilla L. Yang

Nature Communications, 2019, 10, 3468
doi: 10.1038/s41467-019-11429-w

Targeted protein degradation is a promising drug development paradigm. Here we leverage this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a reversible-covalent inhibitor that binds to the hepatitis C virus (HCV) protease active site is conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. An optimized degrader, DGY-08-097, potently inhibits HCV in a cellular infection model, and we demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.

Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

Janice H. Xu, Jerome Eberhardt, Brianna Hill-Payne, Gonzalo E. González-Páez, José Omar Castellón, Benjamin F. Cravatt, Stefano Forli, Dennis W. Wolan, Keriann M. Backus 

bioRxiv, 2019 
doi: 10.1101/721951

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

María Maneiro, Emilio Lence, Marta Sanz-Gaiterob, José M. Otero, Mark J. van Raaij, Paul Thompson, Alastair R. Hawkins and Concepción González-Bello

Org. Chem. Front., 2019
DOI: 10.1039/C9QO00453J 

Targeted irreversible inhibitors bearing electrophiles that become activated towards covalent bond formation upon binding to a specific protein/enzyme is an emerging area in drug discovery. Targeting lysine residues is challenging due to the intrinsically low reactivity of the amino group at physiological pH. Herein we report the first example of a hydroxylammonium derivative that causes a specific covalent modification of an active-site and a sterically inaccessible lysine residue of an enzyme. The described ligands, compounds 1–3, were rationally designed to be activated towards covalent bond formation upon binding to the type I dehydroquinase (DHQ1) enzyme for the development of new anti-virulence agents to combat the widespread resistance to antibiotics. Evidence in atomic detail for the covalent modifications caused by the ligands to the catalytic Lys170 by the formation of a stable secondary amine is provided by the resolution at 1.08–1.25 Å of the crystal structures of DHQ1 from Salmonella typhi enzyme adducts. In addition, the first crystal structure of the addition intermediate adduct at 1.4 Å of a Schiff base formation reaction by using an analog of the natural substrate, compound 4, is also reported. Molecular dynamics simulation studies on non-covalent enzyme/ligand complexes and a two-dimensional QM/MM umbrella sampling simulation study suggested that a direct displacement by Lys170 with the release of NH2OH would be feasible. These studies might open up new opportunities for the development of novel lysine-targeted irreversible inhibitors bearing a methylhydroxylammonium moiety as a latent electrophile.

Targeted Covalent Inhibition of Telomerase

Rick Betori, Yue Liu, Ding Wu, Rama Mishra, Scott Cohen, Stephen J. Kron, Karl Scheidt
ChemRxiv, 2019
doi: 10.26434/chemrxiv.8977457.v1

Telomerase is a ribonuceloprotein complex responsible for maintaining telomeres and protecting chromosomal integrity. The human telomerase reverse transcriptase (hTERT) is expressed in ~90% of cancer cells where it confers the capacity for limitless proliferation. Along with its established role in telomere lengthening, telomerase also serves non-canonical extra-telomeric roles in oncogenic signaling, resistance to apoptosis, and enhanced DNA damage response. Here, we report a new class of natural product-inspired covalent inhibitors of telomerase that target the catalytic active site. We developed rationally designed probe compounds that modulate both the telomeric and extra-telomeric activities of telomerase and present new opportunities to investigate the diverse functions of this unique molecular machine.

Enhancement of the Anti-Aggregation Activity of a Molecular Chaperone Using a Rationally Designed Post-Translational Modification

Philip R. Lindstedt, Francesco A. Aprile, Maria J. Matos, Michele Perni, Jean B. Bertoldo, Barbara Bernardim, Quentin Peter, Gonzalo Jiménez-Osés, Tuomas P. J. Knowles, Christopher M. Dobson, Francisco Corzana, Michele Vendruscolo, and Gonçalo J. L. Bernardes

ACS Cent. Sci. 2019

doi: 10.1021/acscentsci.9b00467

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson’s disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD.

Dual-Reactivity trans-Cyclooctenol Probes for Sulfenylation in Live Cells Enable Temporal Control via Bioorthogonal Quenching

Samuel L. Scinto, Oshini Ekanayake, Uthpala Seneviratne, Jessica E. Pigga, Samantha J. Boyd, Michael T. Taylor, Jun Liu, Christopher W. am Ende, Sharon Rozovsky, and Joseph M. Fox

Journal of the American Chemical Society, 2019 141 (28), 10932-10937
DOI: 10.1021/jacs.9b01164

Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas “ordinary” trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain BLU554 Selectivity for the Hinge Cysteine of FGFR4

Lin, Xiaojing, Yosaatmadja, Yuliana, Kalyukina, Maria, Middleditch, Martin J., Zhang, Zhen, Lu, Xiaoyun, Ding, Ke, Patterson, Adam V., Smaill, Jeff B., Squire, Christopher J.

ACS Med. Chem. Lett., 2019
doi: 10.1021/acsmedchemlett.9b00196

Aberration in FGFR4 signaling drives carcinogenesis and progression in a subset of hepatocellular carcinoma (HCC) patients, thereby making FGFR4 an attractive molecular target for this disease. Selective FGFR4 inhibition can be achieved through covalently targeting a poorly conserved cysteine residue in the FGFR4 kinase domain. We report mass spectrometry assays and cocrystal structures of FGFR4 in covalent complex with the clinical candidate BLU554 and with a series of four structurally related inhibitors that define the inherent reactivity and selectivity profile of these molecules. We further reveal the structure of FGFR1 with one of our inhibitors and show that off-target covalent binding can occur through an alternative conformation that supports targeting of a cysteine conserved in all members of the FGFR family. Collectively, we propose that rotational freedom, steric hindrance, and protein dynamics explain the exceptional selectivity profile of BLU554 for targeting FGFR4.

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition [@RobertoZoncu @DanNomura @OlzmannLab @Clive_chung @HijaiShin]

Clive Yik-Sham Chung, Hijai R. Shin, Charles A. Berdan, Breanna Ford, Carl C. Ward, James A. Olzmann, Roberto Zoncu & Daniel K. Nomura

Nature Chemical Biology, 2019 

doi: 10.1038/s41589-019-0308-4

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

The proteome‐wide potential for reversible covalency at cysteine

Kristine Senkane, Ekaterina Vinogradova, Radu Suciu, Vincent Crowley, Balyn Zaro, Michael Bradshaw ,Ken Brameld, Benjamin Cravatt

Angew. Chem. 2019
doi:10.1002/ange.201905829

Reversible covalency, achieved with, for instance, highly electron‐deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification that persists following unfolding and/or proteolytic processing. So far, reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Here we describe a mass‐spectrometry‐based platform that integrates gel filtration (GF) with activity‐based protein profiling (ABPP) to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small‐molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical probe discovery.

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation

Jessica N. Spradlin, Xirui Hu, Carl C. Ward, Scott M. Brittain, Michael D. Jones, Lisha Ou, Milton To, Andrew Proudfoot, Elizabeth Ornelas, Mikias Woldegiorgis, James A. Olzmann, Dirksen E. Bussiere, Jason R. Thomas, John A. Tallarico, Jeffrey M. McKenna, Markus Schirle, Thomas J. Maimone & Daniel K. Nomura

Nature Chemical Biology, 2019, 15, 747–755
doi: 10.1038/s41589-019-0304-8

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

Nature Chemical Biology, 2019, 15, 737–746

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16—a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases—as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10–40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.

Genetically Encoding Photocaged Quinone Methide to Multitarget Protein Residues Covalently in Vivo

Jun Li, Shanshan Li, Nayyar A. Aslam, Feng ZhengBing Yang, Bing Yang, Rujin Cheng, Nanxi Wang, Sharon Rozovsky, Peng G. Wang, Qian Wang, Lei Wangi

J. Am. Chem. Soc. 2019, 14 (124) 9458-9462

doi: 10.1021/jacs.9b01738

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited cross-linking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.