Tuesday, May 29, 2018

The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition

ACS Chem. Biol., 2018
DOI: 10.1021/acschembio.8b00225

We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues

Monday, May 28, 2018

Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling

Radu M. Suciu, Armand B. Cognetta, III, Zachary E. Potter, and Benjamin F. Cravatt

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00191

Wnt proteins are secreted morphogens that play critical roles in embryonic development and tissue remodeling in adult organisms. Aberrant Wnt signaling contributes to diseases such as cancer. Wnts are modified by an unusual O-fatty acylation event (O-linked palmitoleoylation of a conserved serine) that is required for binding to Frizzled receptors. O-Palmitoleoylation of Wnts is introduced by the porcupine (PORCN) acyltransferase and removed by the serine hydrolase NOTUM. PORCN inhibitors are under development for oncology, while NOTUM inhibitors have potential for treating degenerative diseases. Here, we describe the use of activity-based protein profiling (ABPP) to discover and advance a class of N-hydroxyhydantoin (NHH) carbamates that potently and selectively inhibit NOTUM. An optimized NHH carbamate inhibitor, ABC99, preserves Wnt-mediated cell signaling in the presence of NOTUM and was also converted into an ABPP probe for visualizing NOTUM in native biological systems.

Thursday, May 24, 2018

Organometallic Gold(III) Reagents for Cysteine Arylation

Marco S Messina, Julia M Stauber, Mary A Waddington, Arnold L. Rheingold, Heather D. Maynard, and Alexander M Spokoyny
J. Am. Chem. Soc., 2018

An efficient method for chemoselective cysteine arylation of unprotected peptides and proteins using Au(III) organometallic complexes is reported. The bioconjugation reactions proceed rapidly (<5 min) at ambient temperature in various buffers and within a wide pH range (0.5-14). This approach provides access to a diverse array of S-aryl bioconjugates including fluorescent dye, complex drug molecule, affinity label, poly(ethylene glycol) tags and a stapled peptide. A library of Au(III) arylation reagents can be prepared as air-stable, crystalline solids in one step from commercial reagents. The selective and efficient arylation procedures presented in this work broaden the synthetic scope of cysteine bioconjugation and serve as promising routes for the modification of complex biomolecules.

Tuesday, May 22, 2018

Targeted Covalent Inhibition of Prolyl Oligopeptidase (POP): Discovery of Sulfonylfluoride Peptidomimetics

Salvador Guardiola, Roger Prades, Laura Mendieta, Arwin J. Brouwer, Jelle Streefkerk, Laura Nevola, Teresa Tarragó, Rob M.J. Liskamp, Ernest Giralt

Cell Chemical Biology, 2018

doi: 10.1016/j.chembiol.2018.04.013

Prolyl oligopeptidase (POP), a serine protease highly expressed in the brain, has recently emerged as an enticing therapeutic target for the treatment of cognitive and neurodegenerative disorders. However, most reported inhibitors suffer from short duration of action, poor protease selectivity, and low blood-brain barrier (BBB) permeability, which altogether limit their potential as drugs. Here, we describe the structure-based design of the first irreversible, selective, and brain-permeable POP inhibitors. At low-nanomolar concentrations, these covalent peptidomimetics produce a fast, specific, and sustained inactivation of POP, both in vitro and in human cells. More importantly, they are >1,000-fold selective against two family-related proteases (DPPIV and FAP) and display high BBB permeability, as shown in both lipid membranes and MDCK cells.

Saturday, May 19, 2018

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

Tomonori Tamura, Tsuyoshi Ueda, Taiki Goto, Taku Tsukidate, Yonatan Shapira, Yuki Nishikawa, Alma Fujisawa & Itaru Hamachi

Nature Communications, 9, Article number: 1870, 2018,

 Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M−1 s−1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Wednesday, May 16, 2018

Phage Display of Dynamic Covalent Binding Motifs Enables Facile Development of Targeted Antibiotics

Kelly A. McCarthy, Michael A. Kelly, Kaicheng Li, Samantha Cambray, Azade S. Hosseini , Tim van Opijnen, and Jianmin Gao

J. Am. Chem. Soc.2018140 (19), 6137–6145 DOI: 10.1021/jacs.8b02461 

Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The antibiotic resistance problem at least partially arises from extensive use of broad-spectrum antibiotics. Ideally, for the treatment of infection, one would like to use a narrow-spectrum antibiotic that specifically targets and kills the disease-causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being. In this contribution, we describe a phage display platform that enables rapid identification of peptide probes for specific bacterial strains. The phage library described herein incorporates 2-acetylphenylboronic acid moieties to elicit dynamic covalent binding to the bacterial cell surface. Screening of the library against live bacterial cells yields submicromolar and highly specific binders for clinical strains of Staphylococcus aureus and Acinetobacter baumannii that display antibiotic resistance. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. The phage display platform described here is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics.

Tuesday, May 8, 2018

Chemo- and Regioselective Lysine Modification on Native Proteins

Maria J. Matos, Bruno L. Oliveira, Nuria Martínez-Saez,  Ana Guerreiro, Pedro M. S. D. Cal,
Jean Bertoldo, María Maneiro, Elizabeth Perkins, Julie Howard, Michael J. Deery,
Justin M. Chalker, Francisco Corzana, Gonzalo Jimenez-Oses, and Goncalo J. L. Bernardes

J. Am. Chem. Soc., 2018, 140 (11), pp 4004–4017

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

Job Opportunity: Post Doc Fellow - Novel Covalent Inhibitor Chemistry in Cambridge, United Kingdom

There is a great opportunity to work as an industrial postdoc at Astrazeneca on development of new covalent modifier drugs:


Sunday, May 6, 2018

Natural Product Micheliolide (MCL) Irreversibly Activates Pyruvate Kinase M2 and Suppresses Leukemia

Jing Li, Shanshan Li, Jianshuang Guo, Qiuying Li, Jing Long, Cheng Ma, Yahui Ding,
Chunli Yan, Liangwei Li, Zhigang Wu, He Zhu, Keqin Kathy Li, Liuqing Wen, Quan Zhang,
Qingqing Xue, Caili Zhao, Ning Liu, Ivaylo Ivanov, Ming Luo, Rimo Xi, Haibo Long,
Peng George Wang, and Yue Chen

J. Med. Chem., 2018
DOI: 10.1021/acs.jmedchem.8b00241

Metabolic reprogramming of cancer cells is essential for tumorigenesis in which pyruvate kinase M2 (PKM2), the low activity isoform of pyruvate kinase, plays a critical role. Herein, we describe the identification of a nature-product-derived micheliolide (MCL) that selectively activates PKM2 through the covalent binding at residue cysteine424 (C424), which is not contained in PKM1. This interaction promotes more tetramer formation, inhibits the lysine433 (K433) acetylation, and influences the translocation of PKM2 into the nucleus. In addition, the pro-drug dimethylaminomicheliolide (DMAMCL) with similar properties as MCL significantly suppresses the growth of leukemia cells and tumorigenesis in a zebrafish xenograft model. Cell-based assay with knock down PKM2 expression verifies that the effects of MCL are dependent on PKM2 expression. DMAMCL is currently in clinical trials in Australia. Our discovery may provide a valuable pharmacological mechanism for clinical treatment and benefit the development of new anticancer agents.

Thursday, May 3, 2018

The Taxonomy of Covalent Inhibitors

Alfred Tuley and Walter Fast 
Biochemistry, 2018
DOI: 10.1021/acs.biochem.8b00315

Covalent enzyme inhibitors are widely applied as biochemical tools and therapeutic agents. As a complement to categorization of these inhibitors by reactive group or modification site, we present a categorization by mechanism, which highlights common advantages and disadvantages inherent to each approach. Established categories for reversible and irreversible covalent inhibition are reviewed with representative examples given for each class, including covalent reversible inhibitors, slow substrates, residue-specific reagents, affinity labels (classical, quiescent, and photoaffinity), and mechanism-based inactivators. The relationships of these categories to proteomic profiling probes (activity-based and reactivity-based) as well as complementary approaches such as prodrug and soft drug design are also discussed. A wide variety of strategies are used to balance reactivity and selectivity in the design of covalent enzyme inhibitors. Use of a shared terminology is encouraged to clearly convey these mechanisms, to relate them to prior use of covalent inhibitors in enzymology, and to facilitate the development of more effective covalent inhibitors.

Wednesday, May 2, 2018

Site‐Selective Cysteine–Cyclooctyne Conjugation

Dr. Chi Zhang  Dr. Peng Dai  Dr. Alexander A. Vinogradov  Dr. Zachary P. Gates Prof. Dr. Bradley L. Pentelute

Angew. Chem. Int. Ed. 2018
doi: 10.1002/anie.201800860

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond The Journal of Organic Chemistry, 2020 DOI: 10.1021/acs.joc.2c01530 The α-meth...