Beyond cysteine: recent developments in the area of targeted covalent inhibition

Herschel Mukherjee, Neil P Grimster

Current Opinion in Chemical Biology 2018, 44, 30–38
doi: 10.1016/j.cbpa.2018.05.011

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins.

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

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.

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

DOI: 10.1021/jacs.8b04115

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.

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.

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,
doi:10.1038/s41467-018-04343-0

 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.

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., 2018, 140 (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.