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

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.

Autoregulatory control of mitochondrial glutathione homeostasis.

Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39.

Using a function-first 'scout fragment'-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes.

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 genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity.

Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1.

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1.

Multiplexed proteomic profiling of cysteine reactivity and ligandability in human T cells.

Differential amino acid reactivity with chemical probes can provide valuable information on the functionality and ligandability of proteins in native biological systems. Here, we present a quantitative, multiplexed chemical proteomic protocol for in-depth reactivity and ligandability profiling of cysteines in proteins in quiescent and stimulated T cells. This protocol illuminates dynamic immune state-dependent alterations in cysteine reactivity, revealing chemoselective and stereoselective small-molecule interactions with cysteines in structurally and functionally diverse proteins that lack chemical probes.

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.

Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound.

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells.

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.

2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles.

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SAr).

A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex.

Target engagement assays are crucial for establishing the mechanism-of-action of small molecules in living systems. Integral membrane transporters can present a challenging protein class for assessing cellular engagement by small molecules. The chemical proteomic discovery of alpha-chloroacetamide (αCA) compounds that covalently modify cysteine-54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) is presented.

The Proteome-Wide Potential for Reversible Covalency at Cysteine.

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. 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. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles.

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete.

A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells.

Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target.

Arylation Chemistry for Bioconjugation.

Bioconjugation chemistry has been used to prepare modified biomolecules with functions beyond what nature intended. Central to these techniques is the development of highly efficient and selective bioconjugation reactions that operate under mild, biomolecule compatible conditions. Methods that form a nucleophile-sp carbon bond show promise for creating bioconjugates with new modifications, sometimes resulting in molecules with unparalleled functions.

Direct Access to Versatile Electrophiles via Catalytic Oxidative Cyanation of Alkenes.

Nucleophilic attack on carbon-based electrophiles is a central reactivity paradigm in chemistry and biology. The steric and electronic properties of the electrophile dictate its reactivity with different nucleophiles of interest, allowing the opportunity to fine-tune electrophiles for use as coupling partners in multistep organic synthesis or for covalent modification of proteins in drug discovery. Reactions that directly transform inexpensive chemical feedstocks into versatile carbon electrophiles would therefore be highly enabling.

Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for target engagement challenges in situ.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide using S-adenosyl-L-methionine (SAM) as a methyl donor and, through doing so, can modulate cellular methylation potential to impact diverse epigenetic processes. NNMT has been implicated in a range of diseases, including cancer and metabolic disorders. Potent, selective, and cell-active inhibitors would constitute valuable probes to study the biological functions and therapeutic potential of NNMT.

Divergent unprotected peptide macrocyclisation by palladium-mediated cysteine arylation.

Macrocyclic peptides are important therapeutic candidates due to their improved physicochemical properties in comparison to their linear counterparts. Here we detail a method for a divergent macrocyclisation of unprotected peptides by crosslinking two cysteine residues with bis-palladium organometallic reagents. These synthetic intermediates are prepared in a single step from commercially available aryl bis-halides.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells.

Organometallic palladium reagents for cysteine bioconjugation.

Reactions based on transition metals have found wide use in organic synthesis, in particular for the functionalization of small molecules. However, there are very few reports of using transition-metal-based reactions to modify complex biomolecules, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature and mild pH) and the existence of multiple reactive functional groups found in biomolecules. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions.

Design of New Ligands for the Palladium-Catalyzed Arylation of α-Branched Secondary Amines.

In Pd-catalyzed C-N cross-coupling reactions, α-branched secondary amines are difficult coupling partners and the desired products are often produced in low yields. In order to provide a robust method for accessing N-aryl α-branched tertiary amines, new catalysts have been designed to suppress undesired side reactions often encountered when these amine nucleophiles are used. These advances enabled the arylation of a wide array of sterically encumbered amines, highlighting the importance of rational ligand design in facilitating challenging Pd-catalyzed cross-coupling reactions.

Structural reevaluation of the electrophilic hypervalent iodine reagent for trifluoromethylthiolation supported by the crystalline sponge method for X-ray analysis.

Hypervalent iodine λ3-benziodoxoles are common electrophilic transfer reagents known for their enhanced stability compared to their non-cyclic analogues. Herein we present data showing that chlorobenziodoxole reacts with two different thiolate nucleophiles (thiocyanate and trifluoromethylthiolate), resulting in the formation of stable thioperoxy complexes rather than the expected benziodoxole derivatives. We further report a revised structure for the earlier described electrophilic trifluoromethylthiolation reagent (1), which was previously believed to contain the benziodoxole framework.

Palladium-catalyzed synthesis of N-aryl carbamates.

An efficient synthesis of aryl carbamates was achieved by introducing alcohols into the reaction of palladium-catalyzed cross-coupling of ArX (X = Cl, OTf) with sodium cyanate. The use of aryl triflates as electrophilic components in this transformation allowed for an expanded substrate scope for direct synthesis of aryl isocyanates. This methodology provides direct access to major carbamate protecting groups, S-thiocarbamates, and diisocyanate precursors to polyurethane materials.