Search

We provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Tuning reactivity of sulfur electrophiles is key for advancing click chemistry and chemical probe discovery. To date, activation of the sulfur electrophile for protein modification has been ascribed principally to stabilization of a fluoride leaving group (LG) in covalent reactions of sulfonyl fluorides and arylfluorosulfates. We recently introduced sulfur-triazole exchange (SuTEx) chemistry to demonstrate the triazole as an effective LG for activating nucleophilic substitution reactions on tyrosine sites of proteins. Here, we probed tunability of SuTEx for fragment-based ligand discovery by modifying the adduct group (AG) and LG with functional groups of differing electron-donating and -withdrawing properties. We discovered the sulfur electrophile is highly sensitive to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity. We applied these reactivity principles to identify a large fraction of tyrosine sites (~30%) on proteins (~44%) that can be liganded across >1500 probe-modified sites quantified by chemical proteomics. Our proteomic studies identified non-catalytic tyrosine- and phosphotyrosine-sites that can be liganded by SuTEx fragments, with site specificity, in lysates and live cells to disrupt protein function. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and protein ligand discovery.

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, for the first time, 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.

Background: Patients with rheumatoid arthritis (RA) experience joint swelling and cartilage destruction resulting in chronic pain, functional disability, and compromised joint function. Current RA treatments, including glucocorticoid receptor agonists, produce adverse side effects and lack prolonged treatment efficacy. Cannabinoids (i.e., cannabis-like signaling molecules) exert anti-inflammatory and analgesic effects with limited side effects compared to traditional immunosuppressants, making them excellent targets for the development of new arthritic therapeutics. Monoacylglycerol lipase (MAGL) inhibition reduces inflammation in mouse models of acute inflammation, through cannabinoid receptor dependent and independent pathways. The current study investigated the efficacy of inhibiting synthetic and catabolic enzymes that regulate the endocannabinoid 2-arachidonoylglycerol (2-AG) in blocking paw inflammation, pain-related behaviors, and functional loss caused by collagen-induced arthritis (CIA). Methods: Male DB1A mice subjected to CIA were administered the glucocorticoid agonist dexamethasone (DEX), MAGL inhibitor JZL184 (8 or 40 mg/kg, s.c.), alone or in combination, or diacylglycerol lipase β (DAGLβ) inhibitor KT109 (40 mg/kg, s.c.). CIA-induced deficits were assayed by arthritic clinical scoring, paw thickness measurements, and behavioral tests of pain and paw function. Results: DEX or dual administration with JZL184 reduced paw thickness and clinical scores, and JZL184 dose-dependently attenuated grip strength and balance beam deficits caused by CIA. Traditional measures of pain-induced behaviors (hyperalgesia and allodynia) were inconsistent. The antiarthritic effects of JZL184 (40 mg/kg) were largely blocked by coadministration of the CB(2) antagonist SR144528, and the DAGLβ inhibitor KT109 had no effect on CIA, indicating that these effects likely occurred through CB(2) activation. Conclusions: MAGL inhibition reduced paw inflammation and pain-depressed behavioral signs of arthritis, likely through an endocannabinoid mechanism requiring CB(2). These data support the development of MAGL as a target for therapeutic treatment of inflammatory arthritis.

Sulfonyl-triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment-based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration-dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure-activity relationship studies identified the SuTEx ligand HHS-0701 as a cell-active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Chemoproteomic probes serve as valuable tools to study and perturb protein function on a global scale. Despite advances in methodologies for cysteine and lysine profiling, a large fraction of the human proteome remains inaccessible with current activity-based probes. Here, we describe development and application of sulfur-triazole exchange (SuTEx) chemistry for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can produce sulfonyl probes with high chemoselectivity for tyrosines over other nucleophilic amino acids to investigate thousands of tyrosine sites in lysates and live cells. We discover hyper-reactive tyrosines 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. Our findings illustrate the broad potential for deploying SuTEx to globally investigate tyrosine reactivity, function and post-translational modification state in complex biological systems.

Reaction of ketones with CS2 and 2 equivalents of KH in THF–N,N′-dimethylpropyleneurea solution produces the dianions of 3-oxodithioic acids. These dianions are converted in good yield to 3H-1,2-dithiole-3-thiones by the sequential action of hexamethyldisilathiane and an oxidizing agent such as hexachloroethane.

Oltipraz and related dithiolethiones are an important class of chemopreventive agents. Studies were undertaken to identify cancer chemopreventive dithiolethiones more active than oltipraz. Largely based upon enzyme induction activities in vitro, 17 dithiolethiones, including oltipraz, were analyzed for their ability to induce hepatic phase II enzyme activities in vivo. Of these compounds, 15 produced greater induction of NAD(P)H:quinone reductase and 11 yielded greater induction of glutathione S-transferase than oltipraz. All 17 dithiolethiones were then tested for their ability to inhibit acute hepatotoxicity by aflatoxin B 1 (AFB 1 ), which previously has been shown to be an intermediate predictor of chemopreventive activity. Rats were pretreated with dithiolethiones (0.3 mmol/kg body wt, three times a week per os) and challenged with two acutely toxic doses of AFBI (0.5 mg/kg body wt, once daily for two successive days per os). Inhibition of hepatotoxicity was measured by changes in body weight gain during AFB 1 challenge, reduction in levels of hepatic enzymes in serum and diminution of bile duct cell proliferation. Nine dithiolethiones spanning a range of responses in this toxicity screen were further tested for their ability to prevent AFBI-induced tumorigenicity, as assessed by a reduction in hepatic burden of putative preneoplastic foci. Six dithiolethiones were found to be considerably more effective than oltipraz in preventing AFB 1 -induced tumorigenesis. In general, dithiolethiones that were very effective in inhibition of acute hepatotoxicity were also found to be effective in prevention of hepatic tumorigenesis.

A series of N-hydroxyurea derivatives based on substituted benzoxepin and benzothiepin templates was prepared and the compounds were evaluated for their ability to inhibit 5-lipoxygenase in vitro and ex vivo.

4-Methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz) and several other dithiolethiones protect against the acute toxicities of many xenobiotics and are effective inhibitors of experimental carcinogenesis. These protective effects are mediated, in part, through elevation of glutathione S-transferase, NAD(P)H: quinone reductase and UDP-glucuronosyltransferase activities in the liver and other target tissues. The induction of these phase 2 enzymes by oltiprax results from enhanced transcription. In the present study, the molecular mechanisms of these inductions were analyzed utilizing a construct containing a 41 bp enhancer element derived from the 5'-upstream region of the mouse liver glutathione S-transferase Ya subunit gene ligated to the 5' end of the isolated promoter region of this gene, and inserted into a plasmid containing a human growth hormone reporter gene. When this construct was transfected into murine Hepa 1c1c7 hepatoma cells, the concentrations of 25 dithiolethiones and related analogs required to double growth hormone production were determined and spanned a range nearly three orders of magnitude. Concentrations of dithiolethiones required to double the specific activity of NAD(P)H: quinone reductase were also determined in Hepa 1c1c7 cells. There was a positive correlation (r = 0.78) between the potencies of the 21 active compounds as inducers of both NAD(P)H: quinone reductase activity and growth hormone production. Moreover, no dithiolethiones were inactive in only one system. It is probable, therefore, that the induction of NAD(P)H: quinone reductase and other phase 2 enzymes by oltipraz and other dithiolethiones is mediated entirely through the 41 bp enhancer element.

The asymmetric synthesis of all four of the known natural phlegmarines and one synthetic derivative has been accomplished in 19−22 steps from 4-methoxy-3-(triisopropylsilyl)pyridine. Chiral N-acylpyridinium salt chemistry was used twice to set the stereocenters at the C-9 and C-2′ positions of the phlegmarine skeleton. Key reactions include the use of a mixed Grignard reagent for the second N-acylpyridinium salt addition, zinc/acetic acid reduction of a complex dihydropyridone, and a von Braun cyanogen bromide N-demethylation of a late intermediate. These syntheses confirmed the absolute stereochemistry of all of the known phlegmarines.

Reaction of ketones with CS2 and 2 equivalents of KH in THF–N,N′-dimethylpropyleneurea solution produces the dianions of 3-oxodithioic acids. These dianions are converted in good yield to 3H-1,2-dithiole-3-thiones by the sequential action of hexamethyldisilathiane and an oxidizing agent such as hexachloroethane.