Stable knockout of lanthionine synthase C-like protein-1 (LanCL1) from HeLa cells indicates a role for LanCL1 in redox regulation of deubiquitinating enzymes.

Lanthionine synthase C-like protein-1 (LanCL1) is a glutathione (GSH)-binding protein of uncertain function, widely expressed in mammalian cells. Recent data suggests that LanCL1 has glutathione S-transferase (GST)-like activity, while other reports claim that LanCL1 suppresses mitogen-activated kinase (MAPK) phosphorylation. In the present study, recombinant human LanCL1 had less than 10% the specific activity of GST. When CRISPR-Cas9 was used to stably ablate LanCL1 from HeLa cells, the resulting line was sensitized to H2O2 toxicity. [GSH], [GSSG], [GSH]/[GSSG] and GST activity were unaltered by LanCL1 knockout but glutathione reductase and glutathione peroxidase activities were significantly elevated. LanCL1-KO cells did not differ in basal or H2O2-induced p38-MAPK, ERK p42/p44 or JNK phosphorylation; however, MAPK-targeted transcription factor regulators c-Jun and IκBα were significantly decreased. Because c-Jun and IκBα levels are ubiquitin regulated, experiments addressed the hypothesis that LanCL1 affects ubiquitination dynamics. In the presence of the 26S proteasome inhibitor bortezomib, ubiquitinated proteins accumulated faster in LanCL1-KO cells, suggesting that LanCL1 positively regulates deubiquitination. The activity of ubiquitin C-terminal hydrolase (UCH), a major deubiquitinase (DUB) subclass, was significantly decreased in LanCL1-KO cells while protein levels of A20/TNFAIP3, USP9X and USP10 DUBs were significantly reduced. UCH activity in HeLa cell lysates was lost upon treatment with H2O2 and significantly recovered by addition of recombinant LanCL1 plus GSH. Taken together these data suggest that LanCL1 likely does not act as a GST-like enzyme in vivo, but rather modulates ubiquitin-dependent cell signaling pathways through positive regulation of redox-sensitive DUBs.

Synthesis of 2H-1,2-benzothiazine 1,1-dioxides via heteroannulation reactions of 2-iodobenzenesulfonamide with ketone enolates under S(RN)1 conditions.

[Reaction: see text]. 2-iodobenzenesulfonamide (1a) underwent photostimulated S(RN)1 reactions in liquid NH3 with the potassium enolates derived from acetone, pinacolone, butanone, and 3-methyl-2-butanone to give fair to good yields of 2H-1,2-benzothiazine 1,1-dioxides 2. Reductive dehalogenation of 1a was found to predominate in photoinduced reactions of 1a with 3-pentanone, 2-methyl-3-pentanone, and 2,4-dimethyl-3-pentanone, the extent of reduction being proportional to the number of beta-hydrogen atoms present in the ketone enolate. Isotopic labeling studies with 2,4-dimethyl-3-pentanone-d14 (24) confirmed the major role of the beta-hydrogens in the reduction process. Reactions of 1a with cyclopentanone, cyclohexanone, and cyclooctanone enolates afforded new tricyclic benzothiazine derivatives 26-29. Attempts to extend the heteroannulation reaction to the preparation of 2H-1,2-benzothiazin-3(4H)-one 1,1-dioxides 3 (R = H, Ph) through reactions of 1a with tert-butyl acetate and ethyl phenylacetate enolates resulted only in hydrodehalogenation of 1a. However, oxazoline anion 30, a synthetic equivalent of ethyl phenylacetate, was successfully employed in an alternative S(RN)1-based synthesis of benzothiazine 3 (R = Ph).

Synthesis and anticonvulsant evaluation of some new 2-substituted-3-arylpyrido[2,3-d]pyrimidinones.

A series of 2-substituted-3-arylpyrido[2,3-d]pyrimidinones was prepared for evaluation as potential anticonvulsants. In murine screening, compounds 4a-c having a 2-oxo-2-(4-pyridyl)ethyl group in the 2-position and a 2-substituted phenyl moiety at the 3-position of the pyridopyrimidinone system displayed the most potent anti-seizure activity in both the maximal electroshock (MES) and pentylenetetrazol (scPTZ) tests at doses in the 3-10mg/kg range. Compound 4c showed no agonist activity at the GABA(A) receptor and was unable to block presynaptic sodium and calcium channels in vitro.