Neurotoxicity of ischemic astrocytes involves STAT3-mediated metabolic switching and depends on glycogen usage.

Astrocytic responses are critical for the maintenance of neuronal networks in health and disease. In stroke, reactive astrocytes undergo functional changes potentially contributing to secondary neurodegeneration, but the mechanisms of astrocyte-mediated neurotoxicity remain elusive. Here, we investigated metabolic reprogramming in astrocytes following ischemia-reperfusion in vitro, explored their role in synaptic degeneration, and verified the key findings in a mouse model of stroke. Using indirect cocultures of primary mouse astrocytes and neurons, we demonstrate that transcription factor STAT3 controls metabolic switching in ischemic astrocytes promoting lactate-directed glycolysis and hindering mitochondrial function. Upregulation of astrocytic STAT3 signaling associated with nuclear translocation of pyruvate kinase isoform M2 and hypoxia response element activation. Reprogrammed thereby, the ischemic astrocytes induced mitochondrial respiration failure in neurons and triggered glutamatergic synapse loss, which was prevented by inhibiting astrocytic STAT3 signaling with Stattic. The rescuing effect of Stattic relied on the ability of astrocytes to utilize glycogen bodies as an alternative metabolic source supporting mitochondrial function. After focal cerebral ischemia in mice, astrocytic STAT3 activation was associated with secondary synaptic degeneration in the perilesional cortex. Inflammatory preconditioning with LPS increased astrocytic glycogen content, reduced synaptic degeneration, and promoted neuroprotection post stroke. Our data indicate the central role of STAT3 signaling and glycogen usage in reactive astrogliosis and suggest novel targets for restorative stroke therapy.

Assessment of anti-inflammatory-like, antioxidant activities and molecular docking of three alkynyl-substituted 3-ylidene-dihydrobenzo[d]isothiazole 1,1-dioxide derivatives.

The presence of enyne and benzoisothiazole functions in the molecular architecture of compounds 1, 2 and 3 were expected to provide biochemical activities. In the present work, we first examined the molecular surface contact of three alkynyl-substituted 3-ylidenedihydrobenzo[d] isothiazole 1,1-dioxides. The analysis of the Hirshfeld surfaces reveals that only compound 3 exhibited a well-defined red spots, indicating intermolecular interactions identified as S-O⋯H, C-H⋯O and C-O⋯H contacts. Comparative fingerprint histograms of the three compounds show that close pair interactions are dominated by C-H⋯H-C contact. By UV-visible analysis, compound 1 showed the most intense absorbances at 407 and 441 nm, respectively. The radical scavenging activity explored in the DPPH test, shows that only 1 exhibited low anti-radical activity. Furthermore, cellular antioxidant capacity of benzoisothiazoles 1-3 was investigated with PMA-activated HL-60 cells using chemiluminescence and fluorescence techniques in the presence of L-012 and Amplex Red probe, respectively. Results highlight that compound 1 exhibited moderate anti-ROS capacity while compounds 2 and 3 enhanced ROS production. The cytotoxicity test performed on HL-60 cells, using the MTS assay, confirmed the lack of toxicity of the tested benzoisothiazole 1 compared to 2 and 3 which show low cytotoxicity (≤30%). Anti-catalytic activity was evaluated by following the inhibitory potential of the benzoisothiazoles on MPO activity and depicted benzoisothiazoles-MPO interactions by docking. Both SIEFED and docking studies demonstrated an anti-catalytic activity of the tested benzoisothiazoles towards MPO with the best activity for compound 2.

Synthesis and evaluation of multi-target-directed ligands with BACE-1 inhibitory and Nrf2 agonist activities as potential agents against Alzheimer's disease.

Cumulative evidence suggests that ß-amyloid and oxidative stress are closely related with each other and play key roles in the process of Alzheimer's disease (AD). Multitarget regulation of both pathways might represent a promising therapeutic strategy. Here, a series of selenium-containing compounds based on ebselen and verubecestat were designed and synthesized. Biological evaluation showed that 13f exhibited good BACE-1 inhibitory activity (IC50 = 1.06 µΜ) and potent GPx-like activity (ν0 = 183.0 µM min-1). Aß production experiment indicated that 13f could reduce the secretion of Aß1-40 in HEK APPswe 293T cells. Moreover, 13f exerted a cytoprotective effect against the H2O2 or 6-OHDA caused cell damage via alleviation of intracellular ROS, mitochondrial dysfunction, Ca2+ overload and cell apoptosis. The mechanism studies indicated that 13f exhibited cytoprotective effect by activating the Keap1-Nrf2-ARE pathway and stimulating downstream anti-oxidant protein including HO-1, NQO1, TrxR1, GCLC, and GCLM. In addition, 13f significantly reduced the production of NO and IL-6 induced by LPS in BV2 cells, which confirmed its anti-inflammatory activity as a Nrf2 activator. The BBB permeation assay predicted that 13f was able to cross the BBB. In summary, 13f might be a promising multi-target-directed ligand for the treatment of AD.

Validating Signal Transducer and Activator of Transcription (STAT) Protein-Inhibitor Interactions Using Biochemical and Cellular Thermal Shift Assays.

Signal transducer and activator of transcription (STAT) proteins have important biological functions; however, deregulation of STAT signaling is a driving force behind the onset and progression of inflammatory diseases and cancer. While their biological roles suggest that STAT proteins would be valuable targets for developing therapeutic agents, STAT proteins are notoriously difficult to inhibit using small drug-like molecules, as they do not have a distinct inhibitor binding site. Despite this, a multitude of small-molecule STAT inhibitors have been proposed, primarily focusing on inhibiting STAT3 protein to generate novel cancer therapies. Demonstrating that inhibitors bind to their targets in cells has historically been a very challenging task. With the advent of modern target engagement techniques, such as the cellular thermal shift assay (CETSA), interactions between experimental compounds and their biological targets can be detected with relative ease. To investigate interactions between STAT proteins and inhibitors, we herein developed STAT CETSAs and evaluated known STAT3 inhibitors for their ability to engage STAT proteins in biological settings. While potent binding was detected between STAT proteins and peptidic STAT inhibitors, small-molecule inhibitors elicited variable responses, most of which failed to stabilize STAT3 proteins in cells and cell lysates. The described STAT thermal stability assays represent valuable tools for evaluating proposed STAT inhibitors.

T cell-specific STAT3 deficiency abrogates lupus nephritis.

Signal transducer and activator of transcription (STAT) 3 is a regulator of T-cell responses to external stimuli, such as pro-inflammatory cytokines and chemokines. We have previously shown that STAT3 is activated (phosphorylated) at high levels in systemic lupus erythematosus (SLE) T cells and mediates chemokine-induced migration and T:B cell interactions. Stattic, a small molecular STAT3 inhibitor, can partially ameliorate lupus nephritis in mice. To understand the role of STAT3 better in T-cell pathophysiology in lupus nephritis and its potential as a treatment target, we silenced its expression in T cells using a cd4-driven CRE-Flox model. We found that lupus-prone mice that do not express STAT3 in T cells did not develop lymphadenopathy, splenomegaly, or glomerulonephritis. Moreover, the production of anti-dsDNA antibodies was decreased in these mice compared to controls. To dissect the mechanism, we also used a nephrotoxic serum model of nephritis. In this model, T cell-specific silencing of STAT3 resulted in amelioration of nephrotoxic serum-induced kidney damage. Taken together, our results suggest that in mouse models of autoimmune nephritis, T cell-specific silencing of STAT3 can hamper their ability to help B cells to produce autoantibodies and induce cell tissue infiltration. We propose that STAT3 inhibition in T cells represents a novel approach in the treatment of SLE and lupus nephritis in particular.

Investigation of intermolecular interactions and stability of verubecestat in the active site of BACE1: Development of first model from QM/MM-based charge density and MD analysis.

Alzheimer disease (AD) is a cruel neurodegenerative disorder caused by the deposition of amyloid ß (Aß) peptide inside the brain. The ß-secretase (beta amyloid precursor protein (APP) cleaving enzyme 1, BACE1) is one of the enzymes involved in the cleavage of APP that leads to the Aß formation and it is the primary target for the treatment of AD. Recent report outlines that verubecestat molecule strongly inhibits BACE1; however, its structure, binding mechanism and the stability in the active site of BACE1 are not yet known. The present study aims to determine the structure, binding affinity and the stability of verubecestat molecule in the active site of BACE1 from the molecular docking, quantum mechanics/molecular mechanics (QM/MM)-based charge density analysis and molecular dynamics simulation. Verubecestat molecule was docked at BACE1; it shows high binding affinity towards BACE1. Further, the conformational geometry and the intermolecular interactions of verubecestat in the active site of BACE1 were determined. The molecule forms strong interaction with the neighboring amino acids in the active site of BACE1. The onsite QM/MM-based charge density analysis reveals the nature of charge density distribution and the topological properties of intermolecular interactions of verubecestat molecule in the active site of BACE1. The calculated electrostatic potential (ESP) of verubecestat in the active site of BACE1 displays high negative and positive ESP regions of the molecule. This onsite QM/MM analysis is more relevant to the physiological situation. The molecular dynamics simulation has been performed, which confirms the high stability and compactness of verubecestat in the active site of BACE1. The MM-generalized Born surface area and MM-Poisson Boltzmann surface area free energy calculations of verubecestat-BACE1 also confirm the high binding affinity of verubecestat. Communicated by Ramaswamy H. Sarma.

LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48.

OBJECTIVES: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to ß-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. METHODS: The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. RESULTS: Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ±â€Š1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ±â€Š1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 µM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. CONCLUSIONS: In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.

In vitro characterization of the bioconversion of pomaglumetad methionil, a novel metabotropic glutamate 2/3 receptor agonist peptide prodrug.

To characterize the hydrolysis of the peptide prodrug pomaglumetad methionil (LY2140023; (1R,4S,5S,6S)-4-(L-methionylamino)-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide), to the active drug LY404039 [(1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide], a series of in vitro studies were performed in various matrices, including human intestinal, liver, kidney homogenate, and human plasma. The studies were performed to determine the tissue(s) and enzyme(s) responsible for the conversion of the prodrug to the active molecule. This could enable an assessment of the risk for drug interactions, an evaluation of pharmacogenomic implications, as well as the development of a Physiologically Based Pharmacokinetic (PBPK) model for formation of the active drug. Of the matrices examined, hydrolysis of pomaglumetad methionil was observed in intestinal and kidney homogenate preparations and plasma, but not in liver homogenate. Clearance values calculated after applying standard scaling factors suggest the intestine and kidney as primary sites of hydrolysis. Studies with peptidase inhibitors were performed in an attempt to identify the enzyme(s) catalyzing the conversion. Near complete inhibition of LY404039 formation was observed in intestinal and kidney homogenate and human plasma with the selective dehydropeptidase1 (DPEP1) inhibitor cilastatin. Human recombinant DPEP1 was expressed and shown to catalyze the hydrolysis, which was completely inhibited by cilastatin. These studies demonstrate pomaglumetad methionil can be converted to LY404039 via one or multiple enzymes completely inhibited by cilastatin, likely DPEP1, in plasma, the intestine, and the kidney, with the plasma and kidney involved in the clearance of the circulating prodrug. These experiments define a strategy for the characterization of enzymes responsible for the metabolism of other peptide-like compounds.

[(125)I]Iodo-ASEM, a specific in vivo radioligand for α7-nAChR.

[(125)I]Iodo-ASEM, a new radioligand with high affinity and selectivity for α7-nAChRs (K(i) = 0.5 nM; α7/α4ß2 = 3414), has been synthesized in radiochemical yield of 33 ± 6% from the corresponding di-butyltriazene derivative and at high specific radioactivity (1600Ci/mmol; 59.2 MBq/µmol). [(125)I]Iodo-ASEM readily entered the brains of normal CD-1 mice and specifically and selectively labeled cerebral α7-nAChRs. [(125)I]iodo-ASEM is a new useful tool for studying α7-nAChR.

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5 nM) and TNF-α production in mouse splenocytes (IC50=9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand-enzyme complex.

Structure-based discovery of cellular-active allosteric inhibitors of FAK.

In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyrazolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase inhibitory activities both in cell-free (IC50=0.64µM) and in cellular assays (IC50=7.1µM). These results clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.

Thiourea isosteres as anion receptors and transmembrane transporters.

Compounds containing cyanoguanidine and 3-amino-1,2,4-benzothiadiazine-1,1-dioxide have been studied as anion receptors and transporters. Significant affinity for oxo-anions was observed in organic solution and the receptors were found to function as transmembrane chloride/nitrate antiporters with transport rates enhanced in the presence of valinomycin-K(+) complex.

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes.

Accumulation of the ß-amyloid (Aß) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aß is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by ß- and γ-secretases. Inhibition of Aß production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aß production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aß production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aß42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.

Critical role of the STAT3 pathway in the cardioprotective efficacy of zoniporide in a model of myocardial preservation - the rat isolated working heart.

BACKGROUND AND PURPOSE: Ischemia-reperfusion injury plays an important role in the development of primary allograft failure after heart transplantation. Inhibition of the Na+/H+ exchanger is one of the most promising therapeutic strategies for treating ischemia-reperfusion injury. Here we have characterized the cardioprotective efficacy of zoniporide and the underlying mechanisms in a model of myocardial preservation using rat isolated working hearts. EXPERIMENTAL APPROACH: Rat isolated hearts subjected to 6 h hypothermic (1-4°C) storage followed by 45 min reperfusion at 37°C were treated with zoniporide at different concentrations and timing. Recovery of cardiac function, levels of total and phosphorylated protein kinase B, extracellular signal-regulated kinase 1/2, glycogen synthase kinase-3ß and STAT3 as well as cleaved caspase 3 were measured at the end of reperfusion. Lactate dehydrogenase release into coronary effluent before and post-storage was also measured. KEY RESULTS: Zoniporide concentration-dependently improved recovery of cardiac function after reperfusion. The functional recovery induced by zoniporide was accompanied by up-regulation of p-extracellular signal-regulated kinase 1/2 and p-STAT3, and by reduction in lactate dehydrogenase release and cleaved caspase 3. There were no significant differences in any of the above indices when zoniporide was administered before, during or after ischemia. The STAT3 inhibitor, stattic, abolished zoniporide-induced improvements in functional recovery and up-regulation of p-STAT3 after reperfusion. CONCLUSIONS AND IMPLICATIONS: Zoniporide is a potent cardioprotective agent and activation of STAT3 plays a critical role in the cardioprotective action of zoniporide. This agent shows promise as a supplement to storage solutions to improve preservation of donor hearts.

Preclinical and clinical evidence for the collaborative transport and renal secretion of an oxazolidinone antibiotic by organic anion transporter 3 (OAT3/SLC22A8) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1).

N-({(5S)-3-[4-(1,1-dioxidothiomorpholin-4-yl)-3,5-difluorophenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide (PNU-288034), an oxazolidinone antibiotic, was terminated in phase I clinical development because of insufficient exposure. Analysis of the drug pharmacokinetic and elimination profiles suggested that PNU-288034 undergoes extensive renal secretion in humans. The compound was well absorbed and exhibited approximately linear pharmacokinetics in the oral dose range of 100 to 1000 mg in human. PNU-288034 was metabolically stable in liver microsomes across species, and unchanged drug was cleared in the urine by an apparent active renal secretion process in rat and monkey (two to four times glomerular filtration rate) but not dog. In vitro studies conducted to characterize the transporters involved demonstrated PNU-288034 uptake by human organic anion transporter 3 (OAT3; K(m) = 44 +/- 5 microM) and human multidrug and toxin extrusion protein 1 (hMATE1; K(m) = 340 +/- 55 microM). The compound was also transported by multidrug resistance P-glycoprotein and breast cancer resistance protein. In contrast, human organic cation transporter 2, human OAT1, and hMATE2-K did not transport PNU-288034. Coadministration of PNU-288034 and the OAT3 inhibitor probenecid significantly increased PNU-288034 plasma area under the curve (170%) and reduced both plasma and renal clearance in monkey. Coadministration of PNU-288034 and cimetidine, a MATE1 inhibitor, also reduced plasma clearance in rat to a rate comparable with probenecid coadministration. Collectively, our results demonstrated a strong in vitro-in vivo correlation for active renal secretion coordinated through the vectorial transport process of OAT3 and MATE1, which ultimately resulted in limiting the systemic exposure of PNU-288034.

Coupling of liquid chromatography/tandem mass spectrometry and liquid chromatography/solid-phase extraction/NMR techniques for the structural identification of metabolites following in vitro biotransformation of SUR1-selective ATP-sensitive potassium channel openers.

SUR1-selective ATP-sensitive potassium channel openers (PCOs) have been shown to be of clinical value for the treatment of several metabolic disorders, including type I and type II diabetes, obesity, and hyperinsulinemia. Taking into account these promising therapeutic benefits, different series of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide were developed. In view of the lead optimization process of the series, knowledge of absorption, distribution, metabolism, excretion, and toxicity parameters, and more particularly the metabolic fate of these compounds, is a fundamental requirement. For such a purpose, two selected promising compounds [7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) and 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 157)] were incubated in the presence of phenobarbital-induced rat liver microsomes to produce expected mammal in vivo phase I metabolites. The resulting major metabolites were then analyzed by both mass spectrometry (MS) and NMR to completely elucidate their chemical structures. The two compounds were also further incubated in the presence of nontreated rats and human microsomes to compare the metabolic profiles. In the present study, the combined use of an exact mass liquid chromatography (LC)/tandem MS platform and an LC/solid-phase extraction/NMR system allowed the clarification of some unresolved structural assessments in the accurate chemical structure elucidation process of the selected PCO drugs. These results greatly help the optimization of the lead compounds.

High-throughput approaches towards the definitive identification of pharmaceutical drug metabolites. 2. An example of how unexpected dissociation behaviour could preclude correct assignment of sites of metabolism.

S-oxidation is a common metabolic route for sulfur-containing compounds. Whilst investigating the dissociation of a series of chemically synthesised model S-oxide metabolites, two unexpected losses of 62 m/z units were observed in the collision-induced dissociation (CID) product ion spectrum of protonated 3-dimethylaminomethyl-4-(4-methanesulfinyl-3-methylphenoxy)benzenesulfonamide. A single loss was initially assigned using the low-resolution product ion spectrum, acquired by electrospray ionisation quadrupole ion trap mass spectrometry (ESI-QIT-MS), as methanethial, S-oxide via a charge-remote, four-centred rearrangement. This assignment was consistent with well-documented hydrogen rearrangements in the literature. Further, the loss was not observed for the parent compound. Thus, it was inferred that the site of metabolism was involved in the dissociation and the attractive nature of the four-centred rearrangement meant that the loss of methanethial, S-oxide was a logical assignment. However, deuterium-labelling experiments and accurate mass measurements, performed using electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS), showed that the nominal loss of 62 m/z units occurs via two distinct dissociation pathways. Neither of these losses was of methanethial, S-oxide as initially hypothesised from the low-resolution product ion spectrum of the protonated molecule. Mechanisms consistent with the experimental findings are postulated. An MS(3) spectrum of the fully exchanged, deuterated species supported the proposed mechanisms by suggesting that 3-dimethylaminomethyl-4-(4-methanesulfinyl-3-methylphenoxy)benzenesulfonamide has multiple sites of protonation in the gas phase. The planar structures of the posited product ions are likely to provide the driving force for the rearrangements. The relevance of the observations with regards to pharmaceutical drug metabolite identification is discussed.

A new small-molecule Stat3 inhibitor.

In this issue of Chemistry & Biology, Schust et al. report the discovery of a small molecule (Stattic) that inhibits the binding of a high affinity phosphopeptide for the SH2 domain of Stat3. Stattic is a new tool for studying Stat3 signaling and demonstrates that the SH2 domain is not a dead target.