Furoxans (1,2,5-Oxadiazole-<i>N</i>-Oxides) as Novel NO Mimetic Neuroprotective and Procognitive Agents

Furoxans (1,2,5-oxadiazole-<i>N</i>-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO₂^–, but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-β peptide (Aβ) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies

Proteomic Profiling of Nitrosative Stress: Protein <i>S-</i>Oxidation Accompanies <i>S-</i>Nitrosylation

Reversible chemical modifications of protein cysteine residues by <i>S-</i>nitrosylation and <i>S-</i>oxidation are increasingly recognized as important regulatory mechanisms for many protein classes associated with cellular signaling and stress response. Both modifications may theoretically occur under cellular nitrosative or nitroxidative stress. Therefore, a proteomic isotope-coded approach to parallel, quantitative analysis of cysteome <i>S-</i>nitrosylation and <i>S-</i>oxidation was developed. Modifications of cysteine residues of (i) human glutathione-S-transferase P1-1 (GSTP1) and (ii) the schistosomiasis drug target thioredoxin glutathione reductase (TGR) were studied. Both <i>S-</i>nitrosylation (SNO) and <i>S-</i>oxidation to disulfide (SS) were observed for reactive cysteines, dependent on concentration of added <i>S</i>-nitrosocysteine (CysNO) and independent of oxygen. SNO and SS modifications of GSTP1 were quantified and compared for therapeutically relevant NO and HNO donors from different chemical classes, revealing oxidative modification for all donors. Observations on GSTP1 were extended to cell cultures, analyzed after lysis and in-gel digestion. Treatment of living neuronal cells with CysNO, to induce nitrosative stress, caused levels of <i>S-</i>nitrosylation and <i>S-</i>oxidation of GSTP1 comparable to those of cell-free studies. Cysteine modifications of PARK7/DJ-1, peroxiredoxin-2, and other proteins were identified, quantified, and compared to overall levels of protein <i>S-</i>nitrosylation. The new methodology has allowed identification and quantitation of specific cysteome modifications, demonstrating that nitroxidation to protein disulfides occurs concurrently with <i>S-</i>nitrosylation to protein-SNO in recombinant proteins and living cells under nitrosative stress

2‑Arylidene Hydrazinecarbodithioates as Potent, Selective Inhibitors of Cystathionine γ‑Lyase (CSE)

Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS

Design and Synthesis of Neuroprotective Methylthiazoles and Modification as NO-Chimeras for Neurodegenerative Therapy

Learning and memory deficits in Alzheimer’s disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA_A receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen–glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD

Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe

Hydrogen sulfide (H₂S) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for H₂S, LP2-Cu­(II)-Ln­(III), a heterobinuclear metal complex that uses Cu­(II) decomplexation to control millisecond-scale-lifetime-Tb­(III)- or Eu­(III)-emission intensity. LP2-Cu­(II)-Ln­(III) responded rapidly, selectively, and with high sensitivity to aqueous H₂S. The probe’s potential for biological applications was verified by measuring the H₂S generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by Na₂S-stimulated HeLa cells

Activity of anti-fungal imidazole CYP450 inhibitors on larval and adult <i>Schistosoma mansoni</i> worms.

<p>Survival of schistosomula (A) after 2 d culture and adult worms (B) after 5 d culture for miconazole (black diamond), clotrimazole (black square), and ketoconazole (black triangle). (C) In house SAR on known miconazole analogs against adult worms. Numbers in the parenthesis are survival (%) of adult worms on day 7 in 10 μM of respective compound.</p

Comparison of <i>Schistosoma mansoni</i> CYP450 protein (Sman) with CYP450 proteins from other species.

<p>Multiple alignment of CYP450 proteins from <i>S</i>. <i>mansoni</i> (csm305A); rabbit CYP450 2C5 (1nr6_a); human CYP450 2C9 (1r9o_a); human CYP450 2C19 (4gqs_a); human CYP450 1A1 (4i8v_a); and human CYP450 2b6 (4rrt_a). The residues are shown in one letter code and colored by type: red- negatively charged, blue—positively charged, yellow—Cys, green—hydrophobic, cyan—Gly, ochre—Pro, purple—aromatic. The residues are shown in brighter colors for conserved positions. The ‘P450-signature’ sequence, which forms a channel for electron transfer, and the CYP450 consensus motif responsible for heme-binding and interaction with molecular oxygen and the relevant substrates are boxed. Predicted helices in the secondary structure based on homology modelling of SmCYP450 are indicated by the bold letters A-L based on rabbit CYP450 2C5 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004279#pntd.0004279.ref038" target="_blank">38</a>].</p

Structural modeling of <i>S</i>. <i>mansoni</i> CYP450 (CYP3050A1) and comparison to the structure determined for rabbit CYP450 2C5 (1nr6_a) [38].

<p>The heme is shown is each model as a space-filling projection. The J and J’ helices in rabbit CYP450 2C5, which are absent in <i>S</i>. <i>mansoni</i> CYP450, are highlighted in yellow.</p

CYP450 messenger RNA abundance during the lifecycle of <i>Schistosoma mansoni</i>.

<p>Whole RNA was extracted from different stages of <i>S</i>. <i>mansoni</i> (cercariae, 1-day old schistosomula; juvenile liver worms (23 days post infection), adult males (49 days post infection), adult females (49 days post infection) and eggs) using TRIzol reagent and chloroform/ethanol extraction protocol. cDNA was synthesized from whole RNA and used for qRT-PCR, with reactions done in triplicate. Adult males (= 1) were used as calibrator stage and mRNA abundance was normalized to α-tubulin. Error bars indicate standard error of the mean with n ≥ 3 biological replicates. Numbers indicate fold change relative to adult males and all values are significantly different from adult males; p < 0.05; student t-test. The results indicate that <i>S</i>. <i>mansoni</i> CYP450 is expressed in all stages investigated and that its expression is developmentally regulated.</p

Relaxation of isolated aortic rings by SERMs and NO-SERM.

<p>(<b>A</b>) The EC₅₀ values for relaxation were not significantly different for raloxifene, arzoxifene, DMA, and FDMA (p>0.05, one-way ANOVA and Newman-Keul's post-hoc test), whereas for NO-DMA potency was significantly different from all other SERMs (F_(4,43) = 4.085, p<0.01). The maximal relaxation responses for arzoxifene and FDMA were significantly less than those for DMA and raloxifene (F_{(3, 37)} = 11.77 p<0.05, one-way ANOVA and Newman-Keul's post-hoc test). Each value represents the mean ± S.E.M. (n = 7–13). (<b>B</b>) Removal of the endothelium or inhibition of NOS with L-NAME reduced the maximal relaxation response to DMA (F_{(2, 18)} = 28.22, p<0.001, one-way ANOVA and Newman-Keuls post-hoc test). Each value represents the mean ± S.E.M. (n = 7). (<b>C</b>) The EC₅₀ values for relaxation were significantly increased in the presence of L-NAME or after endothelium removal (F_(2,18) = 7.753, p<0.05, one-way ANOVA and Newman-Keuls post-hoc test). Each value represents the mean ± S.E.M. (n = 7).</p