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1.  Nitric Oxide (NO) Releasing Poly ADP-ribose Polymerase 1 (PARP-1) Inhibitors Targeted to Glutathione S-Transferase P1-Overexpressing Cancer Cells 
Journal of Medicinal Chemistry  2014;57(6):2292-2302.
We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me2N–N(O)=NO– group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity.
doi:10.1021/jm401550d
PMCID: PMC3983374  PMID: 24521039
2.  Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5’ sites 
Cell reports  2014;8(1):284-296.
N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life-cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them, WTAP, METTL14 and KIAA1429, are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps, and their classification into WTAP-dependent and independent sites. WTAP-dependent sites are located at internal positions in transcripts, are topologically static across a variety of systems we surveyed, and are inversely correlated with mRNA stability, consistent with a role in establishing ‘basal’ degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure, and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data sheds new light on proteomic and transcriptional underpinnings of this epitranscriptomic modification.
doi:10.1016/j.celrep.2014.05.048
PMCID: PMC4142486  PMID: 24981863
3.  Comparison of the rhythm control treatment strategy versus the rate control strategy in patients with permanent or long-standing persistent atrial fibrillation and heart failure treated with cardiac resynchronization therapy - a pilot study of Cardiac Resynchronization in Atrial Fibrillation Trial (Pilot-CRAfT): study protocol for a randomized controlled trial 
Trials  2014;15(1):386.
Background
The only subgroups of patients with heart failure and atrial fibrillation in which the efficacy of cardiac resynchronization therapy has been scientifically proven are patients with indications for right ventricular pacing and patients after atrioventricular junction ablation. However it is unlikely that atrioventricular junction ablation would be a standard procedure in the majority of the heart failure patients with cardiac resynchronization therapy and concomitant atrial fibrillation due to the irreversible character of the procedure and a spontaneous sinus rhythm resumption that occurs in about 10% of these patients.
Methods/Design
Pilot-CRAfT is the first randomized controlled trial evaluating the efficacy of a rhythm control strategy in atrial fibrillation patients with cardiac resynchronization therapy devices. The aim of this prospective, single center randomized controlled pilot study is to answer the question whether the patients with cardiac resynchronization therapy and permanent atrial fibrillation would benefit from a strategy to restore and maintain sinus rhythm (that is ‘rhythm control’ strategy) in comparison to rate control strategy. The study population consists of 60 patients with heart failure and concomitant long-standing persistent or permanent atrial fibrillation who underwent a cardiac resynchronization therapy device implantation at least 3 months before qualification. Study participants are randomly assigned to the rhythm control strategy (including electrical cardioversion and pharmacotherapy) or to the rate control group whose goal is to control ventricular rate. The follow-up time is 12 months. The primary endpoint is the ratio of effectively captured biventricular beats. The secondary endpoints include peak oxygen consumption, six-minute walk test distance, heart failure symptom escalation, reverse remodelling of the heart on echo and quality of life.
Trial registration
NCT01850277 registered on 22 April 2013 (ClinicalTrails.gov)
doi:10.1186/1745-6215-15-386
PMCID: PMC4283117  PMID: 25281275
Cardiac resynchronization therapy; Atrial fibrillation; Heart failure; Rhythm control; Cardioversion; Randomized controlled trial
4.  Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit 
Cancer discovery  2014;4(4):452-465.
Although the roles of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here we show that the IKK-related kinases TBK1 and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and IL-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 and MEK inhibitor therapy induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, JAK, and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.
doi:10.1158/2159-8290.CD-13-0646
PMCID: PMC3980023  PMID: 24444711
KRAS; cytokines; TBK1/IKKε; JAK; lung cancer
5.  K-ras 4A and 4B mRNA Levels Correlate with Superoxide in Lung Adenocarcinoma cells, While at the Protein Level, Only Mutant K-ras 4A Protein Correlates with Superoxide 
The K-ras gene is frequently mutated in lung and other cancers. K-ras protein includes two splice variants, K-ras 4A and 4B. While K-ras 4B is more widely expressed, recent evidence implicates K-ras 4A in lung tumorigenesis. We found that K-ras 4A protein has a wide range of expression in a large panel of human lung adenocarcinoma cell lines. In cell lines with mutant K-ras, but not those with wildtype K-ras, the K-ras 4A protein had a strong positive correlation with levels of cellular superoxide. We investigated whether K-ras 4A protein was involved in superoxide production, or alternatively was modulated by elevated superoxide. Experiments with small interfering RNA targeting K-ras 4A did not confirm its role in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to reduce levels of K-ras 4A protein. K-ras 4A and 4B mRNA were also quantified in a number of NSCLC cell lines. 4A mRNA correlated with 4A protein only in K-ras-mutant cells. K-ras 4A mRNA also correlated with superoxide, but with no difference between cell lines with mutant or wildtype K-ras. K-ras 4B mRNA correlated with 4A mRNA and with superoxide, in both K-ras mutant and wildtype cells. The results are consistent with superoxide directly or indirectly up-regulating expression of all K-ras genes, and also increasing the stability of K-ras 4A mutant protein selectively.
doi:10.1016/j.lungcan.2013.01.022
PMCID: PMC3992873  PMID: 23474128
K-ras 4A; lung; adenocarcinoma; superoxide; mutant K-ras; cell lines; siRNA
6.  COVERAGE OF BLOOD VESSELS BY ASTROCYTIC ENDFEET IS REDUCED IN MAJOR DEPRESSIVE DISORDER 
Biological psychiatry  2012;73(7):613-621.
Background
According to clinical studies, depression and cerebrovascular disease influence each other. Despite this evidence, no studies have investigated the relationship between major depressive disorder (MDD) and cerebrovascular disease at the cellular level. Astrocytic processes are a crucial interface between blood vessels and neurons, and astrocyte density is reduced in MDD. This study investigated the coverage of vessels by astrocyte endfeet in the prefrontal cortex in MDD.
Methods
Thirteen pairs of MDD and non-psychiatric control subjects were used for double immunofluorescent staining and confocal image analysis. Frozen sections of gray matter from orbitofrontal area 47 and white matter from the ventro-medial prefrontal cortex were examined. Astrocytic processes (labeled with antibodies for aquaporin-4, AQP4 or glial fibrillary acidic protein, GFAP) were co-localized with blood vessels (labeled with an antibody to collagen IV) to measure the coverage of vessel walls by astrocyte processes.
Results
The coverage of blood vessels by endfeet of AQP4-immunoreactive (IR) astrocytes was significantly reduced by 50 percent in subjects with MDD as compared to controls (ANCOVA: F(1,23)=5.161, p=0.033). This difference was detected in orbitofrontal gray matter but not in white matter. Conversely, the coverage of vessels by GFAP-IR processes did not significantly differ between the groups.
Conclusions
A significant reduction in the coverage of gray matter vessels by AQP4-IR astrocyte processes in MDD suggests alterations in AQP4 functions such as regulation of water homeostasis, blood flow, glucose transport and metabolism, the blood brain barrier, glutamate turnover and synaptic plasticity.
doi:10.1016/j.biopsych.2012.09.024
PMCID: PMC3578083  PMID: 23146357
aquaporin-4; GFAP; glia; neurovascular unit; blood brain barrier; cerebrovascular disease
7.  New surgical modification of fascial closure following endovascular aortic pathology repair 
Introduction
There are clear benefits of percutaneous versus open femoral access for endovascular aortic pathology repair. All closing devices commercially available are expensive. Surgical closure of the femoral artery risks potential prolonged wound healing and as a consequence longer hospital stay. Fascial closure is a technique that remains an interesting option.
Aim
To evaluate the efficacy of the surgical modification of hemostasis control after endovascular repair of aortic pathology.
Material and methods
One hundred sixteen common femoral arteries in a group of 58 patients underwent a minimally invasive procedure. Patients suffering from abdominal, thoracic aorta aneurysms, acute thoracic aorta type B dissections and traumatic aortic injury were treated.
Results
A 1-year period of experience in fascial closure of 116 common femoral arteries was presented in the group of 58 patients undergoing endovascular interventions. Five intraoperative complications were observed and one late. Three primary failures were due to hemorrhage in three arteries, one required open repair and two additional compression after the procedure. Two cases of limb ischemia required surgical correction of artery closure. One limb ischemia was detected 4 weeks later, and was treated conservatively. At 1 year, 92 fascial closures (80%) were in the follow-up and 24 (20%) were lost to follow-up.
Conclusions
This new modification of fascial closure is a safe and cheap method of arterial closure following endovascular repair of selected aortic pathologies. The usage of two suture lines makes this procedure easy and quick. Fascial closure technique is comparable to other techniques in terms of success and complication rates.
doi:10.5114/wiitm.2011.35795
PMCID: PMC3983539  PMID: 24729815
endovascular procedure; fascial closure; closing device
8.  Crosslinking Protein Glutathionylation Mediated by O2-Arylated Bis-Diazeniumdiolate “Double JS-K” 
Chemical research in toxicology  2012;25(12):2670-2677.
Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, e.g., by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semi-quantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein’s thiol group are covalently bound via a crosslinking agent, rather than through a disulfide bond. This form of thiol crosslinking has been shown to occur and confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.
doi:10.1021/tx3003142
PMCID: PMC3524378  PMID: 23106594
9.  Co-transcriptional production of RNA–DNA hybrids for simultaneous release of multiple split functionalities 
Nucleic Acids Research  2013;42(3):2085-2097.
Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.
doi:10.1093/nar/gkt1001
PMCID: PMC3919563  PMID: 24194608
10.  Activation of different split functionalities upon re-association of RNA-DNA hybrids 
Nature nanotechnology  2013;8(4):296-304.
Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications.
doi:10.1038/nnano.2013.44
PMCID: PMC3618561  PMID: 23542902
11.  Listeria monocytogenes-Derived Listeriolysin O Has Pathogen-Associated Molecular Pattern-Like Properties Independent of Its Hemolytic Ability 
There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.
doi:10.1128/CVI.00488-12
PMCID: PMC3535771  PMID: 23136118
12.  Structural modifications modulate stability of glutathione-activated arylated diazeniumdiolate prodrugs 
Bioorganic & medicinal chemistry  2012;20(9):3094-3099.
JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs’ biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound’s in vivo anti-tumor activity.
doi:10.1016/j.bmc.2012.02.045
PMCID: PMC3590845  PMID: 22480849
Nitric oxide; Diazeniumdiolate prodrugs; JS-K; Glutathione; Anti-cancer agents
13.  Thiol Modification By Pharmacologically Active Agents of the Diazeniumdiolate Class 
Promising drug candidates of the diazeniumdiolate (NONOate) chemical family include several types of thiol modification among their mechanisms of action: 1) drugs designed to release nitric oxide (NO) on reaction with the thiol group of glutathione (GSH) arylate the GSH, a step that removes reducing equivalents from the cell; (2) a similar reaction of the drug with the thiol group of a protein changes its structure, leading to potentially impaired function and cell death; (3) the NO generated as a byproduct in the above reactions can undergo oxidation, leading to S-nitrosylation and S-glutathionylation; and (4) diazeniumdiolates can also generate nitroxyl, which reacts with thiol groups to form disulfides or sulfinamides.
doi:10.1615/ForumImmunDisTher.2012006334
PMCID: PMC3622254  PMID: 23585982
thiol; nitric oxide; diazeniumdiolate
14.  Diazeniumdiolated carbamates: A novel class of nitric oxide donors 
Bioorganic & medicinal chemistry  2012;20(6):2025-2029.
We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.
doi:10.1016/j.bmc.2012.01.046
PMCID: PMC3298570  PMID: 22356735
Nitric oxide; Diazeniumdiolate prodrugs; Diazeniumdiolated carbamates; TOM protecting group; Intracellular nitric oxide
15.  Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance☆ 
Redox Biology  2013;1(1):115-124.
JS-K is a nitric oxide (NO)-releasing prodrug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.
Graphical abstract
Highlights
► JS-K is a NO-releasing prodrug, with anticancer activity in vitro and in vivo. ► JS-K metabolism results in the arylation of cellular glutathione (GSH). ► Micromolar levels of JS-K in media significantly deplete the cellular GSH reserve. ► Deregulation of cells' redox balance is a key contributor to JS-K toxicity.
doi:10.1016/j.redox.2012.12.002
PMCID: PMC3757670  PMID: 24024144
ATF, activating transcription factor; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DCF-DA, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSH, glutathione; GSSG, glutathione disulfide (oxidized GSH); HBSS, Hank's balanced salt solution; IPA, Ingenuity Pathway Analysis; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate; LC/MS, liquid chromatography/mass spectrometry; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NO, nitric oxide; NSCLC, non-small cell lung cancer; PARP, poly (ADP-ribose) polymerase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK/JNK, stress activated protein kinase/c-jun N-terminal kinase.; Glutathione; Nitric oxide; Arylated diazeniumdiolate; Leukemia
16.  The Role of Mcl-1 in S. aureus-Induced Cytoprotection of Infected Macrophages 
Mediators of Inflammation  2013;2013:427021.
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.
doi:10.1155/2013/427021
PMCID: PMC3569898  PMID: 23431241
17.  Dose-Dependent Effects of Neonatal SSRI Exposure on Adult Behavior in the Rat 
Brain research  2011;1429:52-60.
Neonatal exposure to antidepressants produces lasting impairments in male sexual behavior. Although perturbation of the serotonin system during neonatal life has been implicated in the long-term behavioral effects of neonatal antidepressant exposure, dose-response studies were necessary to confirm that inhibition of the serotonin transporter during the neonatal period is sufficient to produce impairments in sexual behavior. Therefore, the present study examined the dose-response effects of neonatal citalopram exposure on sexual behavior. In addition, the effects of exposure on anxiety-related behavior were examined since alterations in this behavioral measure could affect sexual behavior. Male Long-Evans rats were injected subcutaneously with citalopram (CTM) in one of three doses (5, 10 or 20 mg/kg/day), or saline (SAL) in a volume of 0.1 ml twice daily (07:00 and 14:00h) from PN8 to PN21. The rats were tested as adults (>PN90) for anxiety-like behavior and exploration in the elevated plus maze test and sexual behavior.
Neonatal citalopram exposure produced persistent reductions in male sexual behavior characterized by significant dose-dependent reductions in the percentage of male rats displaying mounting as well as dose-dependent reductions in the number of mounts and mount latency. Neonatal citalopram exposure also produced significant dose-dependent linear trends for reductions in intromission and ejaculation behavior. However, neonatal SSRI exposure was not found to produce any effects on exploration or anxiety-like behavior in the elevated plus maze test. The present findings support the hypothesis that inhibition of the serotonin transporter during neonatal life by an SSRI is directly responsible for the long-term effects on male sexual behavior.
doi:10.1016/j.brainres.2011.10.025
PMCID: PMC3228278  PMID: 22079319
Neonatal exposure; Citalopram; Sexual behavior; Anxiety
18.  Activation of the c-Jun N-terminal Kinase /Activating Transcription Factor 3 (ATF3) Pathway Characterizes Effective Arylated Diazeniumdiolate-Based Nitric Oxide-Releasing Anticancer Prodrugs 
Journal of medicinal chemistry  2011;54(22):7751-7758.
Improved therapies are needed for non-small cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)–releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against non-small cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue, and structural modification of these into more stable prodrugs. Compound 1 and its homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have anti-tumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.
doi:10.1021/jm2004128
PMCID: PMC3422893  PMID: 22003962
19.  Clinical efficacy of antazoline in rapid cardioversion of paroxysmal atrial fibrillation -- a protocol of a single center, randomized, double-blind, placebo-controlled study (the AnPAF Study) 
Trials  2012;13:162.
Background
Rapid conversion of atrial fibrillation (AF) to sinus rhythm may be achieved by the administration of class IA, IC and III antiarrhythmic drugs or vernakalant hydrochloride. However, that treatment may be related to potential pro-arrhythmia, lack of efficacy or the exceptionally high cost of a compound used. Antazoline is a first generation antihistaminic agent with chinidin-like properties. When administered intravenously, antazoline exerts a strong antiarrhythmic effect on supraventricular arrhythmia, especially on AF, facilitating rapid conversion to sinus rhythm. Despite a relative lack of published data antazoline has been marketed in Poland and widely used in cardiology wards and emergency rooms for many years due to its efficacy, safety and rapid onset of action within minutes of administration.
Methods/design
A randomized, double blind, placebo-controlled, superiority clinical trial was designed to assess clinical efficacy of antazoline in rapid conversion of AF to sinus rhythm. Eligible patients will present AF lasting less than 43 hours, will be in stable cardio-pulmonary condition and will have no prior history of advanced heart failure or significant valvular disease. Long-term antiarrhythmic therapy is not considered an exclusion criterion. Subjects who fulfill selection criteria will be randomly assigned to receive intravenously either antazoline or placebo in divided doses and observed for 1.5 hours after conversion to sinus rhythm or after the last i.v. bolus. Primary end point will be the conversion of AF to sinus rhythm confirmed in an electrocardiogram (ECG) during the observation period. Secondary end points will be comprised of time to conversion and return of AF during the observation period. Special consideration will be given to the observation of any adverse events. A sample size of 80 patients was calculated based on the following assumptions: two-tailed test, a type I error of 0.01, a power of 90%, efficacy of placebo 5%, efficacy of antazoline 50% and 20% drop-out rate to fulfill the criteria of intention-to-treat analysis. Due to the presumed lack of statistical power, the secondary end points and safety endpoints will be considered exploratory.
Clinical trials registry
ClinicalTrials.gov, NCT01527279
doi:10.1186/1745-6215-13-162
PMCID: PMC3502525  PMID: 22967497
Antazoline; Atrial fibrillation; Pharmacological cardioversion; Efficacy; Safety; Randomized clinical trial
20.  First Universities Allied for Essential Medicines (UAEM) Neglected Diseases and Innovation Symposium 
Universities Allied for Essential Medicines organized its first Neglected Diseases and Innovation Symposium to address expanding roles of public sector research institutions in innovation in research and development of biomedical technologies for treatment of diseases, particularly neglected tropical diseases. Universities and other public research institutions are increasingly integrated into the pharmaceutical innovation system. Academic entities now routinely undertake robust high-throughput screening and medicinal chemistry research programs to identify lead compounds for small molecule drugs and novel drug targets. Furthermore, product development partnerships are emerging between academic institutions, non-profit entities, and biotechnology and pharmaceutical companies to create diagnostics, therapies, and vaccines for diseases of the poor. With not for profit mission statements, open access publishing standards, open source platforms for data sharing and collaboration, and a shift in focus to more translational research, universities and other public research institutions are well-placed to accelerate development of medical technologies, particularly for neglected tropical diseases.
doi:10.4269/ajtmh.2012.11-0608
PMCID: PMC3247111  PMID: 22232453
21.  The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline 
ACS medicinal chemistry letters  2010;1(8):386-389.
V-PYRRO/NO is a well studied nitric oxide (NO) prodrug which has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using 14C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in development of further NO prodrugs with increased permeability.
doi:10.1021/ml1000905
PMCID: PMC3013513  PMID: 21212855
Nitric oxide; prodrug; proline; transporter; PROLI/NO; V-PROLI/NO
22.  The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline 
ACS Medicinal Chemistry Letters  2010;1(8):386-389.
V-PYRRO/NO is a well-studied nitric oxide (NO) prodrug that has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using 14C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in the development of further NO prodrugs with increased permeability.
doi:10.1021/ml1000905
PMCID: PMC3013513  PMID: 21212855
Nitric oxide; prodrug; proline; transporter; PROLI/NO; V-PROLI/NO
23.  ‘Click’ Reaction in Conjunction with Diazeniumdiolate Chemistry: Developing High-Load Nitric Oxide Donors 
Organic letters  2010;12(19):4256-4259.
The use of Cu(I)-catalyzed ‘click’ reactions of alkyne-substituted diazeniumdiolate prodrugs with bis- and tetrakisazido compounds is described. The ‘click’ reaction for the bis-azide using CuSO4/Na-ascorbate predominantly gave the expected bis-triazole. However, CuI/diisopropylethylamine predominantly gave uncommon triazolo-triazole products as a result of oxidative coupling. Neither set of ‘click’ condition showed evidence of compromising the integrity of the diazeniumdiolate groups. The chemistry developed has applications in the synthesis of polyvalent and dendritic nitric oxide donors.
doi:10.1021/ol101645k
PMCID: PMC2945422  PMID: 20812718
24.  Deregulated Expression of Superoxide Dismutase-2 Correlates with Different Stages of Cervical Neoplasia 
Disease markers  2011;30(6):275-281.
Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer.
Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO).
Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining.
Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.
doi:10.3233/DMA-2011-0784
PMCID: PMC3825245  PMID: 21725155
Human papillomavirus; Tissue Microarray; Biomarker; Cervical Ne oplasia; Superoxide Dismutase-2
25.  Neonatal Antidepressant Exposure has Lasting Effects on Behavior and Serotonin Circuitry 
A significant fraction of infants born to mothers taking selective serotonin reuptake inhibitors (SSRIs) during late pregnancy display clear signs of antidepressant withdrawal indicating that these drugs can penetrate fetal brain in utero at biologically significant levels. Previous studies in rodents have demonstrated that early exposure to some antidepressants can result in persistent abnormalities in adult behavior and indices of monoaminergic activity. Here, we show that chronic neonatal (postnatal days 8–21) exposure to citalopram (5 mg/kg, twice daily, s.c.), a potent and highly selective SSRI, results in profound reductions in both the rate-limiting serotonin synthetic enzyme (tryptophan hydroxylase) in dorsal raphe and in serotonin transporter expression in cortex that persist into adulthood. Furthermore, neonatal exposure to citalopram produces selective changes in behavior in adult rats including increased locomotor activity and decreased sexual behavior similar to that previously reported for antidepressants that are nonselective monoamine transport inhibitors. These data indicate that the previously reported neurobehavioral effects of antidepressants are a consequence of their effects on the serotonin transporter. Moreover, these data argue that exposure to SSRIs at an early age can disrupt the normal maturation of the serotonin system and alter serotonin-dependent neuronal processes. It is not known whether this effect of SSRIs is paralleled in humans; however, these data suggest that in utero, exposure to SSRIs may have unforeseen long-term neurobehavioral consequences.
doi:10.1038/sj.npp.1300823
PMCID: PMC3118509  PMID: 16012532
neonatal exposure; citalopram; clomipramine; tryptophan hydroxylase; serotonin transporter; behavior

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