Procognitive activity of nitric oxide inhibitors and donors in animal models.

Nitric oxide is a small gaseous molecule that plays important roles in the majority of biological functions. Impairments of NO-related pathways contribute to the majority of neurological disorders, such as Alzheimer's disease (AD), and mental disorders, such as schizophrenia. Cognitive decline is one of the most serious impairments accompanying both AD and schizophrenia. In the present study, the activities of NO donors, slow (spermine NONOate) or fast (DETANONOate) releasers, and selective inhibitor of neuronal nitric oxide synthase N(ω)-propyl-l-arginine (NPLA) were investigated in pharmacological models of schizophrenia and AD. Cognitive impairments were induced by administration of MK-801 or scopolamine and were measured in novel object recognition (NOR) and Y-maze tests. The compounds were investigated at doses of 0.05-0.5 mg/kg. The dose-dependent effectiveness of all the compounds was observed in the NOR test, while only the highest doses of spermine NONOate and NPLA were active in the Y-maze test. DETANONOate was not active in the Y-maze test. The impact of the investigated compounds on motor coordination was tested at doses of 0.5 and 1 mg/kg. Only NPLA at a dose of 1 mg/kg slightly disturbed motor coordination in animals.

TPGS-Galactose-Modified Polydopamine Co-delivery Nanoparticles of Nitric Oxide Donor and Doxorubicin for Targeted Chemo-Photothermal Therapy against Drug-Resistant Hepatocellular Carcinoma.

The lack of cancer cell specificity and the occurrence of multidrug resistance (MDR) are two major obstacles in the treatment of hepatocellular carcinoma (HCC). To tackle these challenges, a novel nanoparticle (NP)-based drug delivery system (DDS) with a core/shell structure consisted of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-galactose (Gal)/polydopamine (PDA) is fabricated. The NP is loaded with doxorubicin (DOX) and a nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN) sensitive to heat to afford NO-DOX@PDA-TPGS-Gal. The unique binding of Gal to asialoglycoprotein receptor (ASGPR) and the pH-sensitive degradation of NP ensure the targeted transportation of NP into liver cells and the release of DOX in HCC cells. The near-infrared (NIR) light further facilitates DOX release and initiates NO generation from BNN due to the photothermal property of PDA. In addition to the cytotoxicity contributed by DOX, NO, and heat, TPGS and NO act as MDR reversal agents to inhibit P-glycoprotein (P-gp)-related efflux of DOX by HepG2/ADR cells. The combined chemo-photothermal therapy (chemo-PTT) by NO-DOX@PDA-TPGS-Gal thus shows potent anti-cancer activity against drug-resistant HCC cells in vitro and in vivo and significantly prolongs the life span of drug-resistant tumor-bearing mice. The present work provides a useful strategy for highly targeted and MDR reversal treatment of HCC.

Effects of Transient Receptor Potential Cation 5 (TRPC5) Inhibitor, NU6027, on Hippocampal Neuronal Death after Traumatic Brain Injury.

Traumatic brain injury (TBI) can cause physical, cognitive, social, and behavioral changes that can lead to permanent disability or death. After primary brain injury, translocated free zinc can accumulate in neurons and lead to secondary events such as oxidative stress, inflammation, edema, swelling, and cognitive impairment. Under pathological conditions, such as ischemia and TBI, excessive zinc release, and accumulation occurs in neurons. Based on previous research, it hypothesized that calcium as well as zinc would be influx into the TRPC5 channel. Therefore, we hypothesized that the suppression of TRPC5 would prevent neuronal cell death by reducing the influx of zinc and calcium. To test our hypothesis, we used a TBI animal model. After the TBI, we immediately injected NU6027 (1 mg/kg, intraperitoneal), TRPC5 inhibitor, and then sacrificed animals 24 h later. We conducted Fluoro-Jade B (FJB) staining to confirm the presence of degenerating neurons in the hippocampal cornus ammonis 3 (CA3). After the TBI, the degenerating neuronal cell count was decreased in the NU6027-treated group compared with the vehicle-treated group. Our findings suggest that the suppression of TRPC5 can open a new therapeutic window for a reduction of the neuronal death that may occur after TBI.

Coronary Stents Decorated by Heparin/NONOate Nanoparticles for Anticoagulant and Endothelialized Effects.

In the treatment of coronary artery disease (CAD), the use of stent implantation often leads to clinical complications such as restenosis, delayed endothelial healing, and thrombosis. Here, we develop a double drug sustained-release coating for the stent surface by grafting heparin/NONOate nanoparticles (Hep/NONOates). The Hep/NONOates and surface modification of the stent were characterized by X-ray photoelectron spectroscopy, attenuated total reflection Fourier-transform infrared spectroscopy, static water contact angle, and scanning electron microscopy (SEM), and the release behaviors of the anticoagulant, heparin (Hep) and the bioactive molecule, nitric oxide (NO) were studied. Furthermore, the blood compatibility and cytotoxicity of the modified stent were evaluated by whole blood adhesion and platelet adhesion tests, hemolysis assay, morphological changes of red blood cells, plasma recalcification time assay, in vitro coagulation time tests, and MTT assay. Finally, the results of a rabbit carotid artery stent implantation experiment showed that the double drug sustained-release coating for the stent can accelerate regeneration of endothelial cells and keep good anticoagulant activity. This study can provide new design ideas based on nanotechnology for improving the safety and effectiveness of drug-eluting stents.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.

Targeting ATR in cancer.

The chemical treatment of cancer started with the realization that DNA damaging agents such as mustard gas present notable antitumoural properties. Consequently, early drug development focused on genotoxic chemicals, some of which are still widely used in the clinic. However, the efficacy of such therapies is often limited by the side effects of these drugs on healthy cells. A refinement to this approach is to use compounds that can exploit the presence of DNA damage in cancer cells. Given that replication stress (RS) is a major source of genomic instability in cancer, targeting the RS-response kinase ataxia telangiectasia and Rad3-related protein (ATR) has emerged as a promising alternative. With ATR inhibitors now entering clinical trials, we here revisit the biology behind this strategy and discuss potential biomarkers that could be used for a better selection of patients who respond to therapy.

In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-nitrosated human serum albumin dimer.

In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane®). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed.

[Human Serum Albumin as Carrier in Drug Delivery Systems].

  Recently, human serum albumin (HSA) has emerged as a versatile carrier for therapeutic agents against diabetes, cancer, and infectious diseases. Market-approved products include fatty acid derivatives of human insulin for diabetes and the paclitaxel-HSA nanoparticle for various cancers such as metastatic breast cancer and advanced pancreatic cancer. In this review, we focus on the next-generation approach including HSA-binding bioactive gas such as nitric oxide (NO) for treating ischemic/reperfusion injury, cancer, and bacterial infection. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated HSA (SNO-HSA), which is why we are investigating whether this albumin form can be therapeutically useful. Recently, we have developed SNO-HSA analogues such as SNO-HSA with many conjugated SNO groups (poly-SNO-HSA) prepared using chemical modification. Unexpectedly, we found striking inverse effects between poly-SNO-HSA and SNO-HSA. Despite the fact that SNO-HSA inhibits apoptosis, poly-SNO-HSA possesses very strong pro-apoptotic effects against tumor cells. Furthermore, poly-SNO-HSA can reduce or even completely eliminate the multidrug resistance often developed by cancer cells. In this review, we put forward the possibility that poly-SNO-HSA can be used as a safe, effective multifunctional antitumor agent.

S-Nitrosated human serum albumin dimer as novel nano-EPR enhancer applied to macromolecular anti-tumor drugs such as micelles and liposomes.

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.

M1 polarization bias and subsequent nonalcoholic steatohepatitis progression is attenuated by nitric oxide donor DETA NONOate via inhibition of CYP2E1-induced oxidative stress in obese mice.

Activation of M1 macrophages in nonalcoholic steatohepatitis (NASH) is produced by several external or endogenous factors: inflammatory stimuli, oxidative stress, and cytokines are known. However, any direct role of oxidative stress in causing M1 polarization in NASH has been unclear. We hypothesized that CYP2E1-mediated oxidative stress causes M1 polarization in experimental NASH, and that nitric oxide (NO) donor administration inhibits CYP2E1-mediated inflammation with concomitant attenuation of M1 polarization. Because CYP2E1 takes center stage in these studies, we used a toxin model of NASH that uses a ligand and a substrate of CYP2E1 for inducing NASH. Subsequently, we used a methionine and choline-deficient diet-induced rodent NASH model where the role of CYP2E1 in disease progression has been shown. Our results show that CYP2E1 causes M1 polarization bias, which includes a significant increase in interleukin-1ß (IL-1ß) and IL-12 in both models of NASH, whereas CYP2E1-null mice or diallyl sulfide administration prevented it. Administration of gadolinium chloride (GdCl3), a macrophage toxin, attenuated both the initial M1 response and the subsequent M2 response, showing that the observed increase in cytokine levels is primarily from macrophages. Based on the evidence of an adaptive NO increase, the NO donor administration in vivo that mechanistically inhibited CYP2E1 catalyzed the oxidative stress during the entire study in NASH-abrogated M1 polarization and NASH progression. The results obtained show the association of CYP2E1 in M1 polarization, and that inhibition of CYP2E1 catalyzed oxidative stress by an NO donor (DETA NONOate [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate]) can be a promising therapeutic strategy in NASH.

Neuroprotective effects of nitric oxide donor NOC-18 against brain ischemia-induced mitochondrial damages: role of PKG and PKC.

In this study we sought to determine whether NO donor NOC-18 can protect brain mitochondria against ischemia-induced dysfunction, particularly opening of mitochondrial permeability transition pore (MPTP), and cell death. We found that inhibition of respiration with NAD-dependent substrates, but not with succinate, was observed after 30 min ischemia indicating that complex I of the mitochondrial respiratory chain is the primary site affected by ischemia. There was no loss of mitochondrial cytochrome c during 30-120 min of brain ischemia. Prolonged, 90 min ischemia substantially decreased calcium retention capacity of brain mitochondria suggesting sensitization of mitochondria to Ca(2+)-induced MPTP opening, and this was prevented by NOC-18 infusion prior to ischemia. NOC-18 did not prevent ischemia-induced inhibition of mitochondrial respiration, however, it partially protected against ischemia-induced necrosis. Protective effects of NOC-18 were abolished in the presence of selective inhibitors of protein kinase G (PKG) and protein kinase C (PKC). These results indicate that pre-treatment with NOC-18 protected brain mitochondria against ischemia-induced MPTP opening by decreasing mitochondrial sensitivity to calcium and partly protected brain cells against necrotic death in PKG- and PKC-depending manner.

Tuning of poly-S-nitrosated human serum albumin as superior antitumor nanomedicine.

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. We have previously demonstrated that poly-nitric oxide (NO) conjugated human serum albumin (Poly-SNO-HSA) has the potential to be a DDS carrier capable of accumulating NO in tumors. However, the stability of Poly-SNO-HSA in the circulation has to be improved, and its optimal molecular size for using the EPR effects has to be evaluated. In the present study, we performed two tuning methods for refining Poly-SNO-HSA, namely, pegylation and dimerization. We observed that pegylation enhanced the stability of Poly-SNO-HSA both in vitro and in vivo, and that dimerization of Poly-SNO-HSA enhanced the antitumor activity via more efficient delivery of NO in Colon 26 tumor-bearing mice. Intriguingly, dimerization resulted in a 10 times higher antitumor activity. These data suggest that pegylation and dimerization of Poly-SNO-HSA are very important tuners to optimize NO stability and accumulation, and thereby effect, in tumors. Thus, polyethylene glycol-Poly-SNO-HSA dimer seems to be a very appealing and safe NO carrier and thereby a strong candidate as an antitumor drug in future development of cancer therapeutics.

Nitrosonifedipine ameliorates the progression of type 2 diabetic nephropathy by exerting antioxidative effects.

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.

Elucidation of the therapeutic enhancer mechanism of poly-S-nitrosated human serum albumin against multidrug-resistant tumor in animal models.

Human serum albumin (HSA) is the most abundant circulating protein and its S-nitrosated form serves as a reservoir of nitric oxide (NO). Previously, we prepared poly-S-nitrosated HSA (Poly-SNO-HSA) by incubation with Traut's Reagent and isopentyl nitrite and evaluated its potential as a novel anticancer agent through apoptosis involving the caspase-3 pathway. Recently, NO donors such as nitroglycerin were reported to revert the resistance to anticancer agents. Therefore, now we have evaluated the effect of the above type of Poly-SNO-HSA on the resistance to doxorubicin (dx) in human myelogenous leukemic cells (K562 cells). P-gp expression and dx accumulation in K562 and dx-resistant K562 cells (K562/dx cells) were quantified using Western blot and FACS analysis, respectively. Compared with parent K562 cells, higher expression of P-gp and lower accumulation of dx were shown in K562/dx cells. Poly-SNO-HSA caused increased dx accumulation in K562/dx cells by decreasing the expressions of P-gp and HIF-1α. Other experiments with the guanylate cyclase inhibitor ODQ and 8-Br-cGMP revealed that also a cGMP signaling pathway is involved in the Poly-SNO-HSA induced increase in dx accumulation. Furthermore, in vivo studies showed that co-treatment with Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx cells-bearing mice. Thus, in addition to its proapoptotic effect Poly-SNO-HSA can in an efficient manner revert drug resistance both in vitro and in vivo, and two pathways for this effect have been identified.

Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines.

BACKGROUND: The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR. METHODS: Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells. RESULTS: NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 µM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1. CONCLUSION: NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors.

Dye-tethered ruthenium nitrosyls containing planar dicarboxamide tetradentate N4 ligands: effects of in-plane ligand twist on NO photolability.

To examine the steric effects of the in-plane ligands in dye-sensitized {RuNO}(6) nitrosyls on their NO photolability, two new ligands, namely, 1,2-Bis(pyridine-2-carboxamido)-4,5-dimethoxybenzene (H(2)(OMe)(2)bpb) and 1,2-Bis(Isoquinoline-1-carboxamido)-4,5-dimethoxybenzene (H(2)(OMe)(2)IQ1, H's are dissociable carboxamide protons) have been designed and synthesized. The syntheses and spectroscopic properties of {RuNO}(6) nitrosyls derived from these two ligands, namely, [((OMe)(2)bpb)Ru(NO)(Cl)] (4-Cl), [((OMe)(2)IQ1)Ru(NO)(Cl)] (5-Cl), [((OMe)(2)bpb)Ru(NO)(Resf)] (4-Resf), and [((OMe)(2)IQ1)Ru(NO)(Resf)] (5-Resf), are reported. The structures of 5-Cl, 4-Resf, and 5-Resf have been determined by X-ray crystallography. Removal of the in-plane ligand twist in the quinoline-based R(2)bQb(2-) ligand frame (because of steric interactions between the extended quinoline ring systems) in both R(2)bpb(2-) and R(2)IQ1(2-) (pyridine and 1-isoquinoline rings, respectively, instead of quinoline rings in the equatorial plane) results in enhanced solution stability, as well as higher quantum yield values for NO photorelease upon exposure to 500 nm light. Both dye-tethered {RuNO}(6) nitrosyls 4-Resf and 5-Resf exhibit greater sensitivity to visible light compared to the chloro-bound species 4-Cl and 5-Cl. In addition, the dye-tethered nitrosyls are fluorescent and hence can be used as trackable NO donors in cellular studies.

One-step preparation of S-nitrosated human serum albumin with high biological activities.

S-Nitrosated human serum albumin (SNO-HSA) is a large molecular weight nitric oxide carrier in human plasma, and because of its many beneficial effects in different tests, it is currently under investigation as a cytoprotective agent. However, making SNO-HSA preparations is a complicated and time-consuming process. We found that binding of caprylic acid (CA) and N-acetyl-l-tryptophan (N-AcTrp) to defatted mercaptalbumin increased S-nitrosation by S-nitrosoglutathione (GS-NO) by making Cys-34 of HSA more accessible and by protecting it against oxidation, respectively. Fortunately, HSA solutions for clinical use contain high concentrations of CA and N-AcTrp as stabilizers. By making use of that fact it was possible to work-out a fast and simple procedure for producing SNO-HSA: incubation of a commercial HSA formulation with GS-NO for only 1 min results in S-nitrosation of HSA. The biological usefulness of such a preparation was tested in a rat ischemia-reperfusion liver injury model. Although our procedure for making SNO-HSA is fast and straightforward, the cytoprotective effect of the preparation was similar to, or better than, that of a preparation made in a more traditional way. The clinical development of SNO-HSA as a strong cytoprotective agent is under way using this method in collaboration with clinicians and industrial developers.

Reactivation of brain acetylcholinesterase by monoisonitrosoacetone increases the therapeutic efficacy against nerve agents in guinea pigs.

Current oxime therapies do not readily cross the blood-brain barrier to reactivate organophosphorus nerve agent-inhibited cholinesterase (ChE) within the CNS. We investigated the ability of monoisonitrosoacetone (MINA), a tertiary oxime, to reactivate ChE inhibited by the nerve agent sarin (GB), cyclosarin (GF), or VX, in peripheral tissues and brain of guinea pigs and determined whether reactivation in the CNS will enhance protection against the lethal effects of these three agents. In the reactivation experiment, animals were pretreated with atropine methylnitrate (1.0mg/kg, i.m.) 15 min prior to subcutaneous (s.c.) challenge with 1.0 x LD(50) of GB, GF, or VX. Fifteen minutes later animals were treated intramuscularly (i.m.) with MINA (ranging from 22.1 to 139.3mg/kg) or 2-PAM (25.0mg/kg). At 60 min after nerve agent, CNS (brainstem, cerebellum, cortex, hippocampus, midbrain, spinal cord, and striatum) and peripheral (blood, diaphragm, heart, and skeletal muscle) tissues were collected for ChE analysis. MINA reactivated nerve agent-inhibited ChE in the CNS and peripheral tissues in a dose-dependent manner in the following order of potency: GB>GF>VX. In a survival experiment, animals were injected i.m. with atropine sulfate (0.5mg/kg), 2-PAM (25.0mg/kg), or MINA (35.0, 60.0, or 100.0mg/kg) alone or in combination 1 min after challenge with varying s.c. doses of GB, GF, or VX to determine the level of protection. The rank order of MINA's efficacy in guinea pigs against nerve agent lethality was the same as for reactivation of inhibited ChE in the CNS. These data show that MINA is capable of reactivating nerve agent-inhibited ChE and that the extent of ChE reactivation within the CNS strongly relates to its therapeutic efficacy.