Combination of NOS- and PDK-Inhibitory Activity: Possible Way to Enhance Antitumor Effects

We have previously demonstrated a high antitumor potential of NOS inhibitor T1023 (1-isobutanoyl-2-isopropylisothiourea hydrobromide): antitumor antiangiogenic activity in several animal tumor models and its ability to synergistically enhance the antitumor effects of bevacizumab, cyclophosphamide and γ-radiation. At the same time, rather rapid adaptation of experimental neoplasias to T1023 treatment was often observed. We attempted to enhance the antitumor activity of this NOS inhibitor by supplementing its molecular structure with a PDK-inhibiting fragment, dichloroacetate (DCA), which is capable of hypoxia-oriented toxic effects. We synthesized compound T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). Its toxic properties, NOS-inhibiting and PDK-inhibiting activity in vivo, and antitumor activity on the mouse Ehrlich carcinoma model (SEC) were investigated in compare with T1023 and Na-DCA. We found that the change of the salt-forming acid from HBr to DCA does not increase the toxicity of 1-isobutanoyl-2-isopropylisothiourea salts, but significantly expands the biochemical and anti-tumor activity. New compound T1084 realizes in vivo NOS-inhibiting and PDK-inhibiting activity, quantitatively, at the level of the previous compounds, T1023 and Na-DCA. In two independent experiments on SEC model, a pronounced synergistic antitumor effect of T1084 was observed in compare with T1023 and Na-DCA at equimolar doses. There were no signs of SEC adaptation to T1084 treatment, while experimental neoplasia rapidly desensitized to the separate treatment of both T1023 and Na-DCA. The totality of the data obtained indicates that the combination of antiangiogenic and hypoxia-oriented toxic effects (in this case, within the molecular structure of the active substance) can increase the antitumor effect and suppress the development of hypoxic resistance of neoplasias. In general, the proposed approach can be used for the design of new anticancer agents.

Endoplasmic Reticulum Stress Promotes iNOS/NO and Influences Inflammation in the Development of Doxorubicin-Induced Cardiomyopathy

Doxorubicin (Dox) is known to cause heart failure in some cancer patients. Despite extensive studies over the past half century, the subcellular basis of Dox-induced cardiomyopathy (DIC) is still elusive. Earlier, we suggested that Dox causes a delayed activation of unfolded protein response (UPR) which may promote mitochondrial Bax activity leading to cardiomyocyte death. As a follow up, using NO donor, S-Nitroso-N-acetyl-d,l-penicillamine (SNAP), and/or NOS inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), we now show that endoplasmic reticulum (ER) stress promotes inflammation through iNOS/NO-induced TLR2 activation. In vivo Dox treatment increased mitochondrial iNOS to promote ER stress as there was an increase in Bip (Grp78) response, proapoptotic CHOP (DDIT3) and ER-mediated Caspase 12 activation. Increased iNOS activity is associated with an increase in TLR2 and TNF-α receptor associated factor 2 (TRAF2). These two together with NF-κB p105/50 expression and a synergistic support through ER stress, promote inflammatory response in the myocardium leading to cell death and ultimately fostering DIC conditions. In the presence of NOS inhibitor, such detrimental effects of Dox were inhibited, suggesting iNOS/NO as key mediators of Dox-induced inflammatory as well as apoptotic responses.

Intrauterine L-NAME Exposure Weakens the Development of Sympathetic Innervation and Induces the Remodeling of Arterial Vessels in Two-Week-Old Rats

Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1–2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/β-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels.

A Review on Heteroaryl Substituted Pyrazolines with their Pharmacological Activity and SAR Studies

In step with the development of organic chemistry, heterocyclic compounds play an important part. Electron-rich nitrogen containing heterocyclic compounds and their derivatives play key role in diverse biological activities. The wide-ranging functionality and stereochemical complexity in a five-member ring structure is shown by the pyrazole nucleus, which has two nitrogen atoms and exhibit aromatic character. The medicinal chemists have been using the relationship between chemical structure and biological activity of a molecule in drug discovery as the driving force to guide synthetic efforts, to design new chemical derivatives. It has been reported that pyrazoline derivatives possess a broad spectrum of biological activities such as antibacterial, antimicrobial, anti-inflammatory, antioxidant, antidiabetic, anticancer, antifungal, antitubercular, antidepressant, anticonvulsant and analgesic activities. They also possess some potent receptor selective biological activities like monoamine oxidases (MAOs) inhibitor, Nitric oxide synthase (NOS) inhibitor, Angiotensin converting enzyme (ACE) inhibitor, cholecystokinin-1 receptor antagonist and estrogen receptor (ER) ligand activity. Hence in this article, we focus on the pyrazole derivatives having heteroaryl substituents and their pharmacological activities along with their structure–activity relationships in order to create opportunities to explore the full potential of these compounds. Keywords: Heterocyclic compounds, Pyrazolines, Claisen-Schmidt condensation, Chalcone.

Na+/K+-ATPase plays a major role in mediating cutaneous thermal hyperemia achieved by local skin heating to 39 ºC

Na+/K+-ATPase is integrally involved in mediating cutaneous vasodilation during an exercise-heat stress, which includes an interactive role with nitric oxide synthase (NOS). Here, we assessed if Na+/K+-ATPase also contributes to cutaneous thermal hyperemia induced by local skin heating, which is commonly employed to assess cutaneous endothelium-dependent vasodilation. Further, we assessed the extent to which NOS contributes to this response. Cutaneous vascular conductance (CVC) was measured continuously at four forearm skin sites in eleven young adults (4 women). After baseline measurement, local skin temperature was increased from 33 to 39 ºC to induce cutaneous thermal hyperemia. Once a plateau in CVC was achieved, each skin site was continuously perfused via intradermal microdialysis with either: 1) lactated Ringer's solution (control), 2) 6 mM ouabain, a Na+/K+-ATPase inhibitor, 3) 20 mM L-NAME, a NOS inhibitor, or 4) a combination of both. Relative the control site, CVC during the plateau phase of cutaneous thermal hyperemia (~50%max) was reduced by the lone inhibition of Na+/K+-ATPase (-19±8%max, P = 0.038) and NOS (-32±4%max, P < 0.001) as well as the combined inhibition of both (-37±9%max, P < 0.001). The magnitude of reduction was similar between NOS inhibition alone and combined inhibition (P = 1.000). The administration of Na+/K+-ATPase and NOS inhibitors fully abolished the plateau of CVC with values returning to pre-heating baseline values (P = 0.439). We show that Na+/K+-ATPase contributes to cutaneous thermal hyperemia during local skin heating to 39 ºC, and this response is partially mediated by NOS.

Molecular Characterization and Expression of Cytochrome P450 Aromatase in Atlantic Croaker Brain: Regulation by Antioxidant Status and Nitric Oxide Synthase During Hypoxia Stress

We have previously shown that nitric oxide synthase (NOS, an enzyme) is significantly increased during hypoxic stress in Atlantic croaker brains and modulated by an antioxidant (AOX). However, the influence of NOS and AOX on cytochrome P450 aromatase (AROM, CYP19a1, an enzyme) activity on vertebrate brains during hypoxic stress is largely unknown. In this study, we characterized brain AROM (bAROM, CYP19a1b) cDNA in croaker and examined the interactive effects of hypoxia and a NOS-inhibitor or AOX on AROM activity. The amino acid sequence of croaker bAROM cDNA is highly homologous (76–80%) to other marine teleost bAROM cDNAs. Both real-time PCR and Northern blot analyses showed that bAROM transcript (size: ∼2.8 kb) is highly expressed in the preoptic-anterior hypothalamus (POAH). Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 4 weeks) caused significant decreases in hypothalamic AROM activity, bAROM mRNA and protein expressions. Hypothalamic AROM activity and mRNA levels were also decreased by pharmacological treatment with N-ethylmaleimide (NEM, an alkylating drug that modifies sulfhydryl groups) of fish exposed to normoxic (DO: ∼6.5 mg/L) conditions. On the other hand, treatments with Nω-nitro-L-arginine methyl ester (NAME, a competitive NOS-inhibitor) or vitamin-E (Vit-E, a powerful AOX) prevented the downregulation of hypothalamic AROM activity and mRNA levels in hypoxic fish. Moreover, NAME and Vit-E treatments also restored gonadal growth in hypoxic fish. Double-labeled immunohistochemistry results showed that AROM and NOS proteins are co-expressed with NADPH oxidase (generates superoxide anion) in the POAH. Collectively, these results suggest that the hypoxia-induced downregulation of AROM activity in teleost brains is influenced by neuronal NOS activity and AOX status. The present study provides, to the best of our knowledge, the first evidence of restoration of AROM levels in vertebrate brains by a competitive NOS-inhibitor and potent AOX during hypoxic stress.

Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs

Dahl salt-sensitive rat (SS) kidneys produce less nitric oxide (NO) than those of salt-resistant rats (SR). Thick ascending limb (THAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in SS THALs primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused THALs, and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35±6 arbitrary units (AU)/min in SR but only 11±2 AU/min in SS THALs (p<0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (p<0.020). The NOS inhibitor L-NAME decreased flow-induced superoxide 36 ± 8% in SS (p<0.02) but had no effect in SR THALs. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR: 44±10 vs SS: 9±2 AU/min; p<0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (p<0.012). L-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in SR but not in SS THALs (p<0.003). We conclude: 1) on NS, flow-induced NO is diminished in SS THALs mainly due to NOS3 uncoupling such that it produces superoxide; and 2) on HS, the difference is enhanced due to failure of SS THALs to increase NOS3 expression.

Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.

The Release of Nitric Oxide Is Involved in the β-Arrestin1-Induced Antihypertensive Effect in the Rostral Ventrolateral Medulla

β-Arrestin1 is a multifunctional scaffold protein with the ability to interact with diverse signaling molecules independent of G protein-coupled receptors. We previously reported that overexpression of β-arrestin1 in the rostral ventrolateral medulla (RVLM) decreased blood pressure (BP) and renal sympathetic nerve activity (RSNA) in spontaneously hypertensive rats (SHRs). Nitric oxide (NO) is widely reported to be involved in central cardiovascular regulation. The goal of this study was to investigate whether NO signaling contributes to the β-arrestin1-mediated antihypertensive effect in the RVLM. It was found that bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1) into the RVLM of SHRs significantly increased NO production and NO synthase (NOS) activity. Microinjection of the non-selective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10 nmol) into the RVLM prevented the β-arrestin1-induced cardiovascular inhibitory effect. Furthermore, β-arrestin1 overexpression in the RVLM significantly upregulated the expression of phosphorylated neuronal NOS (nNOS) by 3.8-fold and extracellular regulated kinase 1/2 (ERK1/2) by 5.6-fold in SHRs. The β-arrestin1-induced decrease in BP and RSNA was significantly abolished by treatment with ERK1/2 small interfering RNA (ERK1/2 siRNA). Moreover, ERK1/2 siRNA attenuated the β-arrestin1-induced NO production, NOS activity, and nNOS phosphorylation in the RVLM. Taken together, these data demonstrate that the antihypertensive effect of β-arrestin1 in the RVLM is mediated by nNOS-derived NO release, which is associated with ERK1/2 activation.

Influence of the type of salt-forming acids on the antiradiation activity of T1023 analogs – salts of N-isobutanoyl-S-isopropylisothiourea

According to leading experts, the vast arsenal of radioprotective agents available in the world today does not fully meet modern practical needs, both in the field of radiation protection, and in the prevention and treatment of complications of radiotherapy. The purpose of the study was to evaluate the effect of the salt-forming acids type on the radioprotective activity of NOS inhibitor T1023. The chemical part of this study included methods of chemical synthesis, physicochemical and elemental analysis. Pharmacological part – assessment of acute toxicity using V.B. Prozorovsky express method and the study of radioprotective activity using Till and McCulloch method based on the ability of mice hematopoietic cells to form spleen colonies after irradiation. The number of endogenous hematopoietic spleen colonies were assessed on the 8th day after total exposure to gamma-irradiation at a dose of 6 Gy in six independent experiments. As a result of directed chemical synthesis, six new derivatives of T1023 – salts of N-isobutanoyl-S-isopropylisothiourea have been developed, identified and characterized. The results of studying the safety and radioprotective activity of the synthesized compounds showed that changes in the salt-forming acid don’t significantly influence the toxicity: all studied compounds are in the 3rd class of toxicity and hazard. At the same time, it was found that the replacement of the salt-forming acid significantly influenced the severity of the radioprotective effect. For some of these compounds radioprotective efficacy is comparable to or exceeds the efficacy of the initial compound T1023. It is important to note that these new compounds were used in lower, more save doses than T1023. The results suggest promising further development of NOS inhibitors – isothiourea derivatives as radioprotective agents.