thiourea and etiron

To test the effect of a continuous infusion of the nitric oxide (NO) synthase (S) inhibitor aminoethyl-isothiourea (AE-ITU) on survival time, hemodynamics, and oxygen transport in a porcine model of live group A streptococcal (GAS) sepsis. Furthermore, to examine the role of endothelin-1, histamine, and reactive oxygen species (ROS) in streptococcal shock.. Prospective, randomized trial.. Laboratory at a university hospital.. Twenty-eight pigs with an average weight of 25 kg.. Sixteen animals received a continuous infusion of live Streptococcus pyogenes 1.3 x 10(10) colony forming units/hr: eight received fluids only, and the other eight received an intravenous infusion of AE-ITU 10 mg/kg/hr starting 30 mins before the GAS challenge. Six control pigs received AE-ITU 10 mg/kg/hr iv for 5 hrs. Another six animals received half the dose of GAS over 5 hrs.. GAS infusion caused a rapid increase in pulmonary, hepatic, and systemic vascular resistance, followed by hypotension with a 90% lethality at 4 hrs. Treatment with AE-ITU increased 4-hr survival in septic animals from 1/8 to 8/8 and 5-hr survival from 0/8 to 5/8, prevented hypotension, and increased urine output. AE-ITU attenuated the decrease in cardiac output, liver blood flow, and oxygen delivery, and hepatic arterial blood flow as a fraction of cardiac output increased (all p < .05). Plasma nitrate/nitrite levels decreased in all animals. Inducible NOS and endothelial constitutive NOS activities in liver, gut, and lung were not increased during sepsis, nor were they decreased after AE-ITU. Plasma levels of endothelin-1 and methylhistamine increased in all septic animals and were not modified by AE-ITU. AE-ITU prevented the increase in monocyte ROS production caused by GAS. In control animals, AE-ITU caused an increase in mean arterial pressure, liver blood flow, and oxygen delivery.. In this model of porcine GAS-induced septic shock, which was not associated with enhanced NO production, infusion of the NOS inhibitor AE-ITU prolonged survival, prevented hypotension, and improved cardiac contractility, organ perfusion, and tissue oxygenation. These beneficial effects of AE-ITU might be a result of the combined effect of ROS scavenging and modulation of local NO production, thus improving the balance of vasodilator and vasoconstrictor forces and reducing oxidative stress.

Topics: Animals; Disease Models, Animal; Female; Hemodynamics; Isothiuronium; Male; Nitric Oxide; Random Allocation; Shock, Septic; Streptococcal Infections; Streptococcus pyogenes; Survival Rate; Swine; Thiourea

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.

Topics: Binding Sites; Crystallography, X-Ray; Endothelium; Heme; Humans; Isothiuronium; Kinetics; Models, Molecular; Nitric Oxide Synthase; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Thiazoles; Thiourea

In an attempt to identify potent inhibitors of inducible (type II) NO synthase (iNOS) for use in cell culture systems, we found that two S-substituted isothioureas were very potent in cell culture but one such compound also interfered with the induction of NO synthase. S-Ethylisothiourea (EITU) and S-aminoethylisothiourea (AEITU) were found to be much more potent than NG-methylarginine, NG-nitroarginine methy lester, or aminoguanidine as inhibitors of NO production by cultured RAW 264.7 cell macrophages activated by lipopolysaccharide (LPS). The approximate EC50 values as inhibitors of NO production, assessed by 24-h accumulation in cell culture media, were 10 microM (EITU), 30 microM (AEITU), 300 microM (NG-methylarginine), and 1000 microM (aminoguanidine). EITU was found to inhibit NO production by activated macrophages without interfering with the induction of iNOS. More specifically, EITU failed to influence transcription of iNOS mRNA (Northern blot analysis), translation of iNOS protein (pulse experiments), or degradation of translated iNOS protein (pulse-chase experiments). In contrast, however, AEITU interfered markedly with the induction of iNOS by mechanisms attributed to inhibition of translation of iNOS mRNA into functional protein as well as acceleration of degradation of already translated iNOS protein. These observations indicate that AEITU should not be used in cell culture experiments where the intent is solely to assess the consequences of inhibition of iNOS catalytic activity.

Topics: Animals; beta-Aminoethyl Isothiourea; Blotting, Western; Cell Line; Enzyme Induction; Enzyme Inhibitors; Enzyme Stability; Isothiuronium; Macrophage Activation; Macrophages; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Biosynthesis; Thiourea; Transcription, Genetic

The inhibitory potency of different classes of nitric oxide synthase (NOS) inhibitors (amino acid-based substances, guanidines, isothioureas, imidazoles and indazoles) versus peripheral neuronal NOS in the pig gastric fundus was investigated by studying their influence on electrically induced relaxations in non-adrenergic noncholinergic conditions. Circular muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine, and tone was raised with 5-hydroxytryptamine. Electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s) induced short-lasting relaxations. The inhibitory effect of 1-phenylimidazole could not be evaluated because it nearly abolished the 5-hydroxytryptamine-induced tone of the tissues. 7-Nitroindazole, imidazole, 2-iminobiotin and aminoguanidine did not inhibit the electrically induced relaxations, while the other 9 substances tested were able to do so. The influence of the incubation period was tested by studying the inhibitory effect after incubation for 10 up to 60 min. For N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-N6-(1-iminoethyl)-lysine (L-NIL), S-methyl-L-thiocitrulline and S-isopropyl isothiourea there was a moderate increase in the inhibitory effect up to 30 min of incubation so that they were incubated for 30 min to study their inhibitory potency. For L-thiocitrulline, S-methyl isothiourea and S-ethyl isothiourea, an incubation period of 60 min was used. The 9 substances concentration-dependently inhibited the electrically induced relaxations with a maximal inhibitory effect of approximately 80% except for S-methyl isothiourea (Emax of 53%). The overall order of potency was: S-isopropyl isothiourea > S-ethyl isothiourea > or = S-methylL-thiocitrulline > or = L-NNA > L-NIO > L-NAME > S-methyl isothiourea > L-thiocitrulline > L-NIL. While the potency for S-isopropyl isothiourea (EC50: 3.1 x 10(-5) M, n = 6) to S-methyl isothiourea (EC50: 11.5 x 10(-5) M, n = 5) was in the same range, the potency of L-thiocitrulline and L-NIL was clearly lower. This study showed several compounds to be potent inhibitors of peripheral neuronal NOS in the pig gastric fundus while some compounds, that were reported to inhibit brain neuronal NOS were not effective. The EC50 values found for the effective substrates in this functional study may be a guideline for the concentrations required to evaluate the role of NO in NANC neurotransmission in gas

Topics: Animals; Biotin; Citrulline; Electric Stimulation; Enzyme Inhibitors; Gastric Fundus; In Vitro Techniques; Isothiuronium; Lysine; Male; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; Ornithine; Swine; Thiourea

S-Ethylisothiourea was a potent competitive inhibitor of human nitric oxide synthase (NOS), with Ki values of 17, 36, and 29 nM for the inducible (i), endothelial (e), and neuronal (n) isozymes, respectively. Unlike some potent inhibitors of NOS, no time dependence was observed. S-Ethylisothiourea was not a detectable substrate for eNOS. S-Ethylisothiourea was also a potent inhibitor of mouse iNOS (Ki value of 5.2 nM), and its binding perturbed the spectrum of iNOS consistent with its altering the environment of the bound heme. The optimum binding of S-ethyl- and S-isopropylisothiourea relative to 70 other analogs suggested that these alkyl substitutions fit into a small hydrophobic pocket. Most isothioureas were 2-6-fold selective for the human iNOS (Ki for iNOS versus Ki for eNOS), with one being 19-fold selective. The cyclized mimics of S-ethylisothiourea, 2-NH2-thiazoline, and 2-NH2-thiazole, were also competitive inhibitors of human NOS. A third structural class of inhibitors, bisisothioureas, were, in general, the most selective in their inhibition of human iNOS. S,S'-(1,3-Phenylenebis(1,2-ethanediyl))bisisothiourea was 190-fold selective (Ki value of 0.047 microM against iNOS versus 9.0 microM against eNOS). These results demonstrate that potent and selective inhibition of human NOS isozymes is achievable.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Endothelium, Vascular; Enzyme Induction; Humans; Isoenzymes; Isothiuronium; Kinetics; Mice; Models, Chemical; Neurons; Nitric Oxide Synthase; Species Specificity; Structure-Activity Relationship; Thiazoles; Thiourea