arl-17477 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.

Topics: Amidines; Analysis of Variance; Animals; Anticoagulants; Benzylamines; Blotting, Western; Brain; Calcium; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Edetic Acid; Enzyme Inhibitors; Inhibitory Concentration 50; Ischemic Attack, Transient; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors

In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.

Topics: Amidines; Animals; Brain Ischemia; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Endothelin-1; Enzyme Inhibitors; Gerbillinae; Humans; Male; Neuroprotective Agents; Nitric Oxide Synthase; Rats; Reperfusion Injury; Time Factors

We have previously shown that the non-specific nitric oxide synthase inhibitor L-NAME blocks the behavioural effects of phencyclidine, but not d-amphetamine. To characterise the specificity of these effects, we used the specific neuronal nitric oxide synthase inhibitor AR-R 17477 in two rat models of psychosis: the prepulse inhibition of the acoustic startle response and locomotor activity. In biochemical assays, AR-R 17477 was shown to be selective for the neuronal nitric oxide synthase isoform. Test drugs were given subcutaneously. AR-R 17477 (0.5, 1 and 5 mg/kg) antagonised phencyclidine-induced hyperlocomotion, while higher doses (10 and 20 mg/kg) were less efficaceous. AR-R 17477 (1 mg/kg) antagonised phencyclidine-induced deficit in prepulse inhibition of the acoustic startle response, while a higher dose (15 mg/kg) was less active. AR-R 17477 did not affect startle amplitude or prepulse inhibition of the acoustic startle response, did not affect locomotion and did not induce any changes in gross behaviour (sniffing, rearing, etc.) as determined in a subjective observation study. AR-R 17477 (1 mg/kg) did not alter the effect of d-amphetamine in prepulse inhibition of the acoustic startle response. Using radiotelemetry in rats, L-NAME (10 mg/kg subcutaneously) increased blood pressure and decreased heart rate while AR-R 17477 (10 mg/kg) did not have any significant effect on these parameters. The results show that a neuronal nitric oxide synthase inhibitor antagonises the effects of phencyclidine on prepulse inhibition of the acoustic startle response and locomotor activity, without exhibiting significant behavioural effects of its own and suggest that our earlier results with L-NAME depended upon an inhibition of neuronal nitric oxide synthase and not on an inhibition of endothelial nitric oxide synthase or inducible nitric oxide synthase. The observed effects are unlikely to be related to an effect on cardiovascular function.

Topics: Amidines; Animals; Antipsychotic Agents; Disease Models, Animal; Enzyme Inhibitors; Hallucinogens; Inhibitory Concentration 50; Male; Motor Activity; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phencyclidine; Rats; Rats, Sprague-Dawley; Reflex, Startle

We tested whether AR-R 17477, a selective inhibitor of neuronal nitric oxide synthase, reduces brain injury in rats subjected to permanent focal ischemia.. Randomized within cohort; nonblinded study.. University basic science laboratory.. Halothane-anesthetized male Wistar rats (n = 53).. Rats were treated with either intravenous saline (diluent) or AR-R 17477 (1 or 3 mg/kg) 30 mins before or 60 mins after the onset of permanent focal cerebral ischemia. Infarction volume was determined at 18 or 48 hrs of ischemia.. Pretreatment with 1 mg/kg AR-R 17477 was associated with a decreased infarct volume (2,3,5-triphenyltetrazolium chloride staining) in the striatum (saline, 81+/-7 mm3; AR-R 17477, 55+/-3 mm3) but not in the cortex at 18 hrs of occlusion (saline, 302+/-29 mm3; AR-R 17477, 237+/-36 mm3). However, this therapeutic effect of AR-R 17477 was no longer evident if the rats were allowed to survive for 48 hrs before analysis of infarction volume. In fact, in this separate cohort of animals, three of eight AR-R 17477-treated and five of eight saline-treated rats died before completing 48 hrs of ischemia. Efficacy of AR-R 17477 was completely absent (even at 18 hrs of ischemia) when drug treatment was delayed until 1 hr after the onset of ischemia. Infarction volume at 18 hrs of ischemia was similar between rats treated with saline, 1 mg/kg (cortex, 229+/-43 mm3; striatum, 67+/-8 mm3) or 3 mg/kg AR-R 17477 (cortex, 284+/-34 mm3; striatum, 75+/-5 mm3). In addition, only one of eight rats treated with 3 mg/kg AR-R 17477 at 1 hr of ischemia survived 48 hrs of occlusion, compared with three of eight rats treated with saline.. Neuronally generated nitric oxide is a mediator of brain injury during permanent focal ischemia in rats. However, severity of the ischemic insult appears to limit the therapeutic efficacy of the specific neuronal nitric oxide synthase inhibitor, AR-R 17477.

Topics: Amidines; Animals; Blood Pressure; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Enzyme Inhibitors; Injections, Intravenous; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxygen Consumption; Rats; Rats, Wistar