Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

Flupentixol and trifluperidol reduce interleukin-1 beta and interleukin-2 release by rat mixed glial and microglial cell cultures.

Neuroleptics penetrate into the brain, where they act not only on neurons but probably also on glial cells. In the available literature, there are no reports on the effect of neuroleptics on cytokine release in glia cultures. The aim of this study was to evaluate the effect of neuroleptics on the release of proinflammatory cytokines (IL-1beta and IL-2) by mixed glial and microglial cell cultures. Trifluperidol at 20 and 2 muM reduced IL-beta secretion by mixed glial cultures after 3 days of exposure. Trifluperidol at 20, 2 and 0.2 muM diminished IL-beta secretion after 1 day of incubation. Trifluperidol at 20 and 2 muM reduced IL-2 release after 1 and 3 days of exposure. Flupentixol at 20 and 2 muM reduced IL-1beta by mixed glial cell cultures after 3 days of exposure. Flupentixol at 20, 2 and 0.2 muM caused diminution of IL-1beta release after 1 day of exposure. Flupentixol at 20 and 2 muM reduced IL-2 release after 1 day of incubation. Flupentixol at 20, 2 and 0.2 muM diminished IL-2 release after 3 days of exposure. Flupentixol at 20, 10, 2 and 0.2 muM reduced IL-1beta release by microgial cell cultures. Flupentixol at 20, 10 and 2 muM reduced release of IL-2 by microglial cells after 1 day of exposure. The results of the present study suggest that neuroleptics have an inhibiting effect on the release of glial cytokines, but clinical significance this results remains to be elucidated.

Flupentixol and trifluperidol reduce secretion of tumor necrosis factor-alpha and nitric oxide by rat microglial cells.

Tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which are produced by activated microglial cells, are involved in the neuropathogenesis of many diseases affecting the central nervous system (CNS). There is a need to develop drugs that inhibit neurotoxic processes in neurodegenerative diseases. The aim of this study was to evaluate the effect of two neuroleptics, flupentixol and trifluperidol, on the release of pro-apoptotic TNF-alpha and NO by LPS-activated rat microglial cells. Flupentixol and trifluperidol reduced the TNF-alpha and NO release by cultured microglia exposed to LPS for 6 and 24h. The results suggest that flupentixol and trifluperidol, which are well-known antipsychotic drugs, may be used in the treatment of CNS diseases associated with excessive TNF-alpha and NO release.

Stable expression and characterization of the human brain potassium channel Kv2.1: blockade by antipsychotic agents.

We have cloned the cDNA encoding the voltage-dependent K+ channel Kv2.1 from human brain (hKv2.1). RNase protection and RT-PCR (reverse transcriptase-PCR) experiments reveal abundant Kv2.1 transcripts in human brain with virtually no expression detectable in human heart. hKv2.1 has been stably transfected into a human glioblastoma cell line, and transformed cells display large, slowly activating outward currents. The kinetics, steady-state activation and inactivation parameters, and external tetraethylammonium sensitivity were all similar to those described previously for hKv2.1 channels transiently expressed in Xenopus oocytes or other mammalian cell lines. A number of dopamine receptor antagonist/antipsychotic agents were shown to block hKv2.1. Trifluoperizine, trifluperidol and pimozide produced time-dependent blockade of hKv2.1 with IC50 values of approx. 1-2 microM. The diphenylbutylpiperidine fluspirilene was shown to be 4-5-fold more potent than the other agents tested inhibiting hKv2.1 current with an IC50 value of 297 nM. The block produced by fluspirilene was both time- and frequency-dependent. Furthermore, fluspirilene (1 microM) shifted the midpotential of the hKv2.1 steady-state inactivation curve by approx. 15 mV in the hyperpolarizing direction. These results demonstrate the usefulness of this transfection system for the pharmacological characterization of hKv2. 1. Fluspirilene proved to be a relatively potent blocker of hKv2.1 and may provide a useful starting point for the development of more potent and selective agents active against this brain K+ channel.

Characterization of haloperidol and trifluperidol as subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonists using [3H]TCP and [3H]ifenprodil binding in rat brain membranes.

[3H]TCP and [3H]ifenprodil binding to N-methyl-D-aspartate (NMDA) receptors in rat forebrain membranes was used to compare the inhibition of haloperidol and trifluperidol with that of ifenprodil and eliprodil. In the [3H]TCP binding assay, inhibition curves of ifenprodil, eliprodil, haloperidol and trifluperidol revealed two affinity states in the presence of glutamate, glycine and spermidine. The potency of these agents to inhibit the high-affinity fraction of the binding agreed with the results of other studies investigating their potency to block glutamate-induced current at recombinant NR1a/NR2B NMDA receptors expressed in Xenopus oocytes. These agents also inhibited [3H]ifenprodil binding in a biphasic manner, whether in the absence or the presence of either the sigma site ligand GBR-12909 or spermidine. Spermidine reduced the fraction of high-affinity sites labeled with [3H]ifenprodil. The only alteration in the affinity was a decrease in the IC50 value of haloperidol to inhibit the high-affinity fraction of [3H]ifenprodil binding. GBR-12909 also reduced the fraction of [3H]ifenprodil sites inhibited by these compounds with high affinity, with no change in the affinity for either fraction. These data suggest that spermidine is neither a competitive antagonist at the fraction of the binding inhibited by these agents with high affinity, nor is this fraction of the binding to sigma sites. Haloperidol and trifluperidol represent a new class of agent that interacts at a site that is labeled by [3H]ifenprodil as well as [3H]TCP in rat brain membranes and that closely reflects ifenprodil's voltage-independent site on the recombinant NR1a/NR2B subtype of the NMDA receptor.

Yeast sterol C8-C7 isomerase: identification and characterization of a high-affinity binding site for enzyme inhibitors.

The yeast gene ERG2 encodes a sterol C8-C7 isomerase and is essential for ergosterol synthesis and cell proliferation. Its striking homology with the so-called sigma1 receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C8-C7 isomerase (ERG2) carries a similar high affinity drug binding domain. Indeed the sigma ligands [3H]haloperidol (Kd = 0.3 nM) and [3H]ifenprodil (Kd = 1.4 nM) bound to a single population of sites in ERG2 wild type yeast microsomes (Bmax values of 77 and 61 pmol/mg of protein, respectively), whereas binding activity was absent in strains carrying ERG2 gene mutations or disruptions. [3H]Ifenprodil binding was inhibited by sterol isomerase inhibitors such as fenpropimorph (Ki = 0.05 nM), tridemorph (Ki = 0.09 nM), MDL28,815 (Ki = 0.44 nM), triparanol (Ki = 1.5 nM), and AY-9944 (Ki = 5.8 nM). [3H]Haloperidol specifically photoaffinity-labeled a protein with an apparent molecular weight of 27400, in agreement with the molecular mass of the sterol C8-C7 isomerase (24900 Da). 9E10 c-myc antibodies specifically immunoprecipitated the c-myc tagged protein after [3H]haloperidol photolabeling, unequivocally proving that the drug binding site is localized on the ERG2 gene product. Haloperidol, trifluperidol, and ifenprodil inhibited the growth of Saccharomyces cerevisiae and reduced the ergosterol content of cells grown in their presence. Our results demonstrate that the yeast sterol C8-C7 isomerase has a polyvalent high-affinity drug binding site similar to mammalian sigma receptors and that in yeast sigma ligands inhibit sterol biosynthesis.

Ocular hypotension induced by topical dopaminergic drugs and phosphodiesterase inhibitors.

The aim of this work was to investigate the ocular hypotensive activity of some topically administered cAMP-phosphodiesterase inhibitors alone and in combination with dopaminergic compounds. Experiments were performed with ocular normotensive rabbits and during transitory induced ocular hyper- or hypotension. An ocular hypotensive effect was observed after instillation of aminophylline, dyphylline, pentoxifylline, caffeine, and iso-caffeine, but not following topical hydroxypropyl-1,3-dimethylxanthine. Dopaminergic compounds were also studied in order to be combined with phosphodiesterase inhibitors as ocular anti-hypertensive treatment. Significant ocular hypotensive activity was observed after topical application of trifluperidol, fluphenazine, thiothixene, and the S(-) enantiomer of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP). Of the cAMP-phosphodiesterase inhibitors that were tested, pentoxifylline was the most interesting compound, with good ocular tolerance, significant reduction in intra-ocular pressure, and potential retinal microvascular benefits. After allowing adequate time for pentoxifylline to reach its maximal activity, trifluperidol or S(-)-3-PPP was also instilled. A more pronounced ocular hypotensive effect was then observed. The findings of this study may suggest that administration of eye-drops combining drugs acting by separate ways on second messengers involved in the regulation of intra-ocular pressure (e.g. cyclic AMP) could be used to reduce intra-ocular pressure during glaucoma.

[The role of the genotype in the cataleptogenic effect of neuroleptics]. / Rol' genotipa v kataleptogennom éffekte neiroleptikov.

The cataleptogenic effects of three neuroleptics from one chemical group was investigated in 8 mice strains (CBA, A/He, C57B1/6, C3H/He, BALB/c, AKR, DD, and CC57Br. Despite significant interstrain, differences in the action of the drugs, haloperidol (0.5 mg/kg) and trifluperidol (0.5 mg/kg) produced much greater cataleptogenic action than fluspirilene (2 mg/kg). At the same time the intensity of catalepsy in various mice strains after haloperidol was not coincident with that after trifluperidol (r = 0.22): CBA mice displayed the maximum catalepsy, but AKR, DD and CC57Br mice, the minimum when haloperidol was given; with trifluperidol, the maximum catalepsy was observed in AKR mice, but absent in DD mice. Fluspirilene induced catalepsy only in CBA and A/He mice. Thus, the presence of catalepsy, a side effect of most neuroleptics, is largely predetermined by hereditary factors.

Improvement of ocular blood flow with dopamine antagonists on ocular-hypertensive rabbit eyes.

The eyedrops of the ocular-hypotensive dopamine antagonists, trifluperidol, moperone, lenperone, and spiperone, were instilled into an ocular-hypertensive rabbit eye. The blood flows in the choroid, retina, iris root-ciliary body, and iris were measured with colored microspheres at various time periods. It was found that all these dopamine antagonists, at a concentration of 0.5%, increased the blood flow in all eye tissues. Dopamine, at a concentration of 3%, produced a biphasic action by decreasing the blood flow initially at 30 min, then increasing it at 120 min and thereafter. But 1.5% dopamine produced a monophasic action which increased the blood flow after 180 min. Since dopamine antagonists are not cholinergics or adrenolytics, they are not supposed to produce the side effects induced by pilocarpine or timolol. It is hoped that they can become satisfactory drugs for glaucoma and ocular hypertension.

[The stimulation of prolactin secretion by the simultaneous inhibition of the dopaminergic and activation of the serotoninergic systems in the hypothalamus]. / Stimuliatsiia sekretsii prolaktina odnovremennoi ingibitsiei dofaminergicheskoi i aktivatsiei serotoninergicheskoi sistem gipotalamusa.

Lactotropic adenohypophyseal function and lactation intensity were studied under simultaneous blockade of dopamine receptors and stimulation of serotonin metabolism in the rat hypothalamus. Blockade of hypothalamic dopamine receptors by tricedil decreased the dopamine level, increased the serotonin content and intensified its metabolism thus increasing the prolactin secretion. Under these conditions, tryptophan application potentiated the above changes in hypothalamic catecholamine and indolamine metabolism, increased the prolactin secretion. The latter is associated with the brain serotoninergic system reactivity to suckling impulses: an important factor in the lactation reflex. Tryptophan maintains a high level of hypothalamic serotonin metabolism, maintaining an increase in the prolactin secretion level and secretory activity of the mammary glands.

Effects of antiglaucoma drugs on the blood flow in rabbit eyes.

Although it is essential that intraocular pressure (IOP) be reduced in glaucoma treatment, it is also vitally important to provide sufficient blood flow to eye tissues so that healthy visual field is maintained. It is possible for an agent to reduce IOP and blood supply to the eye. In that case, glaucoma appears to be under control since IOP has been reduced to within normal range, yet the disease is actually progressing, causing damage to the retina, optic nerve, and other tissues. The 85Sr-microsphere technique was used to study the effects of several antiglaucoma drugs on blood supply to various eye tissues. It was found that pilocarpine, L-timolol, D-timolol and haloperidol are good drugs to use in treating glaucoma because they do not reduce ocular blood flow. D-timolol is particularly good because it does not cause side effects through beta-adrenergic blockade or cholinergic stimulation. On the other hand, trifluperidol and moperone reduce IOP effectively, but also decrease blood supply.

[Action of neurotropic substances on gamma-aminobutyric acid release from brain synaptosomes during K+ depolarization]. / Deistvie neirotropnykh veshchestv na vysvobozhdenie gamma-aminomaslianoi kisloty iz sinaptosom golovnogo mozga na fone K+-depoliarizatsii.

The effects of aminazine (0.25 mM), phthoracyzine (0.5 mM), trifluperidole (0.5 mM) and imipramine (0.5 mM) on GABA release from rat brain synaptosomes depolarized with K+ (50 mM) were investigated. Incubation of synaptosomes with aminazine led to a 2-fold and that with phthoracyzine, trifluperidole and imipramine to a 1.5-fold increase in GABA release from synaptosomes as compared with its basic level. The raising of K+ in the incubation medium to 50 mM brought about a 2-fold augmentation of GABA release. Exposure of synaptosomes to drugs and a higher K+ concentration at a time did not change GABA release as compared to its basic level. Introduction into the incubation medium of the Ga-ionophore A23187 together with 50 mM K+ and trifluperidole or with K+ and imipramine led to the same increase in GABA release from synaptosomes as that produced by the psychotropic drugs as regards native synaptosomes. It is assumed that the lack of the influence of the psychotropic drugs under study of GABA release from synaptosomes depolarized with K+ is caused by blockade of synaptic membrane conductibility for Ca2+.

The effect of trifluperidol on the contractile response of guinea-pig taenia coli to acetylcholine, histamine, potassium and calcium.

In guinea-pig taenia coli, trifluperidol antagonized the contraction induced by all agonists tested in a non-competitive manner. pA2 values for trifluperidol calculated for acetylcholine, histamine and calcium were not different. pD'2 values for trifluperidol calculated for acetylcholine, histamine and potassium did not differ, while the pD'2 for acetylcholine and calcium were significantly different. pD'2 values for trifluperidol calculated for calcium varied with the concentration of the antagonist. Trifluperidol has been considered a non-specific blocking agent in guinea-pig taenia coli. This inhibitory activity has been considered very similar to that of local anaesthetic agents, although calcium was blocked in a non-competitive manner by trifluperidol, differently from anaesthetic agents that block calcium in a competitive way.

Effects of beta adrenergic blocking drugs and trifluperidol on intestinal motility and splanchnic nerve activity in cats.

In cats anaesthetized with chloralose and urethane we studied the effect of the selective beta 1 adrenergic blocking practolol and the non-selective beta 1-2 adrenergic blocking pindolol on the intestinal motility, the efferent sympathetic activity, arterial blood pressure and heart-rate. We compared the effects of trifluperidol on the intestinal tone, the intestinal motility and its duration with those of trifluperidol and practolol combined. It was found that 1-, 2-, and 3 mg/kg of practolol given intravenously had no influence on the spontaneous electric activity of postganglionic fibres of the splanchnic or hypogastric nerves, thus it had no central effect. Accordingly, its site of effect proved to be peripheric. Its administration was associated with a slight decrease of the arterial blood pressure and heart rate. The intestinal tone was instantly increased depending on the does in every case. In two-thirds of our experiments the intestinal motility was restored 1 to 3 minutes following administration depending on the dose. Combined with trifluperidol, practolol produces a further increase in the enhancing activity of trifluperidol on the intestinal tone and motility. It considerably extends the duration of the action of trifluperidol on the intestinal motility. Pindolol increases the intestinal tone and motility dose-dependently in every case, reduces the efferent sympathetic activity, which is inversely proportional to the dose. The action is most pronounced on administration of 0.125 micrograms/kg of pindolol intravenously but it cannot be observed with a dose of 0.5 mg/kg. No significant changes were observed in blood pressure but there was a reduction in heart rate. The action of pindolol is supposed to be both central and peripheral in nature.

Development of tolerance to the stimulatory effect of neuroleptic butyrophenones on pituitary-adrenal activity in rats.

The effects of repeated administration of haloperidol (HAL) and clofluperol (CLO) on the responsiveness of pituitary-adrenal system to these drugs were studied in rats. Chronic treatment of animals with a daily i.p. dose of 5 mumol/kg of the drugs resulted in markedly attenuated responses of plasma corticosterone (CS) to challenge of the same dose of the same drug as compared to the acute treatment which caused a large increase in the plasma CS levels. Animals receiving a chronic HAL treatment showed a complete cross-tolerance to the stimulatory effect of CLO and a nearly complete cross-tolerance to that of chlorpromazine. The adrenal cortices of animals tolerant to HAL responded normally to exogenous ACTH. This result indicates that the tolerance to the stimulatory action of the butyrophenones may occur in the hypothalamo-pituitary axis.

Effect of centrally active drugs on dopamine oxidation by rat brain catecholamine oxidase.

Centrally active drugs of the phenothiazine-, butyrophenone- and iminodibenzyl class markedly decreased the rate of dopamine oxidation in the presence of rat brain catecholamine oxidase.

Atropine stereotypy and its inhibition by intracaudate triperidol in the rat.

One component (rearing) of the stereotyped behavior of the rat was quantitated using erigometer, a new device developed in this laboratory. Intraperitoneal injection of atropine, but not methylatropine, caused dose-related stereotyped behavior in the rat and this effect was antagonized by intracaudate injection of triperidol in a dose-related manner. It is concluded that stereotypy can be evoked also in the case when the striatal dopaminergic tone is normal or even below normal: it is the equilibrium of the striatal cholinergic-dopaminergic systems which must be shifted towards dopamine, which is necessary for the development of this behavioral manifestation.

[Tryptophan uptake by glia and synaptosomes of rabbit cerebral cortex]. / Zakhvat triptofana kletkami glii i sinaptosomami kory golovnogo mozga krolika.

The authors studied the engulfment of L-tryptophane-14C by gliacytes and synaptosomes of the rabbit cerebral cortex. The system of engulfment of the gliacytes was characterized by a high affinity to tryptophane (Km = 0.8 micrometer). Engulfment of tryptophane by synaptosomes had a lower affinity (Km = 50 micrometer). Psychotropic substances--chlorpromazine and imipramine produced an inhibitory influence on glial engulfment. The leading role of gliacytes in the trophic provision of the neurons and the normal course of neurodynamic processes is confirmed.