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.

In vitro metabolism of haloperidol and sila-haloperidol: new metabolic pathways resulting from carbon/silicon exchange.

The neurotoxic side effects observed for the neuroleptic agent haloperidol have been associated with its pyridinium metabolite. In a previous study, a silicon analog of haloperidol (sila-haloperidol) was synthesized, which contains a silicon atom instead of the carbon atom in the 4-position of the piperidine ring. In the present study, the phase I metabolism of sila-haloperidol and haloperidol was studied in rat and human liver microsomes. The phase II metabolism was studied in rat, dog, and human hepatocytes and also in liver microsomes supplemented with UDP-glucuronic acid (UDPGA). A major metabolite of haloperidol, the pyridinium metabolite, was not formed in the microsomal incubations with sila-haloperidol. For sila-haloperidol, three metabolites originating from opening of the piperidine ring were observed, a mechanism that has not been observed for haloperidol. One of the significant phase II metabolites of haloperidol was the glucuronide of the hydroxy group bound to the piperidine ring. For sila-haloperidol, the analogous metabolite was not observed in the hepatocytes or in the liver microsomal incubations containing UDPGA. If silanol (SiOH) groups are not glucuronidated, introducing silanol groups in drug molecules could provide an opportunity to enhance the hydrophilicity without allowing for direct phase II metabolism. To provide further support for the observed differences in metabolic pathways between haloperidol and sila-haloperidol, the metabolism of another pair of C/Si analogs was studied, namely, trifluperidol and sila-trifluperidol. These studies showed the same differences in metabolic pathways as between sila-haloperidol and haloperidol.

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.

Persistent cerebellar deterioration in a patient with lobar pneumonia under lithium, carbamazepine, and trifluperidol treatment.

We report on a patient with schizoaffective disorder who was on combination therapy of lithium, carbamazepine, and the neuroleptic trifluperidol. He experienced a lobar pneumonia and developed an acute and persistent cerebellar deterioration which was most likely due to lithium toxicity, while the serum lithium level was within the therapeutic range. The combination of lithium, carbamazepine, and neuroleptics is common, and is generally considered to be safe. The reported case suggests that this regimen might increase the risk of intoxication with potentially disabling side-effects.

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.

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 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.

[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.

Neuroleptic malignant syndrome with trifluperidol.

In a case of NMS subsequent to administration of trifluperidol to a young man, the patient responded remarkably quickly to bromocriptine. The authors point out the deficiencies in the diagnostic criteria, which need further refinement for a better understanding of the syndrome.

Plasma catecholamine levels in the postoperative period in complication-free and "paralytic" ileus patients.

Plasma catecholamine concentrations were compared in a group of postoperative "paralytic" ileus patients and in another group of patients, who had undergone medium-size abdominal operations followed by uneventful recovery. The plasma epinephrine level was significantly in the former group, whereas no such difference was observed in the norepinephrine concentration. The data appear to confirm that the epinephrine released from the adrenal medulla appreciably contributes to the development of "paralytic" ileus. The therapeutically effective major tranquillizer and alpha-receptor blocking drug, trifluperidol, was found to reduce both epinephrine and norepinephrine levels in "paralytic" ileus patients. The decrease of the plasma epinephrine level was the higher, the greater its initial concentration. These findings seem to support the decisive role of increased catecholamine release in the development of postoperative motor inhibition ("postoperative" ileus) and also explain the success of sympatholytic treatment in such cases, i.e. the return of normal peristalsis.

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+.

Determination of benperidol in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the quantitative determination of benperidol in human plasma using haloperidol as internal standard is described. The method involves liquid-liquid extraction, separation of the substances on a reversed-phase column C18 followed by ultraviolet detection at 254 nm. The mobile phase consists of 32% acetonitrile in 0.05 M potassium dihydrogen phosphate buffer (pH 2.8). The detection limit is 0.5-1.0 ng/ml using 2- or 4-ml plasma samples.