Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 µM/SC84227, 0.60 ± 0.02 µM/SC97202, 6.26 ± 1.64 µM/SC97208, 0.71 ± 0.019 µM/Harmine and 0.63 ± 0.23 µM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

Involvement of the cholinergic system of CA1 on harmane-induced amnesia in the step-down passive avoidance test.

ß-carboline alkaloids such as harmane (HA) are naturally present in the human food chain. They are derived from the plant Peganum harmala and have many cognitive effects. In the present study, effects of the nicotinic system of the dorsal hippocampus (CA1) on HA-induced amnesia and exploratory behaviors were examined. One-trial step-down and hole-board paradigms were used to assess memory retention and exploratory behaviors in adult male mice. Pre-training (15 mg/kg) but not pre-testing intraperitoneal (i.p.) administration of HA decreased memory formation but did not alter exploratory behaviors. Moreover, pre-testing administration of nicotine (0.5 µg/mouse, intra-CA1) decreased memory retrieval, but induced anxiogenic-like behaviors. On the other hand, pre-test intra-CA1 injection of ineffective doses of nicotine (0.1 and 0.25 µg/mouse) fully reversed HA-induced impairment of memory after pre-training injection of HA (15 mg/kg, i.p.) which did not alter exploratory behaviors. Furthermore, pre-testing administration of mecamylamine (0.5, 1 and 2 µg/mouse, intra-CA1) did not alter memory retrieval but fully reversed HA-induced impairment of memory after pre-training injection of HA (15 mg/kg, i.p.) which had no effect on exploratory behaviors. In conclusion, the present findings suggest the involvement of the nicotinic cholinergic system in the HA-induced impairment of memory formation.

Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors.

The beta-carboline, harmane (0.1 - 1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I(1)-receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I(1)-receptor in the RVLM. This is the first report of an endogenous ligand for I(1)-receptors that has central effects on blood pressure.

Influence of flumazenil on the learning-enhancing effect of ambocarb in rats.

The effects of flumazenil (Ro 15-1788) and a new beta-carboline, ambocarb (AMB), on learning were investigated using the multichoice maze. The drugs, administered either alone or simultaneously, were injected once a day before training for eight days. AMB, administered alone, improved the performance and decreased the working errors, whilst flumazenil had no effect on performance during its sole administration but weakly prevented the learning-improving effect of AMB. More significantly, flumazenil antagonized the motor activity depressed by AMB. In the study ex vivo, flumazenil decreased and AMB increased the apparent affinity of [3H]flunitrazepam to the central benzodiazepine receptors. Flumazenil reversed the action of AMB on the central benzodiazepine receptors, but failed to reduce significantly the modulative effects of AMB on [3H]muscimol and [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding. These data indicate that flumazenil, due to its action on the central benzodiazepine receptors, more effectively reverses the inhibition of motor activity than the performance-improving effect of AMB.

Pharmacological characteristics of abnormal behavior induced by harmine with special reference to tremor in mice.

Harmine, a hallucinogen with potent monoamine oxidase inhibitory properties, induced abnormal behavior, including tremor, scratching, head twitch and cage biting, in the mouse. A dose-dependent tremor was produced by all routes of administration of harmine. Although oxotremorine tremor was markedly suppressed by atropine, harmine tremor was unaffected by cholinergic drugs, remarkably inhibited by dopaminergic drugs, antidepressants and diazepam, mildly diminished by p-chlorophenylalanine, markedly augmented by 5-hydroxytryptophan and mildly increased by alpha-methyl-p-tyrosine. These findings suggest that a catecholaminergic (particularly dopaminergic) and serotonergic system imbalance plays an important role in the manifestation of harmine tremor. In view of these characteristics, harmine tremor may be useful as an effective experimental model for the evaluation of antiparkinsonism drugs, along with oxotremorine tremor because of the different mechanism of occurrence. In addition, harmine tremor appears to be useful in characterizing the properties of antidepressant drugs.

Intensification of central catecholaminergin and serotonergic processes by the hypothalamic factors MIF and TRF and by angiotensin II.

The present work deals with the action of MIF (melanocyte stimulating hormone release-inhibiting factor), TRF (thyrotropin releasing factor), and angiotensin II on the behavioral effects of L-DOPA and of D, L-5-hydroxytrytophan (5-HTP) in mice. The influence of MIF and TRF on the antagonistic effect of L-DOPA of harmine tremors in rabbits was also studied. MIF and TRF, injected i.p., intensify the effects of L-DOPA in mice. The minimal dose of MIF required to induce a +3 response is 0.1 microgram/kg; TRF is active at 500 micrograms/kg. When MIF or TRF are injected into the brain, potentiation of L-DOPA is obtained with exceedingly small quantities of MIF (0.1 pg); the effective dose of TRF is 1 microgram. The behavioral effects of 5-HTP are potentiated by TRF only, at doses of 0.1 microgram/kg, i.p. When TRF is administered intracerebrally, the active dose per mouse is 0.1 ng. Harmine (5 mg/kg i.v.) induced, in the rabbit, sustained whole body tremors; if L-DOPA (5 mg/kg) is administered i.v. at the peak of the harmine effect, tremors subside. When the rabbit is pretreated with MIF administered i.p. 1 -2 hr before harmine, in doses devoid of an antitumor effect per se (10 micrograms/kg), the L-DOPA antagonism appears at lower dose. Also dopamine (5-10 mg/kg i,v.) proved effective in abating harmine tremors; previous treatment with MIF (50 micrograms/kg) potentiated the antagonistic effect of dopamine. According to the prevailing theories on the mechanism of neurotransmission, some hypotheses will be discussed to explain the observed potentiation: impaired uptake, impaired degradation, interference with the turnover of the boiamines, supersensitivity of the receptors.