Time-dependent effects of striatal interleukin-2 on open field behaviour in rats.

There is evidence that immune messengers like cytokines can modulate emotional and motivated behaviours and are involved in psychiatric conditions like anxiety, and depression. Previously, we showed that cytokine gene expression (interleukin (IL)-2 mRNA) in specific brain tissues (striatum, prefrontal cortex) correlated with anxiety-like behaviour (open arm time) in an elevated plus-maze in rats. In a subsequent experiment, a single striatal IL-2 injection showed behavioural trends with the lower dose (1 ng) acting in a behavioural suppressive way, whereas the highest dosage (25 ng) led to activation and anxiolytic-like behaviour. Here, to support and extend our previous findings, we tested Wistar outbred rats after a single unilateral (balanced brain sites) IL-2 injection into the ventral/dorsal striatum followed by an open field test acutely and 24 h later. Analyses for horizontal locomotion showed no differences between groups. However, rats with IL-2-treatment (0.1 ng) showed a dose-dependent avoidance effect (i.e. reduced centre time) compared to the 1 ng group and vehicle controls 24 h later. In addition, suppression of free rearing activity was shown for both IL-2 doses (0.1; 1 ng) compared to saline in the acute test, and partly 24 h later. Thus, in experiment 2, we tested for proactive drug mechanisms to test for delayed effects of IL-2 as observed in experiment 1. In a new sample, rats were returned to their home cages after a striatal IL-2 injection (0.1; 0.01; 0 ng), and tested 24 h and 48 h after the injection in an open field. Neither for the first (24 h) nor for the second exposure (48 h later) did the analyses show any significant behavioural effects. We therefore suggest that emotional-related behaviour can be modulated by striatal IL-2 for at least 24 h. However, such IL-2 effects can only be observed if a mild stressful environmental challenge (i.e., forced open field exposure) is followed immediately after injection. In conclusion, proactive drug effects may be excluded for striatal IL-2 effects on open field behaviour.

Tripeptides from synthetic amino acids block the Tat-TAR association and slow down HIV spread in cell cultures.

Non-natural amino acids with aromatic or heteroaromatic side chains were incorporated into tripeptides of the general structure Arg-X-Arg and tested as ligands of the HIV RNA element TAR. Some of these compounds could compete efficiently with the association of TAR and Tat and downregulated a TAR-controlled reporter gene in HeLa cells. Peptide 7, which contains a 2-pyrimidinyl-alkyl chain, also inhibited the spread of HIV-1 in cell cultures. NMR studies of 7 bound to HIV-2-TAR gave evidence for contacts in the bulge region.

Neurochemical and behavioral consequences of striatal injection of 5,7-dihydroxytryptamine.

It is known that central serotonin (5HT) is involved in anxiety, but the behavioral results of many studies have been inconsistent. A prevalent research approach is to destroy 5HT neurotoxically. Such lesions were mostly generated by injecting 5,7-dihydroxytryptamine into ventricles or raphé nuclei, leading to rather global losses of 5HT in the brain. However, there is evidence for differential effects of 5HT in different brain structures regarding anxiety. Therefore, we decided to study the effects of injecting 5,7-dihydroxytryptamine into the forebrain. We chose the ventral striatum as the site of injection, since there is evidence that 5HT may be involved in anxiety there. We administered the neurotoxin bilaterally in adult rats, and analyzed neurochemical and behavioral consequences in three experiments. The first one showed that the toxin dose-dependently (10-50 microg) depleted 5HT in the ventral striatum, neostriatum, frontal cortex, and amygdala. Besides 5HT, dopamine was also partly depleted there. This dopaminergic lesion was prevented in a second experiment, where rats were pre-treated systemically with the dopamine reuptake inhibitor nomifensine. In the final experiment, the functional consequences of such 5HT lesions were tested, which yielded moderate anxiogenic effects in the elevated plus maze and in the open field. Also, there were lesion effects on aversively motivated ultrasonic vocalization during an active avoidance test. In contrast, active avoidance performance itself and general activity in the open field were not affected. Lesion effects became discernible there when challenging rats with MDMA. The psycho-stimulatory effectiveness of this drug, which acts largely via the availability of 5HT in the brain, was reduced to degrees that depended on the size of 5HT lesion. These results are discussed with respect to factors such as severity of lesion, anatomical specificity, and the role of 5HT in anxiety.

Neamine dimers targeting the HIV-1 TAR RNA.

Natural aminoglycoside antibiotics, such as neomycin, target bacterial ribosomal RNA. Neomycin also binds strongly to HIV TAR and RRE RNA through the predominant interactions of its neamine core. In the search for antiviral agents targeting multiple binding sites for aminoglycosides in RNA, we report here the synthesis of new neamine dimers and a trimer in which the neamine cores are connected by different linking chains attached at the 4'- and/or 5-positions. Inhibition of TAR-Tat complexation by these oligomers was studied via fluorimetric binding assays performed under two ionic strengths. All dimers strongly inhibit TAR-Tat association, with IC50 values 17-85 times better than the value obtained with neomycin. These results demonstrate that modifying neamine at the 4'- or the 5-position is a promising strategy in the search for antiviral agents.

New inhibitors of the Tat-TAR RNA interaction found with a "fuzzy" pharmacophore model.

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles.

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.

Enantioselective synthesis of non-natural aromatic alpha-amino acids.

We present two complementary methods for the stereoselective synthesis of non-natural alpha-amino acids with aromatic or heteroaromatic side chains. One approach is based on the chemical transformation of methionine, whereas the other applies the stereoselective Myers alkylation of glycine. The resulting product types differ in the linker length between glycine and the aromatic substituent. Since methionine and pseudoephedrine are available in both absolute configurations, R- or S-configured enantiopure amino acids with either C(2) or C(3) linkers can be obtained on gram scales. In each case the key step of the synthesis is hydroboration of the unsaturated building blocks 9 and 17, followed by palladium-catalyzed Suzuki cross-coupling with aryl halides. Attention must in certain cases be paid to the stereochemical integrity when basic Suzuki conditions are applied. Our initial difficulties are reported as well as the final "racemization-proof" procedures. The protecting groups chosen for the alpha-amino acids should be compatible with solid-phase peptide synthesis. This was confirmed by the successful synthesis of a series of tripeptides.