Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3beta inhibition, and viral replication: a screening approach for new bipolar disorder drugs derived from the valproic acid core structure - PubMed (original) (raw)

Comparative Study

. 2005 May;67(5):1426-33.

doi: 10.1124/mol.104.009308. Epub 2005 Feb 1.

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Comparative Study

Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3beta inhibition, and viral replication: a screening approach for new bipolar disorder drugs derived from the valproic acid core structure

B J Eickholt et al. Mol Pharmacol. 2005 May.

Abstract

Inositol-1,4,5-trisphosphate (InsP3) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP3 depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3beta (GSK-3beta). In addition, the structural requirements of VPA-related compounds to cause InsP3 depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP3 depletion, in vivo teratogenic potency, HIV replication, and GSK-3beta activity in vitro. We found four compounds that function to deplete InsP3 in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP3 depletion. No relationship was found between InsP3 depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3beta activity. Structural requirements of VPA congers to maintain InsP3 depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP3 depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP3 depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP3-depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment.

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Figures

Fig. 1

Fig. 1

The structure of valproic acid and its congeners. Compounds are referred to by roman numeral prefix (I–IX) except for VPA and its amide derivative VPD. Compounds were chosen by broad category to result in a test set with highest structural diversity in branching (VIII), saturation (I–VI and IX), side-chain length, derivatization of the carboxylic acid function, and chiral aspects (_R_- and _S-_enantiomers: I and II, III and IV, V and VI).

Fig. 2

Fig. 2

Characterization of InsP3-depletion efficacy and teratogenicity of VPA analogs and current bipolar disorder treatments. D. discoideum cells were treated with lithium, VPA, or compounds derived from the chemical structure of VPA (Fig. 1). A, cells were grown overnight in complete medium in the presence of VPA or one of its analogs at 0.5 mM, a concentration found in patient plasma undergoing VPA treatment, or with lithium at 10 mM. Changes in InsP3 levels were subsequently measured by isotope dilution assay (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK; see Materials and Methods), and compared with vehicle-only (▪) control or currently used bipolar disorder treatments (▧). Results represent four experiments assayed in triplicate (± S.E.M.). B and C, comparison of relative efficacy of VPA congeners to cause InsP3 depletion expressed as a percentage of that found for VPA or lithium (underline indicates increased InsP3 levels). Compounds were tested for teratogenic rating in an in vivo model. Arbitrary scale of teratogenic ratings for these drugs, from not teratogenic (0) to highly teratogenic (+++++), as described under Materials and Methods (★, inferred from Phiel et al., 2001; Gurvich et al., 2004). RM, racemic mixture; ND, not determined.

Fig. 3

Fig. 3

Enlargement of growth cones from DRG neurons after treatment with InsP3-depleting drugs. Rat DRGs were cultured for 24 h in the presence of 0.5 mM VPA, analogs showing InsP3 depletion in D. discoideum, or one analog showing no effect on InsP3 levels in D. discoideum (Fig. 2A), and afterward, cells were fixed and stained before growth-cone size quantification. A, representative growth cones are shown for vehicle-only control (top left), 0.5 mM VPA (top right), or VPA with _myo_-inositol (2 mM) (bottom left) or prolyl oligopeptidase inhibitor (133 μM) (bottom right). B, quantification of growth-cone size for five VPA derivatives and VPA after treatment (▪) with the indicated drug or with the drug plus inositol (□) or PO inhibitor (▧). Data represent two to four independent experiments containing approximately 25 growth cones per experiment (± S.E.M.); ★, p < 0.05.

Fig. 4

Fig. 4

Direct inhibition studies of GSK-3β activity by VPA and its 10 congeners. Drugs (3 mM) were tested for inhibitory effect on mammalian GSK-3β with vector only control (−, dimethyl sulfoxide). Results are expressed as a percentage of activity in the absence of drug ± S.D.

Fig. 5

Fig. 5

Characterization of bipolar disorder treatments, VPA analogs, and other treatments of HIV replication. Human 293T cells were infected with GFP-labeled HIV particles modified to block cell lysis in the presence of the indicated drugs. Cells were analyzed for the presence of GFP-labeled HIV after 48 h using FACS analysis (see Materials and Methods). A, cell infection rates were quantified by measuring GFP-labeled cells, shown for control (1.1%), compound IX (1.1%), and VPA (2.5%) treatments with increased FL1 levels (gray shaded area, control). Quantification of HIV infection is provided as increase in infection rates over control (vehicle only) for B. B, the indicated compounds at low (0.5 mM) and high concentrations (3 mM) or lithium (3 and 10 mM) or an inhibitor of GABA transaminases (VGB, vigabatrin: 30 and 100 μM). C, the inhibition of histone deacetylases by VPA was mimicked using a range of concentrations of trichostatin A (TSA). Decreased infection rates reflect toxicity of the compounds on target cells at high drug concentrations. Data represent FACS analysis of at least two independent experiments (± S.E.M.).

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