BMC Pharmacology - Latest Articles

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

Rebecca Roof — 2009-05-22T00:00:00Z

Background: Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action. Results: Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity. Conclusion: Though it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.

Rapamycin weekly maintenance dosing and the potential efficacy of combination sorafenib plus rapamycin but not atorvastatin or doxycycline in tuberous sclerosis preclinical models

Nancy Lee — 2009-04-15T00:00:00Z

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by hamartomatous growths in the brain, skin, kidneys, lungs, and heart, which lead to significant morbidity. TSC is caused by mutations in the TSC1 or TSC2 genes, whose products, hamartin and tuberin, form a tumor suppressor complex that regulates the PI3K/Akt/mTOR pathway. Early clinical trials show that TSC-related kidney tumors (angiomyolipomas) regress when treated with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (also known as sirolimus). Although side effects are tolerable, responses are incomplete, and tumor regrowth is common when rapamycin is stopped. Strategies for future clinical trials may include the investigation of longer treatment duration and combination therapy of other effective drug classes. Results: Here, we examine the efficacy of a prolonged maintenance dose of rapamycin in Tsc2+/- mice with TSC-related kidney tumors. Cohorts were treated with rapamycin alone or in combination with interferon-gamma (IFN-g). The schedule of rapamycin included one month of daily doses before and after five months of weekly doses. We observed a 94.5% reduction in kidney tumor burden in Tsc2+/- mice treated (part one) daily with rapamycin (8 mg/kg) at 6 months ≤ age < 7 months, (part 2) weekly with rapamycin (16 mg/kg) at 7 months ≤ age < 12 months, and (part 3) daily with rapamycin (8 mg/kg) at 12 months ≤ age < 13 months; but we did not observe any improvement with combination IFN-g plus rapamycin in this study. We also used a Tsc2-/- subcutaneous tumor model to evaluate other classes of drugs including sorafenib, atorvastatin, and doxycycline. These drugs were tested as single agents and in combination with rapamycin. Our results demonstrate that the combination of rapamycin and sorafenib increased survival and may decrease tumor volume as compared to rapamycin treatment alone while sorafenib as a single agent was no different than control. Atorvastatin and doxycycline, either as single agents or in combination with rapamycin, did not improve outcomes as compared with controls. Conclusion: Our results indicate that prolonged treatment with low doses of mTOR inhibitors may result in more complete and durable TSC-related tumor responses, and it would be reasonable to evaluate this strategy in a clinical trial. Targeting the Raf/Mek/Erk and/or VEGF pathways in combination with inhibiting the mTOR pathway may be another useful strategy for the treatment of TSC-related tumors.

N-acetylcysteine amide decreases oxidative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes

Rong Shi — 2009-04-15T00:00:00Z

Background: While doxorubicin (DOX) is widely used in cancer chemotherapy, long-term severe cardiotoxicity limits its use. This is the first report of the chemoprotective efficacy of a relatively new thiol antioxidant, N-acetylcysteine amide (NACA), on DOX-induced cell death in cardiomyocytes. We hypothesized that NACA would protect H9c2 cardiomyocytes from DOX-induced toxicity by reducing oxidative stress. Accordingly, we determined the ability of NACA to mitigate the cytotoxicity of DOX in H9c2 cells and correlated these effects with the production of indicators of oxidative stress. Results: DOX at 5 μM induced cardiotoxicity while 1) increasing the generation of reactive oxygen species (ROS), 2) decreasing levels and activities of antioxidants and antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase) and 3) increasing lipid peroxidation. NACA at 750 μM substantially reduced the levels of ROS and lipid peroxidation, as well as increased both GSH level and GSH/GSSG ratio. However, treating H9c2 cells with NACA did little to protect H9c2 cells from DOX-induced cell death. Conclusion: Although NACA effectively reduced oxidative stress in DOX-treated H9c2 cells, it had minimal effects on DOX-induced cell death. NACA prevented oxidative stress by elevation of GSH and CYS, reduction of ROS and lipid peroxidation, and restoration of antioxidant enzyme activities. Further studies to identify oxidative stress-independent pathways that lead to DOX-induced cell death in H9c2 are warranted.

Haloperidol changes mRNA expression of a QKI splice variant in human astrocytoma cells

Lin Jiang — 2009-03-31T00:00:00Z

Background: The quaking homolog, KH domain RNA binding (mouse) (QKI) is a candidate gene for schizophrenia. Disturbed QKI mRNA expression is observed in the prefrontal cortex of patients, and some of these changes correlate to treatment with antipsychotic drugs.To test if low doses of antipsychotic drugs can modify QKI mRNA expression, human astrocytoma (U343) and oligodendroglioma (HOG) cell lines were treated with five different antipsychotic drugs including Haloperidol, Aripiprazole, Clozapine, Olanzapine and Risperidone. Messenger RNA expression levels of splice variants QKI-5, QKI-6 and QKI-7 were measured by Real-Time PCR. Results: Haloperidol treatment (0.2 μM) doubled QKI-7 mRNA levels in U343 cells after 6 hours (p-value < 0.02). The effect was dose dependent, and cells treated with ten times higher concentration (2 μM) responded with a five-fold and three-fold increase in QKI-7, 6 and 24 hours after treatment, respectively (p-values < 0.0001). Conclusion: The results in U343 cells suggest that QKI-7 mRNA expression in human astrocytes is induced by Haloperidol, at concentrations similar to plasma levels relevant to clinical treatment of schizophrenia. The molecular mechanism of action of antipsychotic drugs after binding to receptors is not well known. We hypothesize that QKI regulation is involved in this mechanism.

Preferential uptake of the non steroid anti-inflammatory drug diclofenac into inflamed tissues after a single oral dose in rats

A. Schweitzer — 2009-03-16T00:00:00Z

Background: Diclofenac is a nonsteroidal anti-inflammatory drug which is available as prescription (RX) and over-the-counter (OTC) medication for the systemic and topical treatment of painful and inflammatory conditions such as arthritis and back pain. This study was undertaken to investigate the distribution and retention of diclofenac and/or its metabolites in inflamed tissues, using the carrageenan-induced inflammation model and quantitative whole body autoradiography in rats. Methods: [14C]diclofenac sodium was administrated as a single 2 mg/kg oral dose 1 h after injection of carrageenan into one front and one hind footpads and subcutaneously into the dorsum of the neck of rats. A control animal received saline injections. Three carrageenan-treated rats and one control rat were sacrificed at 1, 4, 8, and 24 h after [14C]diclofenac sodium administration (total of 4 rats/time point). The carcasses were immediately snap-frozen and prepared for cryosectioning. Lengthwise whole-body sections (40 μm thick), including all major tissues, were obtained from different levels across the body. The tissue concentrations of total radiolabeled components were determined using quantitative autoradioluminography. Results: The radioactivity patterns demonstrated that diclofenac and/or its metabolites preferentially distributed into the inflamed tissues. In unharmed tissues the distribution was similar in control and treated animals. The exposure, based on the areas under the tissue concentration versus time (AUC0-tlast), was 26 and 53 fold higher in the inflamed neck and inflamed footpads of treated animals than in control rats; the exposures in unharmed tissues were similar in the treated and control rats, and the AUC0-tlast was 17 fold higher in the inflamed paws than in the non inflamed footpads of the carrageenan-treated rats. The higher exposure in the inflamed tissues may be explained partly to the fact that the elimination of total radiolabeled components from inflamed tissues (t1/2 = 6 h) appeared lower than from the corresponding unharmed tissues (t1/2 = 2 h). Conclusion: This animal study demonstrated that diclofenac and/or its metabolites were rapidly and preferentially taken up and retained in inflamed tissues. Although there were theoretical considerations that mildly acidic NSAID may show some preferential distribution in inflamed tissues there was no clear experimental proof for diclofenac until the present study.

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins

Rima Soli — 2009-03-10T00:00:00Z

Background: K+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels. Results: Four multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins.Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins.Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors.Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences. Conclusion: The phylogenetic results were in perfect agreement with those obtained by the CART algorithm.Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.

The mode of action of dimeticone 4% lotion against head lice, Pediculus capitis.

Ian Burgess — 2009-02-20T00:00:00Z

Background: Treatment of head lice using physically acting preparations based on silicones is currently replacing insecticide use due to widespread resistance to neurotoxic agents. It has been postulated that some products act by asphyxiation, although the limited experimental evidence and the anatomy of the louse respiratory system suggest this is unlikely. Results: Observation over several hours of lice treated using 4% high molecular weight dimeticone in a volatile silicone base showed that, although rapidly immobilised initially, the insects still exhibited small movements of extremities and death was delayed. One common effect of treatment is inhibition of the louse's ability to excrete water by transpiration through the spiracles. Inability to excrete water that is ingested as part of the louse blood meal appears to subject the louse gut to osmotic stress resulting in rupture. Scanning electron microscopy coupled with X-ray microanalysis to detect silicon showed dimeticone lotion is deposited in the spiracles and distal region of the tracheae of lice and in some cases blocks the lumen or opening entirely. Conclusion: This work raises doubts that lice treated using dimeticone preparations die from anoxia despite blockage of the outer respiratory tract because movements can be observed for hours after exposure. However, the blockage inhibits water excretion, which causes physiological stress that leads to death either through prolonged immobilisation or, in some cases, disruption of internal organs such as the gut.

AICAR activates the Pluripotency Transcriptional Network in Embryonic Stem Cells and induces KLF4 and KLF2 Expression in Fibroblasts

Luigi Adamo — 2009-02-12T00:00:00Z

Background: Pluripotency, the property of a cell to differentiate into all cellular types of a given organism, is central to the development of stem cell-based therapies and regenerative medicine. Stem cell pluripotency is the result of the orchestrated activation of a complex transcriptional network characterized by the expression of a set of transcription factors including the master regulators of pluripotency Nanog and Oct4. Recently, it has been shown that pluripotency can be induced in somatic cells by viral-mediated expression of the transcription factors Oct3/4, Sox2, Klf4, and c-Myc. Results: Here we show that 5-Aminoimidazole-4-carboxamide-1-b-riboside (AICAR) is able to activate the molecular circuitry of pluripotency in mouse embryonic stem cells (mESC) and maintain Nanog and Oct4 expression in mESC exposed to the differentiating agent retinoic acid. We also show that AICAR is able to induce Klf4, Klf2 and Myc expression in both mESC and murine fibroblasts. Conclusion: AICAR is able to activate the molecular circuitry of pluripotency in mESC and to induce the expression of several key regulators of pluripotency in somatic cells. AICAR is therefore a useful pharmacological entity for studying small molecule mediated induction of pluripotency.

Conformational changes in alpha 7 acetylcholine receptors underlying allosteric modulation by divalent cations

James McLaughlin — 2009-01-13T00:00:00Z

Allosteric modulation of membrane receptors is a widespread mechanism by which endogenous and exogenous agents regulate receptor function. For example, several members of the nicotinic receptor family are modulated by physiological concentrations of extracellular calcium ions. In this paper, we examined conformational changes underlying this modulation and compare these with changes evoked by ACh. Two sets of residues in the α7 acetylcholine receptor extracellular domain were mutated to cysteine and analyzed by measuring the rates of modification by the thiol-specific reagent 2-aminoethylmethane thiosulfonate. Using Ba2+ as a surrogate for Ca2+, we found a divalent-dependent decrease the modification rates of cysteine substitutions at M37 and M40, residues at which rates were also slowed by ACh. In contrast, Ba2+ had no significant effect at N52C, a residue where ACh increased the rate of modification. Thus divalent modulators cause some but not all of the conformational effects elicited by agonist. Cysteine substitution of either of two glutamates (E44 or E172), thought to participate in the divalent cation binding site, caused a loss of allosteric modulation, yet Ba2+ still had a significant effect on modification rates of these residues. In addition, the effect of Ba2+ at these residues did not appear to be due to direct occlusion. Our data demonstrate that modulation by divalent cations involves substantial conformational changes in the receptor extracellular domain. Our evidence also suggests the modulation occurs via a binding site distinct from one which includes either (or both) of the conserved glutamates at E44 or E172.

Flexible modulation of agonist efficacy at the human A3 adenosine receptor by the imidazoquinoline allosteric enhancer LUF6000

Zhan-Guo Gao — 2008-12-12T00:00:00Z

Background: A series of 1H-imidazo- [4,5-c]quinolin-4-amine derivatives, represented by LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo [4,5-c]quinolin-4-amine), are allosteric modulators of the human A3 adenosine receptor (AR). Here we studied the modulation by LUF6000 of the maximum effect (Emax) of structurally diverse agonists at the A3 AR stably expressed in CHO cells. Results: In an assay of [35S]GTPγS binding, the Emax of the A3 AR agonist Cl-IB-MECA at the A3 AR was lower than that of the non-selective AR agonist NECA. LUF6000 exerted an Emax-enhancing effect at a concentration of 0.1 μM or higher, and was shown to increase the Emax of Cl-IB-MECA and other low-efficacy agonists to a larger extent than that of the high-efficacy agonist NECA. Interestingly, LUF6000 converted a nucleoside A3 AR antagonist MRS542, but not a non-nucleoside antagonist MRS1220, into an agonist. LUF6000 alone did not show any effect. Mathematical modeling was performed to explain the differential effects of LUF6000 on agonists with various Emax. A simple explanation for the observation that LUF6000 has a much stronger effect on Cl-IB-MECA than on NECA derived from the mathematical modeling is that NECA has relatively strong intrinsic efficacy, such that the response is already close to the maximum response. Therefore, LUF6000 cannot enhance Emax much further. Conclusion: LUF6000 was found to be an allosteric enhancer of Emax of structurally diverse agonists at the A3 AR, being more effective for low-Emax agonists than for high-Emax agonists. LUF6000 was demonstrated to convert an antagonist into an agonist, which represents the first example in G protein-coupled receptors. The observations from the present study are consistent with that predicted by mathematical modeling.