Molecular Pharmacology Fast Forward

[Article] Mechanisms and functional significance of inhibition of neuronal T-type calcium channels by isoflurane

Orestes, P., Bojadzic, D., Chow, R., Todorovic, S. M — 2008-12-02

Previous data have indicated that T-type calcium channels (low-voltage activated, LVA, T-channels) are potently inhibited by volatile anesthetics. Although the interactions of T-channels with a number of anesthetics have been described, the mechanisms by which these agents modulate channel activity, and the functional consequences of such interactions, are not well studied. Here, we used patch-clamp recordings to explore the actions of a prototypical volatile anesthetic, isoflurane (Iso), on recombinant human Ca_V3.1 and Ca_V3.2 isoforms. We also performed behavioral testing of anesthetic end-points in mice lacking Ca_V3.2. Iso applied at resting channel states blocked current through both isoforms in a similar manner at clinically relevant concentrations (1 minimum alveolar concentration, MAC). Inhibition was more prominent at depolarized membrane potentials (-65 mV versus -100 mV) as evidenced by hyperpolarizing shifts in channel availability curves and a 2.5-fold decrease in IC₅₀ values. Iso slowed recovery from inactivation and enhanced deactivation in both Ca_V3.1 and Ca_V3.2 in a comparable manner, but caused a depolarizing shift in activation curves and greater use-dependent block of Ca_V3.2 channels. In behavioral tests, Ca_V3.2 knockout (KO) mice showed significantly decreased MAC in comparison to wild type (WT) littermates. KO and WT mice did not differ in loss of righting reflex (LORR), but mutant mice displayed a delayed onset of anesthetic induction. We conclude that state-dependent inhibition of T-channel isoforms in the central and peripheral nervous system may contribute to isoflurane's important clinical effects.

[Accelerated Communication] D5 Dopamine Receptors are Required for Dopaminergic Activation of Phospholipase C

Sahu, A., Tyeryar, K. R, Vongtau, H. O, Sibley, D. R., Undieh, A. S — 2008-12-01

Dopamine activates phospholipase C in discrete regions of the mammalian brain, and this action is thought to be mediated through a D₁-like receptor. While multiple lines of evidence exclude a role for the D₁ subtype of D₁-like receptors in the phosphoinositide response, the D₅ subtype has not been similarly examined. Here, mice lacking D₅ dopamine receptors were tested for dopamine agonist-induced phosphoinositide signaling both in vitro and in vivo. The results show that hippocampal, cortical and striatal tissues of D₅ receptor knockout mice significantly or completely lost the ability to produce inositol phosphate or diacylglycerol messengers following stimulation with dopamine or several selective D₁-like receptor agonists. Moreover, endogenous inositol-1,4,5-trisphosphate stimulation by the phospholipase C-selective D₁-like agonist, SKF83959, was robust in wildtype animals but undetectable in the D₅ receptor mutants. Hence, D₅ receptors are required for dopamine and selective D₁-like agonists to induce phospholipase C-mediated phosphoinositide signaling in the mammalian brain.

[Article] Molecular Basis for the Selective Interaction of Synthetic Agonists with the Human Histamine H1-Receptor compared to the Guinea Pig H1-Receptor

Strasser, A., Wittmann, H.-J., Kunze, M., Elz, S., Seifert, R. — 2008-12-01

Previous studies revealed that phenylhistamines and histaprodifens possess higher potency and affinity at guinea pig histamine H₁-receptor (gpH₁R) than at human histamine H₁-receptor (hH₁R). However, recently, we identified an imidazolylpropylguanidine (UR-AK57) with higher potency and efficacy at hH₁R compared to gpH₁R. The aim of this study was to reveal the molecular basis for the species-differences of synthetic ligands. We studied 11 novel phenylhistamines and phenoprodifens. H₁R species isoforms were expressed in Sf9 insect cells, and [³H]mepyramine competition binding- and GTPase assays were performed. We identified bulky phenylhistamines with higher potency and affinity at hH₁R compared to gpH₁R. Molecular dynamics simulations of ligand-H₁R interactions revealed four potential binding-modes for phenylhistamines possessing an additional histamine moiety, with the terminal histamine moiety showing a high flexibility in the binding-pocket. Strikingly, there are similarities in ligand properties between bulky phenylhistamines and UR-AK57. Comparison of bulky phenylhistamine binding-mode with binding-mode of UR-AK57 suggests that only one of these four binding-modes should be established. The higher poteny is explained by more effective van der Waals interaction of the compounds with Asn^2.61 (hH₁R) relative to Ser^2.61 (gpH₁R). Additionally, two stable binding-modes for phenoprodifens with different orientations in the binding pocket were identified. Depending on phenoprodifen orientation, the highly conserved Trp^6.48, part of the toggle switch involved in receptor activation, was found in an inactive or active conformation, respectively. Collectively, we identified the first phenylhistamines with higher potency at hH₁R than at gpH₁R and obtained insight into the binding-mode of bulky phenylhistamines and imidazolylpropylguanidines.

[Article] Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity

2008-12-01

Activators of M₁ muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M₁-active compounds has resulted in nonselective activation of the highly related M₂-M₅ mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M₁ with low micromolar potencies and induced five-fold or greater leftward shifts of the ACh concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, VU0090157 and VU0029767, induced progressive shifts in ACh affinity at M₁ that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M₁ activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M₁ receptor with decreased affinity for ACh as well as in their ability to affect responses of the allosteric M₁ agonist, TBPB. Furthermore, these two compounds were distinct in their abilities to potentiate M₁-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M₁ is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M₁ potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.

[Article] Analysis of DNA methylation and histone modification profiles of liver-specific transporters

Imai, S., Kikuchi, R., Kusuhara, H., Yagi, S., Shiota, K., Sugiyama, Y. — 2008-12-01

Tissue-specific expression of transporters is tightly linked with their physiological functions through the regulation of the membrane transport of their substrates. We hypothesized that epigenetic regulation underlies the tissue-specific expression of mouse liver-specific transporters (Oatp1b2/Slco1b2, Ntcp/Slc10a1, Bsep/Abcb11, and Abcg5/g8). We examined their DNA methylation and histone modification profiles near the transcriptional start site (TSS) in the liver, kidney, and cerebrum. Genome-wide DNA methylation profiling with D-REAM and subsequent bisulfite genomic sequencing demonstrated that the CpG dinucleotides around the TSS of Oatp1b2 (from -515 to +149 CpGs), Ntcp (from -481 to +495 CpGs), Bsep (from -339 to +282 CpGs), and Abcg5/g8 (from -161 to +5 CpGs for Abcg5, i.e. from -213 to -48 CpGs for Abcg8) were hypomethylated in the liver and hypermethylated in the kidney and cerebrum. The opposite pattern was observed for Pept2/Slc15a2 (from -638 to +4 CpGs), which was expressed in the kidney and cerebrum but not in the liver. These DNA methylation profiles are consistent with the tissue distribution of these transporters. A chromatin immunoprecipitation assay demonstrated that the histone H3 associated with Oatp1b2, Ntcp, Bsep, and Abcg5/g8 promoters was hyperacetylated in the liver but was acetylated very little in the kidney and cerebrum, whereas the upstream region of Pept2 was hyperacetylated only in the kidney and cerebrum. These results suggest the involvement of epigenetic systems in the tissue-specific expression of mouse transporters Oatp1b2, Ntcp, Bsep, Abcg5/g8, and Pept2.

[Article] Knock-In Mouse Lines Expressing either Mitochondrial or Microsomal CYP1A1: Differing Responses to Dietary Benzo[a]pyrene as Proof-of-Principle

2008-12-01

Historically CYP1A1 protein is known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH₂-terminal processing that exposes a cryptic targeting signal. Intriguingly, microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH₂-terminus. Cyp1a1(mtt/mtt) mice encode an NH₂-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying Leu7Asn and Leu17Asn mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having Arg34Asp and Lys39Ile mutations which abolish the mitochondrial targeting signal. Following dioxin or β-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.

[Article] Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane

2008-12-01

We show here that the rat vasopressin V_1b receptor simultaneously activates both the G_q/11-IP and G_s-cAMP pathways when transiently expressed in CHO, HEK-293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The non-mammalian analog arginine-vasotocin and the selective V_1b agonist d[Cha⁴]vasopressin also activated the cAMP and IP pathways, although d[Cha4]vasopressin elicited the two responses with equivalent potencies. We determined that the V_1b receptor is present as homodimers at the plasma membrane. Treatment of V_1b-transfected HEK-293 cells with methyl-β-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC₅₀ of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC₅₀. These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V_1b receptor forms dimers that signal differentially through the G_q/11 and G_s proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.

[Article] The reelin and GAD67 promoters are activated by epigenetic drugs that facilitate the disruption of local repressor complexes

Kundakovic, M., Chen, Y., Guidotti, A., Grayson, D. R — 2008-11-24

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNMT-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, HDAC inhibitors, activate these two genes with a comparable dose- and time-dependence. In parallel, both groups of drugs decrease DNMT1, DNMT3A and DNMT3B protein levels, and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes, containing all three DNMTs, MeCP2 and HDAC1, from the corresponding promoters and increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

[Article] Relaxin family peptide receptor (RXFP1) coupling to G{alpha}i3 involves the C-terminal Arg752 and localization within membrane raft microdomains

2008-11-24

The relaxin family peptide receptors (RXFP) 1 and 2 are targets for the relaxin family peptides relaxin and insulin-like peptide 3 (INSL3) respectively. Although both receptors and peptides share a high degree of sequence identity, the cAMP signaling pathways activated by the two systems are quite distinct. Relaxin activation of RXFP1 initially results in accumulation of cAMP via G_s but this is modulated by inhibition of cAMP through G_oB. With time, RXFP1 recruits coupling to G_i3 causing additional cAMP accumulation via a G_i3-Gβ-PI3K-PKC pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via G_s, modulated by cAMP inhibition through G_oB. Thus the aim of this study was to identify the cause of differential G-protein coupling between these highly similar receptors. Construction of chimeric receptors revealed that G_i3 coupling is dependent upon the transmembrane region of RXFP1 and independent of the receptor ectodomain or ligand bound. Generation of C-terminal truncated receptors identified the terminal 10 amino acids of the RXFP1 C-terminus as essential for G_i3 signaling, and point mutations revealed an obligatory role for Arg⁷⁵². RXFP1-mediated G_i3, but not G_s or G_oB, signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to G_i3 after only 3 min of receptor stimulation. Therefore RXFP1 coupling to the G_i3-Gβ-PI3K-PKC pathway requires the terminal 10 amino acids of the RXFP1 C-terminus and membrane raft localisation, and the observed delay in this pathway occurs downstream of G_i3.

[Article] Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms

2008-11-24

The L-type calcium channel (LTCC) isoforms Ca_v1.2 and Ca_v1.3 display similar 1,4-Dihydropyridine binding properties and are both expressed in mammalian brain. Recent work implicates Ca_v1.3 channels as interesting drug targets, but no isoform selective modulators exist. It is also unknown to which extent Ca_v1.1 and Ca_v1.4 contribute to L-type specific dihydropyridine (DHP) binding activity in brain. To address this question and to determine if DHPs can discriminate between Ca_v1.2 and Ca_v1.3 binding pockets we combined radioreceptor assays and quantitative PCR (qPCR). We bred double mutants (Ca_v-DM) from mice expressing mutant Ca_v1.2 channels (Ca_v1.2DHP^-/-) lacking high affinity for DHPs and from Ca_v1.3 knockouts (Ca_v1.3^-/-). (+)-[³H]isradipine binding to Ca_v1.2DHP^-/- and Ca_v-DM brains was reduced to 15.1 and 4.4% of wildtype, respectively, indicating that Ca_v1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca_v1.1 and Ca_v1.4 ₁ subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low affinity binding (127-fold K_d increase) to the mutated Ca_v1.2 channels. Inhibition of (+)-[³H]isradipine binding to Ca_v1.2DHP^-/- (predominantly Ca_v1.3) and wildtype (predominantly Ca_v1.2) brain membranes by unlabeled DHPs revealed a 3-4-fold selectivity of nitrendipine and nifedipine for the Ca_v1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, due to their very low expression, Ca_v1.1 and Ca_v1.4 are unlikely to serve as drug targets to treat CNS diseases.

[Article] Pharmacological analysis of the novel, rapid and potent inactivation of the human 5-HT7 receptor by risperidone, 9-OH-risperidone and other "inactivating antagonists"

Knight, J. A, Smith, C., Toohey, N., Klein, M. T., Teitler, M. — 2008-11-21

In a previous publication, using h5-HT₇ receptor-expressing HEK293 cells, we reported the rapid, potent inactivation of the h5-HT₇ receptor stimulation of cAMP production by three antagonists: risperidone, 9-OH-risperidone, and methiothepin (Smith et al., 2006). In order to better understand the drug-receptor interaction producing the inactivation we a) expanded the list of inactivating drugs; b) determined the inactivating potencies and efficacies by performing concentration-response experiments and c) determined the potencies and efficacies of the inactivators as irreversible binding site inhibitors. Three new drugs were found to fully inactivate the h5-HT₇ receptor: lisuride, bromocryptine, and metergoline. As inactivators these drugs diplayed potencies of 1, 80, and 321nM, respectively. Pre-treatment of 5-HT₇-expressing HEK cells with increasing concentrations of the inactivating drugs risperidone, 9-OH-risperidone, methiothepin, lisuride, bromocryptine, and metergoline potently inhibited radiolabeling of the h5-HT₇ receptor, with IC₅₀ values of 9, 5.5, 152, 3, 73, and 10nM, respectively. Surprisingly, maximal concentrations of risperidone and 9-OH-risperidone inhibited only 50% of the radiolabeling of h5-HT₇ receptors. These results indicate that risperidone and 9-OH-risperidone may be producing the inactivating effect through a different mechanism than lisuride, bromocryptine, metergoline, and methiothepin. These results are not interpretable using the conventional model of GPCR function. This complex appears capable of assuming a stable inactive conformation due to the interaction of certain antagonists. The rapid, potent inactivation of the receptor-G-protein complex by antagonists implies a constitutive, pre-existing complex between the h5-HT₇ receptor and a G-protein.

[Article] Multiple Roles of Charged Amino Acids in Cytoplasmic Loop 7 For Expression and Function of the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Conseil, G., Rothnie, A. J., Deeley, R. G., Cole, S. P.C. — 2008-11-17

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, a same charge amino acid, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu¹¹⁶⁹, Glu¹¹⁷⁰ and Glu¹¹⁷² were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable to wild-type MRP1 while the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, ¹¹⁶⁹AAQA). Same charge mutant D1183E was also not expressed while expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A and ¹¹⁶⁹AAQA were accompanied by changes in orthovanadate-induced trapping of [³²P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

[Accelerated Communication] Refinement of the binding site and mode of action of the anticonvulsant retigabine on KCNQ K+ channels

2008-11-17

The discovery of retigabine has provided access to alternative anticonvulsant compounds with a novel mode of action. Acting as potassium channel opener, retigabine exclusively activates neuronal KCNQ-type K⁺ channels, mainly by hyperpolarizingly shifting the voltage-dependence of channel activation. So far, only parts of the retigabine-binding site have been described, including Trp-265 and Gly-340 (according to KCNQ3 numbering) within transmembrane segments S5 and S6, respectively. Using a refined chimeric strategy, we additionally identified a Leu-314 within the pore region of KCNQ3 as crucial for the retigabine effect. Both Trp-265 and Leu-314 are likely to interact with the retigabine molecule, representing the upper and lower margins of the putative binding site. Guided by a structural model of KCNQ3, which was constructed based on the Kv1.2 crystal structure, further residues affecting retigabine-binding could be proposed and were experimentally verified as mediators for the action of the compound. These results strongly suggest that besides Trp-265 and Leu-314 it is highly likely that another S5 residue, Leu-272, which is conserved in all KCNQ subunits, contributes to the binding site in KCNQ3. More importantly, also Leu-338, extending from S6 of the neighboring subunit is apparently involved in lining the hydrophobic binding pocket for the drug. This pocket, which is formed at the interface of two adjacent subunits, may only be present in the open state of the channel, consistent with the idea that retigabine stabilizes an open channel conformation.

[Minireview] Regulation of the mammalian nervous system by microRNAs

ZENG, Y. — 2008-11-12

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself extensively controlled by a variety of genetic regulatory mechanisms. microRNAs, a class of small, non-coding RNAs, are critical contributors to the regulation of gene expression in the nervous system. Emerging evidence indicates that microRNAs regulate both the development and function of the nervous system. Deficiency in microRNA function has also been implicated in a number of neurological disorders. Understanding the roles of microRNAs will provide new insights into the complexity and operation of the nervous system.

[Article] Thermostable Variants of Cocaine Esterase for Long-Time Protection against Cocaine Toxicity

2008-11-12

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction, as CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine yet identified. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only a few minutes at physiological temperature (37 °C). Here we report thermostable variants of CocE developed through rational design using a novel computational approach followed by in vitro and in vivo studies. This integrated computational-experimental effort has yielded a CocE variant with a ~30-fold increase in plasma half-life both in vitro and in vivo. The novel design strategy can be used to develop thermostable mutants of any protein.

[Article] Mutagenic mapping suggests a novel binding mode for selective agonists of M1 muscarinic acetylcholine receptors

Lebon, G., Langmead, C. J., Tehan, B. G., Hulme, E. C. — 2008-11-11

Point mutations and molecular modelling have been used to study the activation of the M₁ muscarinic acetylcholine receptor (M1 mAChR ) by the functionally-selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them to N-desmethylclozapine (NDMC) and ACh. Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although reduced by mutations of Tyr408. Signalling by AC-42, 77-LH-28-1 and NDMC was reduced by Leu102Ala and abolished by Asp105Glu, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105, to activate the M₁ mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by Trp101Ala , and their signalling activities abolished by Tyr82Ala. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M₁ mAChR. We suggest the hypothesis that the side-chain of Trp101 undergoes conformational isomerisation, opening a novel binding site for the aromatic side-chain of the AC-42 analogues. This may allow the positively-charged piperidine nitrogen of the ligands to access the neighbouring Asp105 carboxylate to activate signalling, following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not appear to use this mechanism. Subtype-specific differences in the free energy of rotation of the side-chain and indole ring of Trp101 might underlie the M₁-selectivity of the AC-42 analogues. Trp conformational isomerisation may open up new avenues in selective muscarinic receptor drug design.

[Perspective] Functional Selectivity of GPCR Ligand Stereoisomers: New Pharmacological Opportunities (Relates to article by Woo et al., Fast Forward 10 Nov 08)

Seifert, R., Dove, S. — 2008-11-10

It is now well established that any given ligand for a G-protein coupled receptor (GPCR) does not simply possess a single defined efficacy. Rather, a ligand possesses multiple efficacies, depending on the specific down-stream signal transduction pathway analyzed. This diversity may be based on ligand-specific GPCR conformations and is often referred to as "Functional Selectivity". It has been known for a century that stereoisomers of catecholamines differ in their potency, and in some systems, also in their efficacy. However, the molecular basis for efficacy differences of GPCR ligand stereoisomers has remained poorly defined. In an elegant study published in this issue of Molecular Pharmacology, Woo et al. show that stereosiomers of the β₂-adrenoceptor selective agonist fenoterol differentially activate G_s- and G_i-proteins in native rat cardiomyocytes. This study is so important because it is the first report to show that even the subtle structural differences within a ligand stereoisomer pair are sufficient to discriminate between GPCR conformations with distinct G-protein coupling properties. The study highlights of how important it is to examine the "more active" (eutomer) and the "less active" (distomer) stereoisomer to understand the mechanisms of action and the cellular effects of GPCR ligands. The study by the Xiao group will ignite a renaissance of the analysis of ligand stereoisomers, using sensitive pharmacological and biophysical assays. The available literature supports the notion that meticulous analysis of ligand stereoisomers is a goldmine for understanding mechanisms of GPCR activation, analysis of signal transduction pathways, development of new therapies for important diseases and drug safety.

[Article] Stereochemistry of an Agonist Determines Coupling Preference of {beta}2-Adrenoceptor to Different G Proteins in Cardiomyocytes

2008-11-10

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that while most β₂-adrenoceptor agonists activate both G_s and G_i proteins, fenoterol, a full agonist of β₂-adrenoceptor, selectively activates G_s protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,Risomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R-isomers. Importantly, while R,R-fenoterol and R,R-methoxyfenoterol preferentially activate G_s signaling, their S,R-isomers were able to activate both G_s and G_i proteins as evidenced by the robust pertussis toxin-sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G_s, G_i2 and G_i3 proteins. The inefficient G_i signaling with the R,R-isomers is not caused by the inability of the R,R-isomers to trigger the PKA-mediated phosphorylation of the β₂-adrenoceptor, since the R,R-isomers also markedly increased phosphorylation of the receptor at serine262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.

[Article] The Role of the Mammalian Copper Transporter 1 in the Cellular Accumulation of Platinum-based Drugs

Larson, C. A., Blair, B. G., Safaei, R., Howell, S. B. — 2008-11-07

The mammalian Copper Transporter 1 (CTR1) is responsible for the uptake of copper (Cu) from the extracellular space. In this study we used an isogenic pair of CTR1^+/+ and CTR1^-/- mouse embryo fibroblasts to examine the contribution of CTR1 to the influx of cisplatin (DDP), carboplatin (CBDCA), oxaliplatin (L-OHP) and transplatin. Exposure to DDP triggered the rapid degradation of CTR1 suggesting that its contribution to influx was likely to be on the initial phase of drug entry. Loss of CTR1 decreased the initial binding of DDP to cells and reduced influx measured over the first 5 min of drug exposure by 81%. Loss of CTR1 almost completely eliminated the initial influx of CBDCA and reduced the initial uptake of L-OHP by 68% but had no effect on the influx of transplatin. Loss of CTR1 rendered cells resistant to even high concentrations of DDP when measured in vitro, and re-expression of CTR1 in the CTR1^-/- cells restored both DDP uptake and cytotoxicity. The growth of CTR1^-/- tumor xenografts in which CTR1 levels were restored by infection with a lentivirus expressing wild type CTR1 was reduced by a single maximum tolerated dose of DDP in vivo whereas the CTR1^-/- xenografts failed to respond at all. We conclude that CTR1 mediates the initial influx of DDP, CBDCA and L-OHP and is a major determinant of responsiveness to DDP both in vitro and in vivo.

[Article] Estimation of Relative Microscopic Affinity Constants of Agonists for the Active State of the Receptor in Functional Studies on M2 and M3 Muscarinic Receptors

Tran, J. A., Chang, A., Matsui, M., Ehlert, F. J. — 2008-11-07

In prior work, we have shown that it is possible to estimate the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist simply through the analysis of their respective concentration-response curves. In this report, we show analytically and through receptor modeling that this product, termed intrinsic relative activity (RA_i), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RA_i estimates of selected muscarinic agonists with a relative estimate of the product of observed affinity and intrinsic efficacy determined independently through the method of partial receptor inactivation. There was good agreement between these two estimates when agonist-mediated inhibition of forskolin-stimulated cAMP accumulation was measured in Chinese hamster ovary cells stably expressing the human M₂ muscarinic receptor. Similarly, there was also good agreement between the two estimates when agonist activity was measured on the ileum from M₂ muscarinic receptor knockout mice, a convenient assay for M₃ receptor activity. The RA_i estimates of agonists in the mouse ileum were similar to those estimated at the human M₃ receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M₁ and M₄ selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M₃ muscarinic receptor component. Our results show that the RA_i estimate is useful a useful, receptor-dependent measure of agonist activity and ligand-directed signaling.

[Article] Involvement of Transient Receptor Potential Vanilloid Subtype 1 (TRPV1) in Analgesic Action of Methylsalicylate

Ohta, T., Imagawa, T., Ito, S. — 2008-11-05

Methylsalicylate (MS) is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite the common usage of MS as a pain reliever, the underlying molecular mechanism is not fully understood. Here we characterized the action of MS on transient receptor potential V1 (TRPV1). In human embryonic kidney (HEK) 293 cells expressing human TRPV1 (hTRPV1), MS evoked increases of [Ca²⁺]_i, which declined regardless of its continuous presence, indicative of marked desensitization. TRPV1 antagonists dose-dependently suppressed the MS-induced [Ca²⁺]_i increase. MS simultaneously elicited an inward current and increase of [Ca²⁺]_i in the voltage-clamped cells, suggesting that MS promoted Ca²⁺ influx through the activation of TRPV1 channels. MS reversibly inhibited hTRPV1 activation by polymodal stimuli such as capsaicin, protons, heat, anandamide and 2-aminoethoxydiphenyl borate. Because both the stimulatory and inhibitory actions of MS were exhibited in capsaicin- and allicin-insensitive mutant channels, MS-induced hTRPV1 activation was mediated by distinct channel regions from capsaicin and allicin. In cultured rat sensory neurons, MS elicited a [Ca²⁺]_i increase in cells responding to capsaicin. MS significantly suppressed nocifensive behavior induced by intraplantar capsaicin in rats. The present data indicate that MS has both stimulatory and inhibitory actions on TRPV1 channels, and suggest that the latter action may partly underlie the analgesic effects of MS independent of inhibition of COXs in vivo.

[Accelerated Communication] Nuclear xenobiotic receptor PXR locks co-repressor SMRT onto the CYP24A1 promoter to attenuate vitamin D3 activation

Konno, Y., Kodama, S., Moore, R., Kamiya, N., Negishi, M. — 2008-11-03

We have studied the molecular mechanism by which the nuclear xenobiotic receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) regulate transcription of the vitamin D₃ 24-hydroxylase (CYP24A1) gene. In the absence of vitamin D₃, PXR activates the CYP24A1 gene by directly binding to and trans-activating vitamin D response elements (VDREs) within its promoter. Vitamin D₃ activates the CYP24A1 promoter by dissociating the co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) from the vitamin D receptor (VDR) on those VDREs. PXR strongly represses vitamin D₃ activation of the CYP24A1 gene, in which PXR indirectly binds to and prevents vitamin D₃-dependent dissociation of SMRT from the CYP24A1 promoter. The degree of the PXR-mediated locking of SMRT depends on the relative concentration of vitamin D₃ to the human PXR activator rifampicin; SMRT increased its dissociation as this ratio increased. CAR is also found to prevent dissociation of SMRT from the CYP24A1 promoter. Thus, our present study defines the novel molecular mechanism by which PXR and CAR mediate drug interactions with vitamin D₃ to regulate the CYP24A1 gene. Pxr^+/+ and Pxr^-/- mice were chronically treated with mouse PXR activator PCN to evaluate the hypothesis that induction of the Cyp24a1 gene is responsible for the loss of bone mineral density often observed in patients chronically treated with PXR activating drugs. PCN-dependent loss of mineral density is observed in the metaphyseal bones of only the Pxr^+/+ mice. This loss, however, does not correlate with the expression levels of the Cyp24a1 gene in these mice.

[Article] Preferential Physical and Functional Interaction of Pregnane X Receptor with the SMRT{alpha} Isoform

Li, C.-W., Dinh, G. K., Chen, J. D. — 2008-10-31

The silencing mediator for retinoid and thyroid hormone receptors (SMRT) serves as a platform for transcriptional repression elicited by several steroid/nuclear receptors and transcription factors. SMRT exists in two major splicing isoforms: and , with SMRT contains only an extra 46-amino acid sequence inserted immediately downstream from the C-terminal corepressor motif. Currently little is known about potential functional differences between these two isoforms. Here we show that the pregnane X receptor (PXR) interacts more strongly with SMRT than with SMRT both in vitro and in vivo. Interestingly, the PXR-SMRT interaction is also resistant to PXR ligand-induced dissociation, in contrast to the PXR-SMRT interaction. Consistently, SMRT inhibits PXR activity more efficiently than does SMRT in transfection assays, while they possess comparable intrinsic repression activity and association with histone deacetylase. We further show that the mechanism for the enhanced PXR-SMRT interaction involves both the 46-amino acid insert and the C-terminal corepressor motif. Specifically, the first five amino acids of the SMRT insert are essential and sufficient for the enhanced binding of SMRT to PXR. Furthermore, we demonstrate that Tyr 2354 and Asp 2355 residues of the SMRT insert are most critical for the enhanced interaction. Additionally, expression data show that SMRT is more abundantly expressed in most human tissues and caner cell lines, and together these data suggest that SMRT may play a more important role than SMRT in the negative regulation of PXR.

[Article] Molecular Characterization of Novel and Selective Peroxisome Proliferator-Activated Receptor {alpha} Agonists with Robust Hypolipidemic Activity In Vivo

2008-10-29

The nuclear receptor PPAR is recognized as the primary target of the fibrate class of hypolipidemic drugs and mediates lipid lowering in part by activating a transcriptional cascade that induces genes involved in the catabolism of lipids. We report here the characterization of three novel PPAR agonists with therapeutic potential for treating dyslipidemia. These structurally related compounds display potent and selective binding to human PPAR and support robust recruitment of coactivator peptides in vitro. These compounds markedly potentiate chimeric transcription systems in cell-based assays and strikingly lower serum triglycerides in vivo. The transcription networks induced by these selective PPAR agonists were assessed by transcriptional profiling of mouse liver after acute and chronic treatment. The induction of several known PPAR target genes involved with fatty acid metabolism were observed, reflecting the expected pharmacology associated with PPAR activation. We also noted the downregulation of a number of genes related to immune cell function, the acute phase response, and glucose metabolism; suggesting that these compounds may have anti-inflammatory action in the mammalian liver. Whereas these compounds are efficacious in acute preclinical models, extended safety studies and further clinical testing will be required before the full therapeutic promise of a selective PPAR agonist is realized.

[Article] SKA-31, a New Activator of KCa2 and KCa3.1 Potassium Channels, Potentiates the EDHF Response and Lowers Blood Pressure

2008-10-27

Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) calcium-activated K⁺ channels are critically involved in modulating calcium-signaling cascades and membrane potential in both excitable and non-excitable cells. Activators of these channels constitute useful pharmacological tools as well as potential new drugs for the treatment of ataxia, epilepsy, and hypertension. We here used the neuroprotectant riluzole as a template for the design of KCa2/3 channel activators that are potent enough for in vivo studies. Out of a library of 41 benzothiazoles we identified two compounds, SKA-20 (anthra[2,1-d]thiazol-2-ylamine) and SKA-31 (naphtho[1,2-d]thiazol-2-ylamine), which are 10-20 times more potent than riluzole and activate KCa2.1 with EC₅₀s of 430 nM and 2.9 µM, KCa2.2 with EC₅₀s of 1.9 µM, KCa2.3 with EC₅₀s of 1.2 µM and 2.9 µM, and KCa3.1 with EC₅₀s of 115 nM and 260 nM. Likewise, SKA-20 and SKA-31 activated native KCa2.3 and KCa3.1 channels in murine endothelial cells and the more "drug-like" SKA-31 (half-life 12 hours) potentiated endothelium-derived hyperpolarizing factor-mediated dilations of carotid arteries from KCa3.1^+/+ mice but not from KCa3.1^-/- mice. Administration of 10 and 30 mg/kg SKA-31 lowered mean arterial blood pressure by 4 and 6 mmHg in normotensive mice and by 12 mmHg in angiotensin-II-induced hypertension. These effects were absent in KCa3.1-deficient mice. In conclusion, with SKA-31 we have designed a new pharmacological tool to define the functional role of KCa2/3 channel activation in vivo. The blood pressure lowering effect of SKA-31 suggests KCa3.1 channel activation as a new therapeutic principle for the treatment of hypertension.

[Article] Allosteric LFA-1 inhibitors modulate natural killer cell function

Weitz-Schmidt, G., Chreng, S., Riek, S. — 2008-10-27

Natural killer (NK) cells are thought to play an important role in a variety of disease pathologies including transplant rejection and autoimmunity. None of the therapeutic modalities currently available are known to potently interfere with NK cell activity. Here we demonstrate for the first time that low molecular weight inhibitors of the integrin lymphocyte function-associated antigen-1 (LFA-1) readily block NK cell adhesion, activation and NK cell-mediated cytolysis in vitro, in contrast to other immunosuppressive agents. These effects were independent of the type of allosteric mechanism by which LFA-1 inhibition was achieved. In addition, we describe a simple, non-radioactive whole blood assay which should be suitable to monitor NK cell activation in clinical practice. Taken together, our study underlines the importance of LFA-1 in NK cell effector functions and indicates that allosteric LFA-1 inhibitors may become important tools to further elucidate the therapeutic potential of NK cell modulation in immunological diseases.

[Article] The Gq and G12 Families of Heterotrimeric G Proteins Report Functional Selectivity

Zhang, L., Brass, L. F., Manning, D. R. — 2008-10-24

Receptors coupled to the G_q and G₁₂ families of heterotrimeric G proteins have surfaced rarely in the context of functional selectivity, and always indirectly. We explore here the differential engagement of G_q and G₁₃ by the thromboxane A₂ receptor TP, via agonist-effected [³⁵S]GTPS binding when the G proteins themselves are used as reporters. We find for TP introduced into human embryonic kidney (HEK) 293 cells and for the receptor expressed normally in human platelets an agonist-selective engagement of G_q versus G₁₃. Pinane thromboxane A₂ (PTA₂) activates G_q in preference to G₁₃, while 8-iso-prostaglandin F₂ (8-iso-PGF₂) activates G₁₃ in preference to G_q. U46619, in contrast, exhibits no preference. Reserve of receptor in relation to G protein, and of G protein in relation to downstream events, is apparent in some instances, but does not have a bearing on selectivity. Activation of G proteins by PTA₂ is right-shifted from binding of the ligand to receptor, a manifestation of which is a bimodal action: PTA₂ is an antagonist at low concentrations and an agonist at higher concentrations. We posit two populations of TP, or two intrinsic sites of ligand binding, and selectivity evident not only in terms of the G proteins activated but properties of antagonism versus agonism.

[Article] Phenylephrine-induced cardiomyocyte injury is triggered by superoxide generation through uncoupled eNOS and ameliorated by DY9836, a novel calmodulin antagonist

2008-10-24

The pathophysiological relevance of eNOS-induced superoxide production in cardiomyocyte injury following prolonged PE exposure remains unclear. The aims of this study were to define the mechanism of O₂^.- production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist, DY-9836, {3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole}, to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96h to PE led to translocation from membrane to cytosol of eNOS and breakdown of caveolin-3 and dystrophin. When NO and O₂^.- production were monitored in PE-treated cells by DAF-FM and DHE, respectively, Ca²⁺-induced NO production elevated by 5.7-fold (p<0.01) after 48 h PE treatment and the basal NO concentration markedly elevated (16-fold; p<0.01) after 96 h PE treatment. On the other hand the O₂^.- generation at 96 h was closely associated with an increased uncoupled eNOS level. Co-incubation with DY-9836 (3 µM) during the last 48 h inhibited the aberrant O₂^.- generation near completely and NO production by 72% (P<0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin-3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TUNEL staining was also attenuated by DY-9836 treatment. These results suggest that O₂^.- generation by uncoupled eNOS likely triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O₂^.- and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

[Minireview] Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors

Mustafi, D., Palczewski, K. — 2008-10-22

Biological membranes are densely packed with membrane proteins that occupy about half of their volume. In almost all cases, membrane proteins in the native state lack the higher-order symmetry required for their direct study by diffraction methods. Despite many technical difficulties, numerous crystal structures of detergent solubilized membrane proteins have been determined that illustrate their internal organization. Among such proteins, class A G protein-coupled receptors (GPCRs) have become amenable to crystallization and high resolution x-day diffraction analyses. The derived structures of native and engineered receptors not only provide insights into their molecular arrangements, but also furnish a framework for designing and testing potential models of transformation from inactive to active receptor signaling states and for initiating rational drug design.

[Article] Inhibition of Arachidonic Acid and Iron-Induced Mitochondrial Dysfunction and Apoptosis by Oltipraz and Novel 1,2-Dithiole-3-thione Congeners

Shin, S. M., Kim, S. G. — 2008-10-22

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz), a prototype drug candidate containing a 1,2-dithiole-3-thione moiety, has been widely studied as a cancer chemopreventive agent. Oltipraz and other novel 1,2-dithiole-3-thione congeners have the capability to prevent insulin resistance via AMP-activated protein kinase (AMPK) activation. Arachidonic acid (AA, a proinflammatory fatty acid) exerts a deleterious effect on mitochondria and promotes reactive oxygen species (ROS) production. This study investigated whether AA alone or in combination with iron (catalyst of auto-oxidation) causes ROS-mediated mitochondrial impairment, and if so, whether oltipraz and synthetic 1,2-dithiole-3-thiones protect mitochondria and cells against excess ROS produced by AA+iron. Oltipraz treatment effectively inhibited mitochondrial permeability transition promoted by AA+iron in HepG2 cells, thereby protecting cells from ROS-induced apoptosis. Oltipraz was found to attenuate apoptosis induced by rotenone (complex I inhibitor), but not that by antimycin A (complex III inhibitor), suggesting that the inhibition of AA-induced apoptosis by oltipraz might be associated with the electron transport system. AMPK activation by oltipraz contributed to cell survival, which was supported by the reversal of oltipraz's restoration of mitochondrial membrane potential by concomitant treatment of compound C. By the same token, an AMPK activator inhibited AA+iron-induced mitochondrial permeability transition with an increase in cell viability. Moreover, new 1,2-dithiole-3-thiones with the capability of AMPK activation protected cells from mitochondrial permeability transition and ROS overproduction induced by AA+iron. Our results demonstrate that oltipraz and new 1,2-dithiole-3-thiones are capable of protecting cells from AA+iron-induced ROS production and mitochondrial dysfunction, which may be associated with AMPK activation.

[Article] Inhibition of P-Glycoprotein-Mediated Taxol Resistance by Reversibly-Linked Quinine Homodimers

Pires, M. M, Emmert, D., Hrycyna, C. A, Chmielewski, J. — 2008-10-22

P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers due to its over-expression. Here rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC₅₀ 1.7 µM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and Bodipy-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t_1/2 20 h), while demonstrating high resistance to non-enzymatic hydrolysis in cell culture media (t_1/2 21 d). Specific inhibition of [¹²⁵I]-IAAP binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. Additionally, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled taxol in MCF-7/DX1 cells and it completely reversed the P-gp mediated taxol-resistance phenotype.

[Article] Opposing Effects of Platelet-activating Factor and Lyso-Platelet-activating Factor on Neutrophil and Platelet Activation

2008-10-17

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored, but rapidly generated via enzymatic acetylation of the precursor, lysoPAF. The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of PKA. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF, and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.

[Article] Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1)

2008-10-16

GPR40 is a G protein-coupled receptor whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of ~3µM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate did not have any inhibitory effect. Furthermore, chemical ligands that are known to activate GPR40 competed for binding in a dose-dependent manner. All the chemical ligands that inhibited the binding promoted phosphorylation of extracellular-regulated kinase (ERK)-1/2 in HEK-293 cells which expressed GPR40, and [Ca²⁺]_i responses in mouse insulinoma (MIN6) cells which natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca²⁺]_i response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach will be of value in studying the pharmacology of GPR40.

[Article] INTRACELLULAR ACTIVATION AND DEACTIVATION OF TASIDOTIN,AN ANALOGUE OF DOLASTATIN 15: CORRELATION WITH CYTOTOXICITY

2008-10-16

Tasidotin, an oncolytic drug in Phase II clinical trials, is a peptide analogue of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl terminal alcohol of dolastatin 15 is replaced with tert-butylamine. As expected, from studies with cemadotin, [³H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [³H]P5 was not the end product of tasidotin metabolism. Large amounts of [³H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably impacted negatively by the extent of degradation of P5 to proline and, assumedly, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC₅₀ of tasidotin and a marked increase in intracellular [³H]tasidotin. BCPP also caused a 4-fold increase in the IC₅₀ of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC₅₀ obtained for P5 in the presence of BCPP.

[Article] Structural basis of NR2B-selective antagonist recognition by NMDA receptors

2008-10-15

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) endowed with unique pharmacological and functional properties. In particular, their high permeability to calcium ions confers on NMDARs a central role in triggering long term changes in synaptic strength. Under excitotoxic pathological conditions, as occurring during brain trauma, stroke or Parkinson's and Huntington's diseases, calcium influx through NMDAR channels can also lead to neuronal injury. This argues for the use of NMDAR antagonists as potential therapeutic agents. To date, the most promising NMDAR antagonists are ifenprodil and derivatives, compounds that act as non-competitive inhibitors selective for NMDARs containing the NR2B subunit. Recent studies have identified the large N-terminal domain (NTD) of NR2B as the region controlling ifenprodil sensitivity of NMDARs. We present here a detailed characterization of the ifenprodil binding site using both experimental and computational approaches. 3D homology modelling reveals that ifenprodil fits well in a closed cleft conformation of the NRB NTD; however, ifenprodil can adopt either one of two possible binding orientations of opposite direction. By studying the effects of cleft mutations, we show that only the orientation in which the phenyl moiety points deep towards the NTD hinge is functionally relevant. Moreover, based on our model, we identify novel NTD NR2B residues that are crucial for conferring ifenprodil sensitivity and provide functional evidence that these residues directly interact with the ifenprodil molecule. This work provides a general insight into the origin of the subunit-selectivity of NMDAR non-competitive antagonists and offer clues for the discovery of novel NR2B-selective antagonists.

[Article] Overlapping Binding Site for the Endogenous Agonist, Small Molecule Agonists and Ago-allosteric Modulators on the Ghrelin Receptor

2008-10-15

A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides - the 28 amino acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone secretagogue GHRP-6 - plus four non-peptide agonists - the original benzolactam L-692,429, the spiroindoline sulfonamide MK-677, and two novel oxindole derivatives SM-130686 and SM-157740. The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09 to Gln substitution in the extracellular segment of TM-III. Similarly, all six agonists were affected by substitutions of PheVI:16, ArgVI:20 and PheVI:23 on the opposing face of TM-VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the non-peptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small molecule agonists - including ago-allosteric modulators - and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor.

[Article] YC-1 stimulates the expression of gaseous monoxide-generating enzymes in vascular smooth muscle cells

Liu, X.-m., Peyton, K. J., Mendelev, N. N., Wang, H., Tulis, D. A., Durante, W. — 2008-10-15

The benzylindazole derivative 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-]quinozalin-1-one (ODQ), or by the protein kinase G inhibitors, KT 5823 and DT-2, and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator BAY 41-2272. However, theYC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors, wortmannin and LY294002. In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor, KT 5720, and mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides while simultaneously stimulating their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified.

[Article] Fibroblast Growth Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-Small Cell Lung Cancer Cells

2008-10-10

Despite widespread expression of EGF receptors (EGFRs) and EGF family ligands in non-small cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent co-expression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. Notably, FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently co-expressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and co-express FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI (RO4383596) that targets FGFRs inhibited basal FRS2 and ERK phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that co-express FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1 or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

[Article] Amitriptyline Activates Cardiac Ryanodine Channels And Causes Spontaneous Sarcoplasmic Reticulum Calcium Release

Chopra, N., Laver, D., Davies, S. S, Knollmann, B. C — 2008-10-09

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's pro-arrhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2) causing premature Ca²⁺ release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured AMT’s effects on RyR2 channels from mice and sheep and on intact mouse cardiomyocytes loaded with the Ca²⁺ fluorescent indicator Fura-2AM. AMT induced trains of long channel openings (bursts) with 60-90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT]-, voltage- and P_o-dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT activated also RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P_o by increasing cytoplasmic [Mg²⁺] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca²⁺ releases and decreased the SR Ca²⁺ content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 µmol/l) compared to its effect in lipid bilayers (5-10 µmol/l). Increasing extracellular [Mg²⁺] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca²⁺ leak may contribute to AMT's pro-arrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.

[Article] Copper regulation of hypoxia-inducible factor -1 activity

Feng, W., Ye, F., Xue, W., Zhou, Z., Kang, Y. J. — 2008-10-08

Previous studies have demonstrated that copper (Cu) up-regulates hypoxia-inducible factor 1 (HIF-1). The present study was undertaken to test the hypothesis that Cu is required for HIF-1 activation. Treatment of HepG2 cells with a Cu chelator tetraethylenepentamine (TEPA) or siRNA targeting Cu chaperone for superoxide dismutase 1 (CCS) suppressed hypoxia-induced activation of HIF-1. Addition of excess Cu to relieved the suppression by TEPA, but not that by CCS gene silencing, indicating the requirement of Cu for activation of HIF-1, which is CCS-dependent. Cu deprivation did not affect production or stability of HIF-1, but reduced HIF-1 binding to the hypoxia-responsive element (HRE) of target genes and to p300, a component of HIF-1 transcriptional complex. Cu likely inhibits factor inhibiting HIF-1 (FIH-1) to ensure the formation of HIF-1 transcriptional complex. This study thus defines that Cu is required for HIF-1 activation through regulation of HIF-1 binding to the HRE and the formation of HIF-1 transcriptional complex.

[Article] Site Selectivity of Competitive Antagonists for the Mouse Adult Muscle Nicotinic Acetylcholine Receptor

Liu, M., Dilger, J. P. — 2008-10-08

The muscle-type nicotinic acetylcholine receptor has two non-identical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site-selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. Previously, we reported the IC₅₀ of the antagonists individually. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing ≥67% receptor inhibition. Metocurine shifted the apparent IC₅₀ of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC₅₀ of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC₅₀ of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L and L. We found L/L = 0.22 (range 0.14-0.34), 20 (9-29), 21 (4-36) and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium and rocuronium respectively. The wide range of L/L for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium) or may indicate that the binding of an antagonist at one site affects the affinity of the second site.

[Article] Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric oxide synthase 2 expression in astrocytes by a novel diindolylmethane analog protects striatal neurons against apoptosis

Carbone, D. L., Popichak, K. A., Moreno, J. A., Safe, S., Tjalkens, R. B. — 2008-10-07

The progressive debilitation of motor functions in Parkinson's Disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Chronic inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-kappa B (NF-B) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuro-inflammatory phenotype has received considerable attention as a potential target for chemotherapy but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate efficacy of a novel anti-inflammatory diindolylmethane-class compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-B-dependent expression of inducible nitric oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), through a mechanism distinct from that described for the thiazoladinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-B with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor gamma (PPAR-) or the nuclear co-repressor, NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected co-cultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing activation of NF-B-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.

[Article] Hydrogen sulfide inhibits rotenone-induced apoptosis via preservation of mitochondrial function

Hu, L.-F., Lu, M., Wu, Z.-Y., Wong, P. T.-H., Bian, J.-S. — 2008-10-06

Hydrogen sulfide (H₂S) has been proposed as a novel neuromodulator, which plays critical roles in the central nervous system (CNS) affecting both neurons and glial cells. However, its relationship with neurodegenerative diseases is unexplored. The present study was undertaken to investigate the effects of H₂S on cell injury induced by rotenone, a commonly used toxin in establishing in vivo and in vitro Parkinson's disease (PD) models, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report here that sodium hydrosulfide (NaHS), an H₂S donor, concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death. NaHS also prevented rotenone-induced p38- and JNK- MAPK phosphorylation and rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (_m) dissipation, cytochrome c release, caspase-9/3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, 5-hydroxydecanoate (5-HD), a selective blocker of mitochondrial ATP-sensitive potassium (mitoK_ATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Thus, we demonstrated for the first time that H₂S inhibited rotenone-induced cell apoptosis via regulation of mitoK_ATP channel/ p38- & JNK-MAPK pathway. Our data suggest that H₂S may have potential therapeutic value for neurodegenerative diseases, such as PD. Key words: neuroprotection; cytochrome c; caspase-9/3; mitoK_ATP channel; p38/JNK MAPK

[Article] STI571-Induced Cell Edge Translocation of Kinase-Active and Kinase-Defective Abl: Requirements of Myristoylation and SH3 Domain

Fujita, A., Shishido, T., Yuan, Y., Inamoto, E., Narumiya, S., Watanabe, N. — 2008-10-03

STI571 is the first successful target-based drug with excellent potency against chronic myelogenous leukemia. Studies on this compound have illuminated potentials and problems of kinase inhibitors in the treatment of cancer. As found in crystal structures, STI571-bound abl is believed to form closed conformation with N-terminal regulatory domains. Here we present evidence of distinct STI571-induced modulation of abl functions using high-resolution live-cell imaging approaches. Within lamellipodia of fibroblast cells, STI571 was found to induce rapid translocation of abl to the lamellipodium tip. Quantitative analysis yielded 0.81 µM and 1.8 µM for EC₅₀ of STI571-induced cell edge translocation of abl-KD-GFP and wild-type abl-GFP, respectively. It also revealed adverse response of drug-resistant abl-T334I to STI571, suggesting that drug binding to abl-GFP triggers translocation. N-myristoylation and the SH3 domain were required for this translocation whereas disruption of intramolecular interactions of these motifs enhanced cell edge association of abl. An intact C-terminal last exon region in abl, but not its F-actin binding, was required for efficient cell edge translocation. Moreover, single-molecule observation revealed an STI571-induced rapid increase in slow diffusive species of abl in both the tip and the body region of lamellipodia. These results suggest that while activated abl translocates to the cell edge at its open state, STI571 can also bind and lock abl in the open and membrane-tethered conformation as long as the SH3 domain and the C-terminal region are intact. High resolution imaging can be a powerful tool for elucidating inhibitor modulation of abl functions under intracellular environment.

[Article] Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor-{kappa}B Signaling Pathway in Human Hepatocellular Carcinoma Cells

Liang, J.-A., Wu, S.-L., Lo, H.-Y., Hsiang, C.-Y., Ho, T.-Y. — 2008-10-03

Vanillin has been reported to exhibit anti-invasive and anti-metastatic activities by suppressing the enzymatic activity of matrix metalloproteinase-9 (MMP-9). However, the underlying mechanism of anti-invasive activity remains unclear so far. Herein we demonstrated that vanillin reduced 12-O-tetradecanoylphorbol-13-acetate-induced MMP-9 gelatinolytic activity and suppressed cell invasion through the down-regulation of MMP-9 gene transcription in HepG2 cells. Vanillin significantly reduced the 6.6-fold invasive capacity of HepG2 cells in non-cytotoxic concentrations and this anti-invasive effect was concentration dependent in the Matrigel invasion assay. Moreover, vanillin significantly suppressed the TPA-induced enzymatic activity of MMP-9 and decreased the induced mRNA level of MMP-9. Analysis on the transcriptional regulation indicated that vanillin suppressed MMP-9 transcription by inhibiting nuclear factor-B (NF-B) activity. Western blot further confirmed that vanillin inhibited NF-B activity through the inhibition of I-B- phosphorylation and degradation. In conclusion, vanillin might be a potent anti-invasive agent that suppressed the MMP-9 enzymatic activity via NF-B signaling pathway.

[Article] Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11)

Hayashi, H., Sugiyama, Y. — 2008-10-01

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously demonstrated that 4-phenylbutyrate (4PBA) treatment, an FDA-approved drug for urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. Conversely, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surface-resident BSEP. Accordingly, to help the development of the medical treatment of cholestasis, the present study investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on short-chain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular weight of the mature form of BSEP/Bsep shifted from 170 kDa to 190 kDa following ubiquitin modification (molecular weight: 8 kDa). Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations, E297G and D482G. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surface-resident BSEP by 4PBA treatment and PFIC2-type mutations.

[Article] THIOPHENECARBOXYLATE SUPPRESSOR OF CYCLIC NUCLEOTIDES DISCOVERED IN A SMALL-MOLECULE SCREEN BLOCKS TOXIN-INDUCED INTESTINAL FLUID SECRETION

Tradtrantip, L., Yangthara, B., Padmawar, P., Morrison, C., Verkman, A. S. — 2008-09-29

We carried out a 'pathway' screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein coupled receptors, adenylyl cyclase and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had IC₅₀ of <5 µM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as non-selective phosphodiesterase activators, though it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl- current in T84 colonic cells in response to cholera and E. coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease and growth inhibition of cAMP-dependent tumors.