Transient Receptor Potential Melastatin 7 (TRPM7) Ion Channel Inhibitors: Preliminary SAR and Conformational Studies of Xenicane Diterpenoids from the Hawaiian Soft Coral Sarcothelia edmondsoni.

Waixenicin A, a xenicane diterpene from the octocoral Sarcothelia edmondsoni, is a selective, potent inhibitor of the TRPM7 ion channel. To study the structure-activity relationship (SAR) of waixenicin A, we isolated and assayed related diterpenes from S. edmondsoni. In addition to known waixenicins A (1) and B (2), we purified six xenicane diterpenes, 7S,8S-epoxywaixenicins A (3) and B (4), 12-deacetylwaixenicin A (5), waixenicin E (6), waixenicin F (7), and 20-acetoxyxeniafaraunol B (8). We elucidated the structures of 3-8 by NMR and MS analyses. Compounds 1, 2, 3, 4, and 6 inhibited TRPM7 activity in a cell-based assay, while 5, 7, and 8 were inactive. A preliminary SAR emerged showing that alterations to the nine-membered ring of 1 did not reduce activity, while the 12-acetoxy group, in combination with the dihydropyran, appears to be necessary for TRPM7 inhibition. The bioactive compounds are proposed to be latent electrophiles by formation of a conjugated oxocarbenium ion intermediate. Whole-cell patch-clamp experiments demonstrated that waixenicin A inhibition is irreversible, consistent with a covalent inhibitor, and showed nanomolar potency for waixenicin B (2). Conformational analysis (DFT) of 1, 3, 7, and 8 revealed insights into the conformation of waixenicin A and congeners and provided information regarding the stabilization of the proposed pharmacophore.

Waixenicin A inhibits cell proliferation through magnesium-dependent block of transient receptor potential melastatin 7 (TRPM7) channels.

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).

Halogenated fatty acid amides and cyclic depsipeptides from an eastern Caribbean collection of the cyanobacterium Lyngbya majuscula.

A lipophilic extract of an eastern Caribbean collection of Lyngbya majuscula yielded two new halogenated fatty acid amides, grenadamides B (1) and C (2), and two new depsipeptides, itralamides A (3) and B (4), along with the known compounds hectochlorin and deacetylhectochlorin. The recently reported depsipeptide carriebowmide (5) was also present in the extract and isolated as its sulfone artifact (6). Compounds 1-4 were identified by spectroscopic methods. The configurations of the amino acid residues of 3, 4, and 6 were determined by LC-MS analyses of diastereomeric derivatives of the acid hydrolysates (advanced Marfey's method). Based on the configurational analysis of 6, in direct comparison with authentic carriebowmide (5), a minor structural revision of 5 is proposed. Compounds 1 and 2 displayed marginal activity against the beet armyworm (Spodoptera exigua). Compounds 1-4 and 6 were assessed for general cell toxicity in human embryonic kidney (HEK293) cells. Only itralamide B (4) displayed significant cytotoxicity, showing an IC(50) value of 6 +/- 1 muM.

The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions.

Overall, the inflammatory potential of lipopolysaccharide (LPS) in vitro and in vivo was investigated using different omics technologies. We investigated the hippocampal response to intracerebroventricular (i.c.v) LPS in vivo, at both the transcriptional and protein level. Here, a time course analysis of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) showed a sharp peak at 4 h and a return to baseline at 16 h. The expression of inflammatory mediators was not temporally correlated with expression of the microglia marker F4/80, which did not peak until 2 days after LPS injection. Of 480 inflammation-related genes present on a microarray, 29 transcripts were robustly up-regulated and 90% of them were also detected in LPS stimulated primary microglia (PM) cultures. Further in vitro to in vivo comparison showed that the counter regulation response observed in vivo was less evident in vitro, as transcript levels in PM decreased relatively little over 16 h. This apparent deficiency of homeostatic control of the innate immune response in cultures may also explain why a group of genes comprising tnf receptor associated factor-1, endothelin-1 and schlafen-1 were regulated strongly in vitro, but not in vivo. When the overall LPS-induced transcriptional response of PM was examined on a large Affymetrix chip, chemokines and cytokines constituted the most strongly regulated and largest groups. Interesting new microglia markers included interferon-induced protein with tetratricopeptide repeat (ifit), immune responsive gene-1 (irg-1) and thymidylate kinase family LPS-inducible member (tyki). The regulation of the former two was confirmed on the protein level in a proteomics study. Furthermore, conspicuous regulation of several gene clusters was identified, for instance that of genes pertaining to the extra-cellular matrix and enzymatic regulation thereof. Although most inflammatory genes induced in vitro were transferable to our in vivo model, the observed discrepancy for some genes potentially represents regulatory factors present in the central nervous system (CNS) but not in vitro.

R-citalopram functionally antagonises escitalopram in vivo and in vitro: evidence for kinetic interaction at the serotonin transporter.

1. Clinical observations with the selective serotonin reuptake inhibitor (SSRI), S-citalopram, indicate that S-citalopram is more efficacious and produces earlier symptom relief than RS-citalopram. Since R-citalopram is at least 20-fold weaker than S-citalopram as inhibitor of the 5-HT transporter (SERT) in preclinical studies, the clinical data suggest an unexpected antagonistic interaction between the two enantiomers. We therefore characterised the interaction of R- and S-citalopram with the SERT in in vivo and in vitro assays. 2. In both behavioural (potentiation of 5-hydroxytryptophan (5-HTP)-induced behaviour) and electrophysiological studies (inhibition of 5-HT-elicited ion currents in Xenopus oocytes expressing the human SERT (hSERT) R-citalopram inhibited the effects of S-citalopram in a dose-dependent manner. With S-citalopram : R-citalopram ratios of 1 : 2 and 1 : 4, 5-HTP potentiation was significantly smaller than with S-citalopram alone. 3. R-citalopram did not antagonise the effects of another SSRI (fluoxetine) in either behavioural or electrophysiological studies. 4. In oocytes, inhibition of hSERT-mediated currents by R-citalopram was almost completely reversible and characterised by fast on- and off-sets of action. In contrast, the off-set for S-citalopram was 35-fold slower than for R-citalopram. 5. Kinetic analysis of the oocyte experiments suggests that S-citalopram binding to SERT induces a long-lasting, inhibited state of the transporter and that coapplication of R-citalopram partially relieves SERT of this persistent inhibition. 6. We propose that the kinetic interaction of R- and S-citalopram with SERT is a critical factor contributing to the antagonistic effects of R-citalopram on S-citalopram in vitro and in vivo.

Development and optimization of a high-throughput bioassay for TRPM7 ion channel inhibitors.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z' factors > or =0.5).

The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation.

The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies.

Isolation and structure determination of malevamide E, a dolastatin 14 analogue, from the marine cyanobacterium Symploca laete-viridis.

A new depsipeptide, malevamide E (1), was isolated from field-collected colonies of the filamentous cyanobacterium Symploca laete-viridis. The gross structure of 1 was determined by spectroscopic analyses, including one- and two-dimensional NMR and accurately measured MS/MS. Chiral HPLC analyses of an acid hydrolysate of 1 allowed the stereochemical assignments of its amino acid residues, which include N-methyl-L-alanine, alpha-N,gamma-N-dimethyl-L-asparagine, N-methyl-L-phenylalanine, L-proline, D-valine, and N-methyl-L-valine. LC-MS/MS analysis of S. laete-viridis fractions established the co-occurrence of malevamide E (1) and its homologue dolastatin 14 (2), which was previously reported in low yield from the sea hare Dolabella auricularia. Malevamide E (1) demonstrated a dose-dependent (2-45 microM) inhibition of store-operated Ca(2+) entry in thapsigargin-treated human embryonic kidney (HEK) cells, indicating an inhibitory effect on Ca(2+) release-activated Ca(2+) (CRAC) channels.

The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function.

Upon injury, astrocytes assume an activated state associated with the release of inflammatory mediators. To model this, we stimulated murine primary astrocytes with a complete inflammatory cytokine mix consisting of TNF-alpha, IL-1beta and IFN-gamma. We analysed the transcriptional response of 480 genes at 4 and 16 h after stimulation on a chip designed to give a representative overview over the inflammation-relevant part of the transcriptome of macrophage-like cells. The list of the 182 genes found to be significantly regulated in astrocytes revealed an intriguing co-ordinate regulation of genes linked to the biological processes of antiviral/antimicrobial defence, antigen presentation and facilitation of leucocyte invasion. The latter group was characterized by very high up-regulations of chemokine genes. We also identified regulations of a thymidylate kinase and an interferon-regulated protein with a tetratricopeptide motive, both up to now only known from macrophages. The transcriptional regulations were confirmed on the protein level by a proteomic analysis. These findings taken together suggest that activated astrocytes in brain behave similarly in many respects to inflamed macrophages in the periphery.

Inhibition of microglial inflammation by the MLK inhibitor CEP-1347.

CEP-1347 is a potent inhibitor of the mixed lineage kinases (MLKs), a distinct family of mitogen-activated protein kinase kinase kinases (MAPKKK). It blocks the activation of the c-Jun/JNK apoptotic pathway in neurons exposed to various stressors and attenuates neurodegeneration in animal models of Parkinson's disease (PD). Microglial activation may involve kinase pathways controlled by MLKs and might contribute to the pathology of neurodegenerative diseases. Therefore, the possibility that CEP-1347 modulates the microglial inflammatory response [tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1)] was explored. Indeed, the MLK inhibitor CEP-1347 reduced cytokine production in primary cultures of human and murine microglia, and in monocyte/macrophage-derived cell lines, stimulated with various endotoxins or the plaque forming peptide Abeta1-40. Moreover, CEP-1347 inhibited brain TNF production induced by intracerebroventricular injection of lipopolysaccharide in mice. As expected from a MLK inhibitor, CEP-1347 acted upstream of p38 and c-Jun activation in microglia by dampening the activity of both pathways. These data imply MLKs as important, yet unrecognized, modulators of microglial inflammation, and demonstrate a novel anti-inflammatory potential of CEP-1347.

Specific modulation of astrocyte inflammation by inhibition of mixed lineage kinases with CEP-1347.

Inflammatory conversion of murine astrocytes correlates with the activation of various MAPK, and inhibition of terminal MAPKs like JNK or p38 dampens the inflammatory reaction. Mixed lineage kinases (MLKs), a family of MAPK kinase kinases, may therefore be involved in astrocyte inflammation. In this study, we explored the effect of the MLK inhibitors CEP-1347 and CEP-11004 on the activation of murine astrocytes by either TNF plus IL-1 or by a complete cytokine mix containing additional IFN-gamma. The compounds blocked NO-, PG-, and IL-6 release with a median inhibitory concentration of approximately 100 nM. This activity correlated with a block of the JNK and the p38 pathways activated in complete cytokine mix-treated astrocytes. Although CEP-1347 did not affect the activation of NF-kappaB, it blocked the expression of cyclooxygenase-2 and inducible NO synthase at the transcriptional level. Quantitative transcript profiling of 17 inflammation-linked genes revealed a specific modulation pattern of astrocyte activation by MLK inhibition, for instance, characterized by up-regulation of the anti-stress factors inhibitor of apoptosis protein-2 and activated transcription factor 4, no effect on manganese superoxide dismutase and caspase-11, and down-regulation of major inflammatory players like TNF, GM-CSF, urokinase-type plasminogen activator, and IL-6. In conclusion, MLK inhibitors like CEP-1347 are highly potent astrocyte immune modulators with a novel spectrum of activity.