The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1–0.4 g at 30 Hz for 10–60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM‐induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM‐induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse‐induced bone loss. J. Cell. Biochem. 106: 306–316, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of vibriocidal compounds from medicinal plants using chromatographic fingerprinting

The aim of the present study was to investigate the vibriocidal activity of bark of Syzygium cumini, leaves of Lawsonia inermis, fruits of Terminalia bellerica and identify the bioactive compounds. The vibriocidal activity of plant extracts was determined in aqueous and organic solvents, and the minimum inhibitory concentration (MIC) against Vibrio spp. using the disk diffusion method was established. The chemical constituents of the plant extracts were analysed by thin layer chromatography (TLC), the vibriocidal compounds were determined by TLC-bioautography and were further confirmed by high performance liquid chromatography (HPLC). Significant inhibitory activity was observed with ethanol extract of plants against the test bacteria while less antibacterial activity was observed in acetone, methanol and aqueous extracts. The MIC of the plant extracts ranged between 2.5 and 20 mg/ml. The TLC, TLC-bioautography and HPLC analysis showed that gallic acid and tannin present in ethanol extracts of S. cumini, tannin present in L. inermis and gallic acid present in T. bellerica may be responsible for the vibriocidal activity. S. cumini, L. inermis and T. bellerica can be used for the treatment of gastroenteritis, diarrhoea and cholera diseases after detailed investigations. We also conclude that the plants rich in gallic acid and tannin can be used as an alternative to search for new vibriocidal drugs.     

Type II NADH dehydrogenase of the respiratory chain of Plasmodium falciparum and its inhibitors

Plasmodium falciparum NDH2 (pfNDH2) is a non-proton pumping, rotenone-insensitive alternative enzyme to the multi-subunit NADH:ubiquinone oxidoreductases (Complex I) of many other eukaryotes. Recombinantly expressed pfNDH2 prefers coenzyme CoQ₀ as an acceptor substrate, and can also use the artificial electron acceptors, menadione and dichlorophenol–indophenol (DCIP). Previously characterized NDH2 inhibitors, dibenziodolium chloride (DPI), diphenyliodonium chloride (IDP), and 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) do not inhibit pfNDH2 activity. Here, we provide evidence that HDQ likely targets another P. falciparum mitochondrial enzyme, dihydroorotate dehydrogenase (pfDHOD), which is essential for de novo pyrimidine biosynthesis.     

Discovery of α-amidosulfones as potent and selective agonists of CB2: Synthesis,SAR, and pharmacokinetic properties

A series of α-amidosulfones were found to be potent and selective agonists of CB₂. The discovery, synthesis, and structure–activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.     

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.Hepatitis C virus (HCV) is a highly variable enveloped virus. It is the sole member of the genus Hepacivirus within the family Flaviviridae (36). Based on sequence homology, patient isolates are classified into seven genotypes and more than 100 subtypes (17, 52).The genome of HCV is a single-stranded RNA molecule of positive polarity with a size of ∼9.6 kb. It encodes a polyprotein of ca. 3,000 amino acids and contains nontranslated regions (NTRs) at both the 5′ and 3′ termini that are required for translation and RNA replication (33). Cellular and two viral proteases, NS2-3 and NS3-4A, liberate the individual viral proteins. The N-terminal portion of the polyprotein contains the structural proteins core and envelope glycoproteins 1 and 2 (E1, E2), which constitute the virus particle. These proteins are cleaved from the polyprotein by the host cell signal peptidase (18, 24). In the case of the core protein, an additional cleavage step mediated by the signal peptide peptidase liberates its mature C terminus (41). Further downstream of the structural proteins the polyprotein harbors p7, a short membrane-associated polypeptide required for virus assembly and release (27, 55), and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Proteins NS3 to NS5B are the minimal components of the membrane-bound replication complexes that catalyze RNA replication (16, 38).Using the novel JFH1-based HCV infection model (35, 61, 65), it has been demonstrated recently that besides the canonical structural proteins core, E1, and E2, NS5A, p7, NS3, and NS2 also are crucial for the production of infectious HCV particles (1, 26, 27, 39, 40, 55, 57). These data highlight that HCV assembly and release is a coordinated process involving both structural and nonstructural proteins. However, how the aforementioned proteins contribute to the production of infectious virus particles remains poorly understood.HCV p7 comprises two helical domains connected by a polar loop. Studies with epitope-tagged p7 variants indicate that both termini of the protein are resident in the lumen of the endoplasmic reticulum (ER) (4) or that, in addition, a second alternative topology with the C terminus exposed to the cytoplasm can be adopted (25). Using such constructs for fluorescent microscopy, a complex localization of p7 was revealed. While most prominent staining generally was observed at the ER (4, 19, 23), pools of p7 also were detected at mitochondria (19) and at the plasma membrane (4). These data suggest that p7 influences virus replication at various sites within infected cells, and that the function and/or localization of p7 is regulated by different trafficking signals that could be exposed in a topology-dependent manner. However, caution is warranted since, due to the lack of antibodies, epitope-tagged p7 variants had to be employed for most analyses, and since localization studies of virus-producing cells with functional p7 still are lacking.One hallmark of p7 is its ability to form cation-selective channels in artificial membranes (20, 46, 49), a property that likely depends on the oligomerization of the protein (7, 21). There are intriguing correlations that link p7''s function as an ion channel protein in vitro to its role in the assembly and release of infectious HCV particles in tissue culture. First, the mutation of the conserved dibasic motif in the polar loop of p7 abrogates ion channel activity and interferes with virus production in tissue culture (20, 27, 55). Second, iminosugars coupled to long alkyl chains like N-nonyl deoxygalactonojirimycin (NN-DGJ) not only interfere with ion channel activity but also repress the release of infectious particles from transfected Huh-7 cells (46, 56). Taken together, these data suggest that the ion channel activity of p7 is crucial for its role in the late steps of the HCV replication cycle, and that this function is amenable to the development of selective inhibitors for antiviral therapy. However, presently it is unknown how mechanistically p7, as an ion channel protein, facilitates HCV assembly and release or if p7 also is a component of virus particles and participates in entry.Besides its function as an ion channel, p7 harbors a signal-like sequence in its C-terminal domain that directs the insertion of the N terminus of NS2 into the lumen of the ER (4). Strikingly, due to structural determinants within the C terminus of E2, p7, and the N terminus of NS2, signalase cleavages at the E2/p7 and the p7/NS2 sites are incomplete, thus yielding E2-p7-NS2 and E2-p7 precursor proteins (3, 18, 34, 42). Although these precursors are not absolutely essential for the production of infectious HCV particles (26, 27), a defined ratio between mature and precursor proteins might play a role to orchestrate optimal virus assembly. Given these circumstances, genetic studies of p7 function are complicated, since mutations may, on the one hand, affect ion channel activity, and on the other hand influence processing at the E2-p7 and p7-NS2 junctions.To circumvent this problem, in this study we developed a complementation system that permits the rescue of genomes with defects in p7 by the ectopic expression of p7 in trans. This enabled us to directly assess the function of p7 in the absence of secondary effects caused by aberrant polyprotein cleavage. Using this approach, we analyzed the role of the native signal sequence of p7 and p7-containing precursor proteins. In addition, we investigated key determinants that are essential for the optimal function of p7 in the course of HCV infectious particle production.     

Arylphthalazines as potent,and orally bioavailable inhibitors of VEGFR-2

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.     

Design and synthesis of N6-substituted-4′-thioadenosine-5′-uronamides as potent and selective human A3 adenosine receptor agonists

On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA (N⁶-(3-Iodo-benzyl)-9-(5′-methylaminocarbonyl-β-d-ribofuranosyl)adenine), which is a potent and selective A₃ adenosine receptor (AR) agonist, were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (K_i = 0.25 nM) at the human A₃AR and was more potent than IB-MECA (K_i = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2-methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A₃AR binding, although small alkyl analogues were more potent.     

A combination of ultrahigh throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonists

The use of ultrahigh throughput screens (uHTS) is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter [Path Hunter technology is a registered trademark of DiscoveRx Corporation.] technology from DiscoveRx to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-μl total reaction volume assayed in a 3456-well plate format. This single addition, homogenous assay which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal to background ratio of approximately 4-fold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed agonism for the GR receptor in primary human monocytes and due to their performance in a physiologically relevant model they likely will have a better chance to evoke clinical efficacy.