Validation and optimization of novel high-throughput assays for human epithelial sodium channels.

The epithelial sodium channel (ENaC) plays a crucial role in salt and water homeostasis and is primarily involved in sodium reabsorption in the kidney and lung. Modulators of ENaC function, particularly within lung epithelia, could offer potential treatments for a number of diseases. As a constitutively active sodium channel, ENaC expression at the cell membrane is highly regulated through rapid turnover. This short half-life of the channel at the membrane and cytotoxicity from overexpression pose a problem for reagent generation and assay development in drug discovery. We have generated an HEK293 stable cell line expressing ENaC ß and γ subunits containing the PY motif trafficking mutations found in Liddle's syndrome to overcome rapid channel turnover at the membrane. A BacMam virus was used to transiently express the ENaC α subunit to reconstitute channel function to reduce the toxicity associated with long-term overexpression. We have configured a 384-well FLIPR membrane potential antagonist assay for high-throughput screening and an IonWorks Quattro electrophysiology antagonist assay that is predictive of potency values derived from primary lung epithelial cell short-circuit measurements. The triage strategy for compound screening and profiling against this target using these assays has resulted in the discovery of novel chemotypes.

Novel amino-piperidines as potent antibacterials targeting bacterial type IIA topoisomerases.

We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.

Novel cyclohexyl-amides as potent antibacterials targeting bacterial type IIA topoisomerases.

As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties.

Development of co-selected single nucleotide polymorphisms in the viral promoter precedes the onset of human immunodeficiency virus type 1-associated neurocognitive impairment.

The long terminal repeat (LTR) regulates gene expression of HIV-1 by interacting with multiple host and viral factors. Cross-sectional studies in the pre-HAART era demonstrated that single nucleotide polymorphisms (SNPs) in peripheral blood-derived LTRs (a C-to-T change at position 3 of C/EBP site I (3T) and at position 5 of Sp site III (5T)) increased in frequency as disease severity increased. Additionally, the 3T variant correlated with HIV-1-associated dementia. LTR sequences derived by longitudinal sampling of peripheral blood from a single patient in the DrexelMed HIV/AIDS Genetic Analysis Cohort resulted in the detection of the 3T and 5T co-selected SNPs before the onset of neurologic impairment, demonstrating that these SNPs may be useful in predicting HIV-associated neurological complications. The relative fitness of the LTRs containing the 3T and/or 5T co-selected SNPs as they evolve in their native patient-derived LTR backbone structure demonstrated a spectrum of basal and Tat-mediated transcriptional activities using the IIIB-derived Tat and colinear Tat derived from the same molecular clone containing the 3T/5T LTR SNP. In silico predictions utilizing colinear envelope sequence suggested that the patient's virus evolved from an X4 to an R5 swarm prior to the development of neurological complications and more advanced HIV disease. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of specific polymorphisms in the LTR, env, and/or tat that could predict the onset of neurological disease and result in alterations in viral function.

Controlled steroid delivery via bioabsorbable stent: safety and performance in a rabbit model.

BACKGROUND: Middle turbinate lateralization, adhesions, and inflammation are causes of suboptimal sinus patency following surgery. A bioabsorbable drug-eluting stent has been developed to maintain sinus patency while providing controlled steroid delivery to the sinus mucosa. The aim of this study was to characterize the in vivo drug delivery efficacy and tolerance of this stent in a rabbit model. METHODS: Bioabsorbable stents coated with mometasone furoate were placed bilaterally in the maxillary sinuses of 31 rabbits via dorsal maxillary sinusotomy. Animals were sacrificed between 5 days and 18 weeks postoperatively. Efficacy was assessed by measuring tissue concentrations of steroid in maxillary sinus and nasal mucosa and by measurement of plasma steroid concentrations. Tolerance was assessed by histological evaluation of the sinus mucosa at different time points. RESULTS: Therapeutic mucosal drug concentrations were attained in a time-dependent fashion (range 175-28,189 ng/g). Plasma drug concentrations were generally near or below the lower limit of quantification (15 pg/mL). Histopathological examination of the mucosa showed no differences in the reaction to steroid-coated stents versus nondrug-coated control stents, with inflammation, epithelial ulceration, and bony reaction ranging from none to mild at all time points. Microscopic fungal hyphae were noted in a small proportion of both treatment and control sinuses, without evidence of associated adverse tissue reaction. CONCLUSIONS: In a rabbit model, mometasone-coated bioabsorbable stents are able to provide local steroid delivery with negligible systemic absorption. Corticosteroid-eluting stents may prove useful following endoscopic sinus surgery in maintaining sinus patency and reducing inflammation.

Use of planar array electrophysiology for the development of robust ion channel cell lines.

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Determinants of the anesthetic sensitivity of neuronal nicotinic acetylcholine receptors.

Some neurotransmitter-gated ion channels are very much more sensitive to general anesthetics than others, even when they are genetically and structurally related. The most striking example of this is the extreme sensitivity of heteromeric neuronal nicotinic acetylcholine receptors to inhalational general anesthetics compared with the marked insensitivity of the closely related homomeric neuronal nicotinic receptors. Here we investigate the role of the alpha subunit in determining the anesthetic sensitivity of these receptors by using alpha(3)/alpha(7) chimeric subunits that are able to form functional homomeric receptors. By comparing the sensitivities of a number of chimeras to the inhalational agent halothane we show that the short (13 amino acids) putative extracellular loop connecting the second and third transmembrane segments is a critical determinant of anesthetic sensitivity. In addition, using site-directed mutagenesis, we show that two particular amino acids in this loop play a dominant role. When mutations are made in this loop, there is a good correlation between increasing anesthetic sensitivity and decreasing acetylcholine sensitivity. We conclude that this extracellular loop probably does not participate directly in anesthetic binding, but rather determines receptor sensitivity indirectly by playing a critical role in transducing anesthetic binding into an effect on channel gating.