Search

Significance Subtype-selective modulation of ion channels is often important, but extremely difficult to achieve for drug development. Using Nav1.7 as an example, we show that this challenge could be attributed to poor design in ion channel assays, which fail to detect most potent and selective compounds and are biased toward nonselective mechanisms. By exploiting different drug binding sites and modes of channel gating, we successfully direct a membrane potential assay toward non–pore-blocking mechanisms and identify Nav1.7-selective compounds. Our mechanistic approach to assay design addresses a significant hurdle in Nav1.7 drug discovery and is applicable to many other ion channels., Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B 2 receptor. In this study, we have examined the relationship between B 2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg 9 removal from BK and its analogs on E 2 receptor binding. The ratios of binding constants ( K i +Arg K i −Arg ) for the Arg 9 containing compounds and the corresponding des-Arg 9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg 9 -BK and Lys°-BK/des-Arg 9 -Lys°-BK.

A new basic antigen with a molecular weight of 36.5 kDa and a pI of 8.65 was purified from short ragweed pollen using ammonium sulfate precipitation, Q-sepharose chromatography and S-sepharose chromatography. The purified protein, herein referred to as Ambrosia artemisiifolia basic antigen (AaBA), migrated as a single band in SDS-polyacrylamide gel electrophoresis and isoelectric focusing, and as a single peak on reverse phase HPLC. AaBA reacted with sera from humans allergic to ragweed pollen including pooled NIH serum. AaBA and two major acidic allergens, Amb a I (antigen E) and Amb a II (antigen K), displayed immunological cross-reactivity inasmuch as they competed significantly with each other in a competition ELISA. This suggests that at least some major epitopes in all three antigens are similar; however, other possibilities like adjacent location of different epitopes cannot be ruled out at this time. Molecular weight, amino acid composition and sequence data suggest that AaBA is very similar to Amb a II whereas it is significantly different from other basic antigens purified from ragweed pollen.

There is strong evidence from studies in humans and animal models to suggest the involvement of energy metabolism defects in neurodegenerative diseases. Uridine, a pyrimidine nucleoside, has been suggested to be neuroprotective in neurological disorders by improving bioenergetic effects, increasing ATP levels and enhancing glycolytic energy production. We assessed whether uridine treatment extended survival and improved the behavioral and neuropathological phenotype observed in G93A-ALS mice. In vitro and in vivo pharmacokinetic analyses in mutant SOD models provided optimal dose and assurance that uridine entered the brain. A dose-ranging efficacy trial in G93A mice was performed using survival, body weight, open-field analysis, and neuropathology as outcome measures. Urinary levels of 8-hydroxy-2'-deoxyguanosine, identifying DNA oxidative damage, were measured and used as a pharmacodynamic biomarker. Uridine administration significantly extended survival in a dose-dependent manner in G93A mice, while improving the behavioral and neuropathological phenotype. Uridine increased survival by 17.4%, ameliorated body weight loss, enhanced motor performance, reduced gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Consistent with a therapeutic effect, uridine significantly reduced urinary 8-hydroxy-2'-deoxyguanosine in G93A mice. These data suggest that uridine may be a therapeutic candidate in ALS patients.

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.

Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.