Stephen C. Miller

Adverse Effects of Low Dose Amiodarone: A Meta-Analysis

OBJECTIVES We sought to assess the odds of experiencing adverse effects with low dose amiodarone therapy compared with placebo. BACKGROUND An estimate of the likelihood of experiencing amiodarone-related adverse effects with exposure to low daily doses of the drug is lacking in the published reports, and little information is available on adverse effect event rates in control groups not receiving the drug. METHODS Data from four published trials involving 1,465 patients were included in a meta-analysis design. The criteria for inclusion were 1) double-blind, placebo-controlled design; 2) absence of a crossover design between patient groups; 3) mean follow-up of at least 12 months; 4) maintenance amiodarone dose < or = 400 mg/day; and 5) presence of an explicit description of adverse effects. Data were pooled after testing for homogeneity of treatment effects across trials, and summary odds ratios were calculated by the Peto-modified Mantel-Haenszel method for each adverse effect. RESULTS The mean amiodarone dose per day ranged from 152 to 330 mg; 738 patients were randomized to receive amiodarone and 727 placebo. Exposure to amiodarone in this dose range, for a minimal duration of 12 months, resulted in odds similar to those of placebo for hepatic and gastrointestinal adverse effects, but in significantly higher odds than those of placebo (p < 0.05) for experiencing thyroid (odds ratio [OR] 4.2, 95% confidence interval [CI] 2.0 to 8.7), neurologic (OR 2.0, 95% CI 1.1 to 3.7), skin (OR 2.5, 95% CI 1.1 to 6.2), ocular (OR 3.4, 95% CI 1.2 to 9.6) and bradycardic (OR 2.2, 95% CI 1.1 to 4.3) adverse effects. A trend toward increased odds of pulmonary toxicity was noted (OR 2.0, 95% CI 0.9 to 5.3), but this did not reach statistical significance (p = 0.07). The unadjusted total incidence of drug discontinuation was 22.9% in the amiodarone group and 15.4% in the placebo group. The odds of discontinuing the drug in the amiodarone group was approximately 1.5 times that of the placebo group (OR 1.52, 95% CI 1.2 to 1.9) (p = 0.003). CONCLUSIONS Compared with placebo, there is a higher likelihood of experiencing several amiodarone-related adverse effects with exposure to low daily doses of the drug. Thus, although low dose amiodarone may be well tolerated, it is not free of adverse effects.

Robust Light Emission from Cyclic Alkylaminoluciferin Substrates for Firefly Luciferase

Firefly luciferase utilizes the chemical energy of ATP and oxygen to convert its substrate, D-luciferin, into an excited-state oxyluciferin molecule. Relaxation of this molecule to the ground state is responsible for the yellow-green light emission. Synthetic cyclic alkylaminoluciferins that allow robust red-shifted light emission with the modified luciferase Ultra-Glo are described. Overall light emission is higher than that of acyclic alkylaminoluciferins, aminoluciferin, and the native substrate D-luciferin.

A synthetic luciferin improves bioluminescence imaging in live mice

Firefly luciferase is the most widely used optical reporter for noninvasive bioluminescence imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI with existing luciferase reporters and enables imaging in the brain that could not be achieved with D-luciferin.

Beyond D-luciferin: expanding the scope of bioluminescence imaging in vivo

The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive imaging in live mice. However, photon emission from the luciferase is crucially dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of bioluminescence imaging.

Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over D-luciferin.

Firefly luciferase adenylates and oxidizes d-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. d-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of d-luciferin in live luciferase-expressing cells. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.

Profiling sulfonate ester stability: identification of complementary protecting groups for sulfonates

Sulfonation is prized for its ability to impart water-solubility to hydrophobic molecules such as dyes. This modification is usually performed as a final step, since sulfonated molecules are poorly soluble in most organic solvents, which complicates their synthesis and purification. This work compares the intrinsic lability of different sulfonate esters, identifying new sulfonate protecting groups and mild, selective cleavage conditions.

Labeling Tetracysteine‐Tagged Proteins with a SplAsH of Color: A Modular Approach to Bis‐Arsenical Fluorophores

Our understanding of protein localization and molecular interactions has been greatly enhanced through the use of fluorescent protein fusions. However, there are many situations in which the large size (27 kDa) of the fluorescent protein interferes with the physiological role of the protein under study. Furthermore, the spectral properties of fluorescent proteins have thus far been restricted to the visible range, and their modest photostability has limited their use in many applications, such as single-molecule studies. Recently, a number of protein-based tags have been described that recruit small-molecule fluorophores through noncovalent or covalent interactions, both in vitro and in living cells. Although these approaches have enabled the use of fluorophores with improved photophysical properties, these fusion proteins present a similarly large change in the size of the modified protein. On the other hand, strategies that utilize much smaller peptide tags—such as hexahistidine (His6) and tetracysteine (TC) motifs—have the potential to allow labeling with minimal perturbation of the protein itself. 2, 8] Of particular interest is that tetracysteine tags can be labeled intracellularly, but these have thus far been practically limited to hydroxylated xanthene or phenoxazine dyes (e.g. , FlAsH and ReAsH). Herein we describe a general strategy that allows the recruitment of any fluorophore to a tetracysteine tag. The simultaneous structural requirements for both fluorescence and the rigid display of arsenic atoms have sharply limited the range of fluorophores that can be targeted to tetracysteine tags. Although fluorescein and resorufin are compatible, their brightness, pH sensitivity, and propensity to photobleach are suboptimal. On the other hand, rhodamines—aminated xanthenes—are pH-insensitive, bright dyes with excellent photostability. However, bis-arsenical rhodamines have been reported to be nonfluorescent, even when bound to a tetracysteine tag. Thus, the scope of compatible dyes is both narrow and difficult to predict. In light of this, we have adopted a modular approach, wherein the bis-arsenical targeting moiety is separated from the fluorophore payload, thereby removing any restriction on its structure. We based our bis-arsenical targeting moiety on a fluorescein derivative, since fluorescein is an inexpensive starting material and the synthesis of FlAsH proceeds in higher yield than that of other bis-arsenicals. To ensure that the fluorescein-based targeting moiety would not interfere with the fluorescence properties of the attached dye, we disrupted the conjugation of ACHTUNGTRENNUNGfluorescein by forming a spirolactam (Scheme 1). Importantly,

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Synthesis of Near-IR Fluorescent Oxazine Dyes with Esterase-labile Sulfonate Esters

Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional compatibility and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the target molecule.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Significance Few organisms are capable of glowing in the dark. Perhaps the best known example is the firefly, a beetle that can emit flashes of yellow-green light when the small molecule d-luciferin is oxidized by the enzyme firefly luciferase. Here we show that a homologous enzyme in fruit flies is also capable of bioluminescence, but only when treated with a rigid synthetic analog of d-luciferin. The discovery of hidden luciferase activity in a nonluminescent insect suggests that the inherent chemistry required for bioluminescence is more common than previously thought, and that the evolution of new enzymatic activities can be directed by the appearance of a new substrate even in the absence of mutation. Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.