Mitochondrial dysfunction is a common mediator of disease and organ injury. Although recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. To examine the impact of the ␤-adrenoceptor (␤-AR) signaling pathway on MB, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes were exposed for 24 h to multiple ␤-AR agonists: isoproter-, and formoterol (selective ␤ 2 -AR agonist). The Seahorse Biosciences (North Billerica, MA) extracellular flux analyzer was used to quantify carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterenol and BRL 37244 did not alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the ␤-AR antagonist propranolol and the ␤ 2 -AR inverse agonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol hydrochloride (ICI-118,551). Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, peroxisome proliferator-activated receptor ␥ coactivator 1␣, and multiple genes involved in the mitochondrial electron transport chain (F0 subunit 6 of transmembrane F-type ATP synthase, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 6, and NADH dehydrogenase [ubiquinone] 1␤ subcomplex subunit 8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified nisoxetine from the Sigma Library of Pharmacologically Active Compounds and two compounds from the ChemBridge DIVERSet that increased mitochondrial respiratory capacity. These data provide compelling evidence for the use and development of ␤ 2 -AR ligands for therapeutic MB.

Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and the mitochondrial permeability transition (MPT). Iron is a critical catalyst for ROS formation, and reactive oxygen species (ROS) play an important role in APAP-induced hepatotoxicity. Previous studies show that APAP disrupts lysosomes, which release ferrous iron (Fe2+) into the cytosol to trigger the MPT and cell killing. Here, our aim was to investigate whether iron released from lysosomes after APAP is then taken up into mitochondria via the mitochondrial electrogenic Ca2+, Fe2+ uniporter (MCFU) to cause mitochondrial dysfunction and cell death. Hepatocytes were isolated from fasted male C57BL/6 mice. Necrotic cell killing was assessed by propidium iodide fluorimetry. Mitochondrial membrane potential (Δψ) was visualized by confocal microscopy of rhodamine 123 (Rh123) and tetramethylrhodamine methylester (TMRM). Chelatable Fe2+ was monitored by quenching of calcein (cytosol) and mitoferrofluor (MFF, mitochondria). ROS generation was monitored by confocal microscopy of MitoSox Red and plate reader fluorimetry of chloromethyldihydrodichlorofluorescein diacetate (cmH2DCF-DA). Administered 1 h before APAP (10 mM), the lysosomally targeted iron chelator, starch-desferal (1 mM), and the MCFU inhibitors, Ru360 (100 nM) and minocycline (4 μM), decreased cell killing from 83% to 41%, 57% and 53%, respectively, after 10 h. Progressive quenching of calcein and MFF began after ~4 h, signifying increased cytosolic and mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. Dipyridyl, a membrane-permeable iron chelator, dequenched calcein and MFF fluorescence after APAP. Starch-desferal, but not Ru360 and minocycline, suppressed cytosolic calcein quenching, whereas starch-desferal, Ru360 and minocycline all suppressed mitochondrial MFF quenching and mitochondrial depolarization. Starch-desferal, Ru360 and minocycline also each decreased ROS formation. Moreover, minocycline 1 h after APAP decreased cell killing by half. In conclusion, release of Fe2+ from lysosomes followed by uptake into mitochondria via MCFU occurs during APAP hepatotoxicity. Mitochondrial iron then catalyzes toxic hydroxyl radical formation, which triggers the MPT and cell killing. The efficacy of minocycline post-treatment shows minocycline as a possible therapeutic agent against APAP hepatotoxicity.

A variety of chroman spiroketals are synthesized via inverse-demand [4 + 2] cycloaddition of enol ethers and ortho-quinone methides (o-QMs). Low temperature o-QM generation in situ allows for the kinetic, diastereoselective construction of these motifs, providing entry to a number of unusual chroman spiroketal natural products.Aliphatic spiroketals are a common substructure of natural products isolated in a large variety from both marine and terrestrial sources. 1 Studies aimed at understanding the origins of their conformational preference have resulted in the general assumption that the spiroketal carbon corresponds to the thermodynamically most stable isomer as determined by stereoelectronic influences. Hence, the biosynthesis of these fluxional natural products usually results in the construction of the most stable diastereomer or a mixture that reflects energy differences between respective diastereomers.For some time, we have speculated that a rare subset consisting of chroman spiroketals (1, 2 2 ,3 and 3 ,4 Figure 1) may possess features that refute this basic tenet and require a kinetic assembly. These uniquely robust chroman spiroketal scaffolds have caused us to consider new methods for their construction.Some time ago, we developed a method that enables the controlled, low-temperature generation of o-quinone methides (o-QMs) (Figure 2, I). 5 The process, which is driven by the relative stability of sequential anions formed along the cascade, begins by formation of an alkoxide. This species intercepts a neighboring carbonyl of a phenolic carbonate and thus liberates a phenoxide that subsequently undergoes β-elimination of a carboxylate and thereby forms a reactive o-QM species, which captures the first nucleophile that it subsequently encounters. This is a useful method for o-QM generation because, since the reactive o-QM intermediate is generated at low temperature in low concentrations, it subsequently participates in very precise, kinetically controlled reactions. 6 Using this procedure, we demonstrated the first examples of diastereoselective o-QM cycloadditions and found that the inverse demand [4 + 2] significantly favors an endo transition state with electron-rich alkenes. 7 In further experiments, we showed that chiral enol ethers containing remote stereocenters, such as those derived from (1S,2R)-2-phenyl-cyclohexanol, will participate in diastereoselective cycloadditions to yield the corresponding chroman acetals with an R configuration (Figure 2, II) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript R 1 , R 2 , and R 3 substituents should also participate in cycloadditions with similar diastereoselectivity (Figure 2, III). Detailed analysis of our proposed transition state allowed us to further speculate that substituents R 1 and R 3 would have a pronounced effect on the stereochemical outcome, whereas the R 2 residue would not, allowing it to be tolerated on the same face as the reaction. Therefore, we began to examine model systems that might eventually be ...

A low-temperature method for generating o-quinone methides is described which permits facile introduction of assorted R substituents onto the aryl ring system at low temperature. The method is useful for the efficient preparation of ortho-ring-alkylated phenols.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi–GDP complex by directly interacting with both Gαi and GDP. Gαi–GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.