Effects of Troponoids on Mitochondrial Function and Cytotoxicity.

The α-hydroxytropolones (αHTs) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target HBV RNase H with submicromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays that assess mitochondrial function. Previous studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial/nuclear genome ratios, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass interfered with these processes. The troponoids had no impact on the mitochondrial DNA/nuclear DNA ratio after 3 days of compound exposure. The patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon the addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines, leading to cytotoxicity, but are not cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by coadministering ROS antagonists to ameliorate increased ROS levels.

Effects of photodeoxygenation on cell biology using dibenzothiophene S-oxide derivatives as O(3P)-precursors.

Photodeoxygenation of dibenzothiophene S-oxide and its derivatives have been used to generate atomic oxygen [O(3P)] to examine its effect on proteins, nucleic acids, and lipids. The unique reactivity and selectivity of O(3P) have shown distinct oxidation products and outcomes in biomolecules and cell-based studies. To understand the scope of its global impact on the cell, we treated MDA-MB-231 cells with 2,8-diacetoxymethyldibenzothiophene S-oxide and UV-A light to produce O(3P) without targeting a specific cell organelle. Cellular responses to O(3P)-release were analyzed using cell viability and cell cycle phase determination assays. Cell death was observed when cells were treated with higher concentrations of sulfoxides and UV-A light. However, significant differences in cell cycle phases due to UV-A irradiation of the sulfoxide were not observed. We further performed RNA-Seq analysis to study the underlying biological processes at play, and while UV-irradiation itself influenced gene expression, there were 9 upregulated and 8 downregulated genes that could be attributed to photodeoxygenation.

Synthesis of triphenylphosphonium dibenzothiophene S-oxide derivatives and their effect on cell cycle as photodeoxygenation-based cytotoxic agents.

Photodeoxygenation of Dibenzothiophene-S-oxide (DBTO) in UV-A light produces atomic oxygen [O(3P)] and the corresponding sulfide, dibenzothiophene (DBT). Recently, DBTO has been derivatized to study the effect of UV-A light-driven photodeoxygenation in lipids, proteins, and nucleic acids. In this study, two DBTO derivatives with triphenylphosphonium groups were synthesized to promote mitochondrial accumulation. The sulfone analogs of these derivatives were also synthesized and used as fluorescent mitochondrial dyes to assess localization in mitochondria of HeLa cells. These derivatives were then used to study the effect of photodeoxygenation on MDA-MB-231 breast cancer cell line using cell viability assays, cell cycle phase determination tests, and RNA-Seq analysis. The DBTO derivatives were found to significantly decrease cell viability only after UV-A irradiation as a result of generating corresponding sulfides that were found to significantly affect gene expression and cell cycle.

Metal coordinating inhibitors of Rift Valley fever virus replication.

Rift Valley fever virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the order Bunyavirales, whose replication depends on the enzymatic activity of the viral L protein. Screening for RVFV inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 47 novel RVFV inhibitors with selective indexes from 1.1-103 and 50% effective concentrations of 1.2-56 µM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against a second Bunyavirus, La Crosse Virus (LACV), and the flavivirus Zika (ZIKV). These data indicate that the α-Hydroxytropolone and N-Hydroxypyridinedione chemotypes should be investigated in the future to determine their mechanism(s) of action allowing further development as therapeutics for RVFV and LACV, and these chemotypes should be evaluated for activity against related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus.

Synthetic Derivatives of Ciclopirox are Effective Inhibitors of Cryptococcus neoformans.

Opportunistic fungal infections caused by Cryptococcus neoformans are a significant source of mortality in immunocompromised patients. They are challenging to treat because of a limited number of antifungal drugs, and novel and more effective anticryptococcal therapies are needed. Ciclopirox olamine, a N-hydroxypyridone, has been in use as an approved therapeutic agent for the treatment of topical fungal infections for more than two decades. It is a fungicide, with broad activity across multiple fungal species. We synthesized 10 N-hydroxypyridone derivatives to develop an initial structure-activity understanding relative to efficacy as a starting point for the development of systemic antifungals. We screened the derivatives for antifungal activity against C. neoformans and Cryptococcus gattii and counter-screened for specificity in Candida albicans and two Malassezia species. Eight of the ten show inhibition at 1-3 µM concentration (0.17-0.42 µg per mL) in both Cryptococcus species and in C. albicans, but poor activity in the Malassezia species. In C. neoformans, the N-hydroxypyridones are fungicides, are not antagonistic with either fluconazole or amphotericin B, and are synergistic with multiple inhibitors of the mitochondrial electron transport chain. They appear to function primarily by chelating iron within the active site of iron-dependent enzymes. This preliminary structure-activity relationship points to the need for a lipophilic functional group at position six of the N-hydroxypyridone ring and identifies positions four and six as sites where further substitution may be tolerated. These molecules provide a clear starting point for future optimization for efficacy and target identification.

Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication.

The Hepatitis B Virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Here, we synthesized and analyzed a library of 57 amide-containing α-hydroxytropolones (αHTs) as potential leads for HBV drug development. Fifty percent effective concentrations ranged from 0.31 to 54 µM, with selectivity indexes in cell culture of up to 80. Activity against the HBV RNaseH was confirmed in semi-quantitative enzymatic assays with recombinant HBV RNaseH. The compounds were overall poorly active against human ribonuclease H1, with 50% inhibitory concentrations of 5.1 to >1,000 µM. The αHTs had modest activity against growth of the fungal pathogen Cryptococcus neoformans, but had very limited activity against growth of the Gram - bacterium Escherichia coli and the Gram + bacterium Staphylococcus aureus, indicating substantial selectivity for HBV. A molecular model of the HBV RNaseH templated against the Ty3 RNaseH was generated. Docking the compounds to the RNaseH revealed the anticipated binding pose with the divalent cation coordinating motif on the compounds chelating the two Mn++ ions modeled into the active site. These studies reveal that that amide αHTs can be strong, specific HBV inhibitors that merit further assessment toward becoming anti-HBV drugs.

A Series of Indole-Thiazole Derivatives Act as GPER Agonists and Inhibit Breast Cancer Cell Growth.

The G protein-coupled estrogen receptor (GPER, GPR30) represents a promising target for the treatment of estrogen receptor α and ß negative breast cancers. Previously reported agonists of GPER have shown that activation of GPER inhibits breast cancer cell proliferation. We report herein a new GPER agonist scaffold based upon in silico pharmacophore screening. Three of these compounds were found to increase cAMP at similar levels as the known GPER-selective agonist G-1. Compound 5 was found to be selective for GPER (over estrogen receptor α and ß) and inhibit breast cancer cell proliferation at levels consistent with G-1. Docking studies go on to suggest that both 5 and G-1 bind within the same binding pocket in GPER and point to possible key residues that are important in GPER activation.