Jennifer A. Wilson

A high-throughput cell-based assay for inhibitors of ABCG2 activity

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 μM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z′ 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (≥ 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 μM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.

Synthesis, Activity, and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one), potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at noncytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogues can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.

Isolation, structural elucidation, and absolute stereochemistry of enigmazole A, a cytotoxic phosphomacrolide from the Papua New Guinea marine sponge Cinachyrella enigmatica.

Enigmazole A (1), a novel phosphate-containing macrolide, was isolated from a Papua New Guinea collection of the marine sponge Cinachyrella enigmatica. The structure of 1, including the absolute stereochemistry at all eight chiral centers, was determined by a combination of spectroscopic analyses and a series of microscale chemical derivatization studies. Compound 1 is comprised of an 18-membered phosphomacrolide that contains an embedded exomethylene-substituted tetrahydropyran ring and an acyclic portion that spans an embedded oxazole moiety. Two additional analogues, 15-O-methylenigmazole A and 13-hydroxy-15-O-methylenigmazole A, were also isolated and assigned. The enigmazoles are the first phosphomacrolides from a marine source and 1 exhibited significant cytotoxicity in the NCI 60-cell line antitumor screen, with a mean GI(50) of 1.7 microM.

Microwave-Based Reaction Screening: Tandem Retro-Diels–Alder/Diels–Alder Cycloadditions of o-Quinol Dimers

We have accomplished a parallel screen of cycloaddition partners for o-quinols utilizing a plate-based microwave system. Microwave irradiation improves the efficiency of retro-Diels-Alder/Diels-Alder cascades of o-quinol dimers which generally proceed in a diastereoselective fashion. Computational studies indicate that asynchronous transition states are favored in Diels-Alder cycloadditions of o-quinols. Subsequent biological evaluation of a collection of cycloadducts has identified an inhibitor of activator protein-1 (AP-1), an oncogenic transcription factor.

Isobutylhydroxyamides from the Pericarp of Nepalese Zanthoxylum armatum Inhibit NF1-Defective Tumor Cell Line Growth

A neurofibromatosis type 1 (NF1)-based bioassay-guided phytochemical investigation on Zanthoxylum armatum collected in Nepal led to the isolation of new timuramides A-D (1-4) and six known sanshools (5-10). The structures of all compounds were established by using modern spectroscopic techniques, including 1D and 2D NMR analysis and comparison with previously reported data. Most of the compounds inhibited growth of an Nf1- and p53-deficient mouse glioma cell line at noncytotoxic concentrations.

Engineering soya bean seeds as a scalable platform to produce cyanovirin‐N, a non‐ARV microbicide against HIV

Summary There is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC50 of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.

Grassypeptolides F and G, Cyanobacterial Peptides from Lyngbya majuscula

Grassypeptolides F (1) and G (2), bis-thiazoline-containing cyclic depsipeptides with a rare β-amino acid, extensive N-methylation, and a large number of d-amino acids, are reported from an extract of the Palauan cyanobacterium Lyngbya majuscula. Both 1 and 2 were found to have moderate inhibitory activity against the transcription factor AP-1 (IC₅₀ = 5.2 and 6.0 μM, respectively).

Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus

BackgroundThe lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.ResultsHere we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking.ConclusionsThe engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses.

Inhibitors of the Oncogenic Transcription Factor AP-1 from Podocarpus latifolius

An activator protein-1 (AP-1) based bioassay-guided phytochemical investigation on Podocarpus latifolius led to the isolation of three new sempervirol-type diterpenes, cycloinumakiol (1), inumakal (2), and inumakoic acid (3), along with three known norditerpenes (4-6). Compounds 4 and 6 were responsible for the observed bioactivity.

Inhibition of ABCG2-mediated drug efflux by naphthopyrones from marine crinoids.

Five new naphthopyrones (1-5) along with the known compounds TMC-256A1, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256C1, comaparvin, 6-methoxycomaparvin, and 6-methoxycomaparvin 5-methyl ether (6-11) were isolated from crinoids of the family Comasteridae. All compounds were tested for their ability to inhibit the multidrug transporter ABCG2, which plays a role in drug resistance. Six of the seven angular naphthopyrones showed moderate activity with <60% inhibition of ABCG2-mediated transport as compared to the positive control fumitremorgin C. None of the linear naphthopyrones inhibited ABCG2-mediated efflux.