Curtis J. Henrich

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.

New inhibitors of ABCG2 identified by high-throughput screening

In order to identify novel inhibitors of the ATP-binding cassette transporter, ABCG2, a high-throughput assay measuring the accumulation of the ABCG2 substrate pheophorbide a in ABCG2-overexpressing NCI-H460 MX20 cells was used to screen libraries of compounds. Out of a library of 7,325 natural products and synthetic compounds from the National Cancer Institute/Developmental Therapeutics Program collection, 18 were found to inhibit ABCG2 at 10 μmol/L. After eliminating flavonoids and compounds of limited availability from the 18 original compounds, 10 of the 11 remaining compounds reversed mitoxantrone resistance in NCI-H460/MX20 cells and prevented ABCG2-mediated BODIPY-prazosin transport in ABCG2-transfected HEK293 cells, confirming an interaction with ABCG2. Based on the activity profiles and the availability of materials, five inhibitors were examined for their ability to compete with [125I]iodoarylazidoprazosin labeling of ABCG2, increase binding of the anti-ABCG2 antibody 5D3, and prevent P-glycoprotein or multidrug resistance protein 1–mediated transport. At a concentration of 20 μmol/L, all of the compounds reduced iodoarylazidoprazosin labeling by 50% to 80% compared with controls. All five compounds also increased 5D3 labeling of ABCG2, indicating that these compounds are inhibitors but not substrates of ABCG2. None of the compounds affected P-glycoprotein–mediated rhodamine 123 transport, whereas three affected multidrug resistance protein-1–mediated calcein transport at 25 μmol/L, suggesting that the compounds are relatively specific for ABCG2. These five novel inhibitors of ABCG2 activity may provide a basis for further investigation of ABCG2 function and its relevance in multidrug resistance. [Mol Cancer Ther 2007;6(12):3271–8]

Botryllamides: Natural Product Inhibitors of ABCG2

ABCG2 is a membrane-localized, human transporter protein that has been demonstrated to reduce the intracellular accumulation of substrates through ATP-dependent efflux. Highly expressed in placental syncytiotrophoblasts, brain microvasculature, and the gastrointestinal tract, ABCG2 has been shown to mediate normal tissue protection as well as limit oral bioavailability of substrate compounds. Development of ABCG2 inhibitors for clinical use may allow increased penetration of therapeutic agents into sanctuary sites and increased gastrointestinal absorption. Previously identified inhibitors have lacked potency or specificity or were toxic at concentrations needed to inhibit ABCG2; none are in clinical development. A previously developed high-throughput assay measuring inhibition of ABCG2-mediated pheophorbide a transport was applied to natural product extract libraries. Among the active samples were extracts from the marine ascidian Botryllus tyreus. Bioassay-guided fractionation resulted in purification of a series of botryllamides. Ten botryllamides were obtained, two of which (designated I and J) were novel. Activity against ABCG2 was confirmed by assessing the ability of the compounds to inhibit ABCG2-mediated BODIPY-prazosin transport in ABCG2-transfected HEK293 cells, compete with [(125)I]-iodoarylazidoprazosin (IAAP) labeling of ABCG2, stimulate ABCG2-associated ATPase activity, and reverse ABCG2-mediated resistance.

Clerodane Diterpenes from Casearia arguta That Act As Synergistic TRAIL Sensitizers

Casearia arguta was investigated as part of the ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, argutins A-H, eight new highly oxygenated clerodane diterpenes, were isolated from the plant Casearia arguta collected in Guatemala. The modified Mosher ester method was utilized to establish the absolute configuration of argutins A and F. Each of the argutins showed varying levels of synergy with TRAIL. Argutin B showed the highest TRAIL sensitization; the synergistic effect of argutin B and TRAIL together was 3-fold greater than argutin B alone.

Actinopolysporins A-C and tubercidin as a Pdcd4 stabilizer from the halophilic actinomycete Actinopolyspora erythraea YIM 90600.

Our current natural product program utilizes new actinomycetes originating from unexplored and underexplored ecological niches, employing cytotoxicity against a selected panel of cancer cell lines as the preliminary screen to identify hit strains for natural product dereplication, followed by mechanism-based assays of the purified natural products to discover potential anticancer drug leads. Three new linear polyketides, actinopolysporins A (1), B (2), and C (3), along with the known antineoplastic antibiotic tubercidin (4), were isolated from the halophilic actinomycete Actinopolyspora erythraea YIM 90600, and the structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. All four compounds were assayed for their ability to stabilize the tumor suppressor programmed cell death protein 4 (Pdcd4), which is known to antagonize critical events in oncogenic pathways. Only 4 significantly inhibited proteasomal degradation of a model Pdcd4-luciferase fusion protein, with an IC50 of 0.88±0.09 μM, unveiling a novel biological activity for this well-studied natural product.

Cryptocaryols A-H, α-pyrone-containing 1,3-polyols from Cryptocarya sp. implicated in stabilizing the tumor suppressor Pdcd4.

A high-throughput cell-based reporter assay designed to identify small-molecule stabilizers of the tumor suppressor Pdcd4 was used to screen extracts in the NCI Natural Products Repository. Bioassay-guided fractionation of an extract from a Papua New Guinea collection of the tropical tree Cryptocarya sp. provided a series of new 5,6-dihydro-α-pyrone-containing 1,3-polyols (1-8), named cryptocaryols A-H. Their structures were assigned from a combination of NMR, MS, and CD studies in conjunction with NMR database comparisons. Compounds 1-8 were found to rescue Pdcd4 from TPA-induced degradation with EC50 concentrations that ranged from 1.3 to 4.9 μM.

A cell-based high-throughput screen to identify synergistic TRAIL sensitizers

We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E’s more specific mechanism of action suggests minimal toxic side effects.

A selective small-molecule nuclear factor-κB inhibitor from a high-throughput cell-based assay for “activator protein-1 hits”

NSC 676914 has been identified as a selective nuclear factor-κB (NF-κB) inhibitor that does not inhibit cell proliferation. This compound was originally identified in a high-throughput cell-based assay for activator protein-1 (AP-1) inhibitors using synthetic compound libraries and the National Cancer Institute natural product repository. NSC 676914 shows activity against NF-κB in luciferase reporter assays at concentrations much less than the IC50 for AP-1. A serum response element reporter used as a specificity control and indicator of cell proliferation was relatively insensitive to the compound. Pretreatment with NSC 676914 is here shown to repress 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced IκB-α phosphorylation and translocation of p65/50 to the nucleus but not the processing of p52 from p100, suggesting the inhibition of NF-κB regulator IKKβ rather than IKKα. Inhibition of NF-κB activation occurred as a consequence of blocking phosphorylation of IKK. Induction of IκB-α phosphorylation by TPA was diminished by pretreatment of NSC 676914 even at 1.1 μmol/L. In contrast, kinases c-Jun-NH2-kinase and extracellular signal-regulated kinases 1 and 2, important for AP-1 activation, showed no significant repression by this compound. Furthermore, a Matrigel invasion assay with breast cancer cell lines and a transformation assay in mouse JB6 cells revealed that TPA-induced invasion and transformation responses were completely repressed by this compound. These results suggest that NSC 676914 could be a novel inhibitor having potential therapeutic activity to target NF-κB for cancer treatment or prevention. [Mol Cancer Ther 2009;8(3):571–81]

Quassinoid Inhibition of AP-1 Function Does Not Correlate with Cytotoxicity or Protein Synthesis Inhibition †

Several quassinoids were identified in a high-throughput screening assay as inhibitors of the transcription factor AP-1. Further biological characterization revealed that while their effect was not specific to AP-1, protein synthesis inhibition and cell growth assays were inconsistent with a mechanism of simple protein synthesis inhibition. Numerous plant extracts from the plant family Simaroubaceae were also identified in the same screen; bioassay-guided fractionation of one extract (Ailanthus triphylla) yielded two known quassinoids, ailanthinone (3) and glaucarubinone (4), which were also identified in the pure compound screening procedure.