Discovery and Synthesis of a Naturally Derived Protein Kinase Inhibitor that Selectively Inhibits Distinct Classes of Serine/Threonine Kinases.

The DNAJB1-PRKACA oncogenic gene fusion results in an active kinase enzyme, J-PKAcα, that has been identified as an attractive antitumor target for fibrolamellar hepatocellular carcinoma (FLHCC). A high-throughput assay was used to identify inhibitors of J-PKAcα catalytic activity by screening the NCI Program for Natural Product Discovery (NPNPD) prefractionated natural product library. Purification of the active agent from a single fraction of an Aplidium sp. marine tunicate led to the discovery of two unprecedented alkaloids, aplithianines A (1) and B (2). Aplithianine A (1) showed potent inhibition against J-PKAcα with an IC50 of ∼1 µM in the primary screening assay. In kinome screening, 1 inhibited wild-type PKA with an IC50 of 84 nM. Further mechanistic studies including cocrystallization and X-ray diffraction experiments revealed that 1 inhibited PKAcα catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC50 values in the range ∼11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.

Biochemical Discovery, Intracellular Evaluation, and Crystallographic Characterization of Synthetic and Natural Product Adenosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase A (PKA) Inhibitors.

The recent demonstration that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.

Identification of natural product modulators of Merkel cell carcinoma cell growth and survival.

Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.

Development of a High-throughput NanoBRET Screening Platform to Identify Modulators of the RAS/RAF Interaction.

Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.

A Molecular Networking Strategy: High-Throughput Screening and Chemical Analysis of Brazilian Cerrado Plant Extracts against Cancer Cells.

Plants have historically been a rich source of successful anticancer drugs and chemotherapeutic agents, with research indicating that this trend will continue. In this contribution, we performed high-throughput cytotoxicity screening of 702 extracts from 95 plant species, representing 40 families of the Brazilian Cerrado biome. Activity was investigated against the following cancer cell lines: colon (Colo205 and Km12), renal (A498 and U031), liver (HEP3B and SKHEP), and osteosarcoma (MG63 and MG63.3). Dose-response tests were conducted with 44 of the most active extracts, with 22 demonstrating IC50 values ranging from <1.3 to 20 µg/mL. A molecular networking strategy was formulated using the Global Natural Product Social Molecular Networking (GNPS) platform to visualize, analyze, and annotate the compounds present in 17 extracts active against NCI-60 cell lines. Significant cytotoxic activity was found for Salacia crassifolia, Salacia elliptica, Simarouba versicolor, Diospyros hispida, Schinus terebinthifolia, Casearia sylvestris var. lingua, Magonia pubescens, and Rapanea guianensis. Molecular networking resulted in the annotation of 27 compounds. This strategy provided an initial overview of a complex and diverse natural product data set, yielded a large amount of chemical information, identified patterns and known compounds, and assisted in defining priorities for further studies.

In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors.

The transfer of the small protein ubiquitin to a target protein is an intricately orchestrated process called ubiquitination that results in modulation of protein function or stability. Proper regulation of ubiquitination is essential, and dysregulation of this process is implicated in several human diseases. An example of a ubiquitination cascade that is a central signaling node in important disease-associated pathways is that of CBLB [a human homolog of a viral oncogene Casitas B-lineage lymphoma (CBL) from the Cas NS-1 murine retrovirus], a RING finger ubiquitin ligase (E3) whose substrates include a number of important cell-signaling kinases. These include kinases important in immune function that act in the T cell receptor and costimulatory pathways, the Tyro/Axl/MerTK (TAM) receptor family in natural killer (NK) cells, as well as growth factor receptor kinases like epidermal growth factor receptor (EGFR). Loss of CBLB has been shown to increase innate and adaptive antitumor immunity. This suggests that small-molecule modulation of CBLB E3 activity could enhance antitumor immunity in patients. To explore the hypothesis that enzymatic inhibition of E3s may result in modulation of disease-related signaling pathways, we established a high-throughput screen of >70,000 chemical entities for inhibition of CBLB activity. Although CBLB was chosen as a proof-of-principle target for inhibitor discovery, we demonstrate that our assay is generalizable to monitoring the activity of other ubiquitin ligases. We further extended our observed in vitro inhibition with additional cell-based models of CBLB activity. From these studies, we demonstrate that a class of natural product-based alkaloids, known as methyl ellipticiniums (MEs), is capable of inhibiting ubiquitin ligases intracellularly.

Identification of potential modulators of osteosarcoma metastasis by high-throughput cellular screening of natural products.

A high-throughput screening assay was developed and applied to a large library of natural product extract samples, in order to identify compounds which preferentially inhibited the in vitro 2D growth of a highly metastatic osteosarcoma cell line (MG63.3) compared to a cognate parental cell line (MG63) with low metastatic potential. Evaluation of differentially active natural product extracts with bioassay-guided fractionation led to the identification of lovastatin (IC50  = 11 µm) and the limonoid toosendanin (IC50  = 26 nm). Other statins and limonoids were then tested, and cerivastatin was identified as a particularly potent (IC50  < 0.1 µm) and selective agent. These compounds potently and selectively induced apoptosis in MG63.3 cells, but not MG63. Assays with other cell pairs were used to examine the generality of these results. Statins and limonoids may represent unexplored opportunities for development of modulators of osteosarcoma metastasis. As cerivastatin was previously approved for clinical use, it could be considered for repurposing in osteosarcoma, pending validation in further models.

The Natural Product Butylcycloheptyl Prodiginine Binds Pre-miR-21, Inhibits Dicer-Mediated Processing of Pre-miR-21, and Blocks Cellular Proliferation.

Identification of RNA-interacting pharmacophores could provide chemical probes and, potentially, small molecules for RNA-based therapeutics. Using a high-throughput differential scanning fluorimetry assay, we identified small-molecule natural products with the capacity to bind the discrete stem-looped structure of pre-miR-21. The most potent compound identified was a prodiginine-type compound, butylcycloheptyl prodiginine (bPGN), with the ability to inhibit Dicer-mediated processing of pre-miR-21 in vitro and in cells. Time-dependent RT-qPCR, western blot, and transcriptomic analyses showed modulation of miR-21 expression and its target genes such as PDCD4 and PTEN upon treatment with bPGN, supporting on-target inhibition. Consequently, inhibition of cellular proliferation in HCT-116 colorectal cancer cells was also observed when treated with bPGN. The discovery that bPGN can bind and modulate the expression of regulatory RNAs such as miR-21 helps set the stage for further development of this class of natural product as a molecular probe or therapeutic agent against miRNA-dependent diseases.

Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase (TDP1).

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme crucial for cleavage of the covalent topoisomerase 1-DNA complex, an intermediate in DNA repair. TDP1 plays a role in reversing inhibition of topoisomerase I by camptothecins, a series of potent and effective inhibitors used in the treatment of colorectal, ovarian, and small-cell lung cancers. It is hypothesized that inhibition of TDP1 activity may enhance camptothecin sensitivity in tumors. Here, we describe the design, development, and execution of a novel assay to identify inhibitors of TDP1 present in natural product extracts. The assay was designed to address issues with fluorescent "nuisance" molecules and to minimize the detection of false-positives caused by polyphenolic molecules known to nonspecifically inhibit enzyme activity. A total of 227,905 purified molecules, prefractionated extracts, and crude natural product extracts were screened. This yielded 534 initial positives (0.23%). Secondary prioritization reduced this number to 117 (0.05% final hit rate). Several novel inhibitors have been identified showing micromolar affinity for human TDP1, including halenaquinol sulfate, a pentacyclic hydroquinone from the sponge Xestospongia sp.

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.

Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro.

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z' values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.

High-throughput screening for identification of inhibitors of EpCAM-dependent growth of hepatocellular carcinoma cells.

The cancer stem cell marker, EpCAM, is an important indicator of Wnt/ß-catenin signaling activation and a functional component of hepatocellular tumor-initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma (HCC) cells. EpCAM(+) and EpCAM(-) HCC cell lines were assessed for differential sensitivity to a Wnt/ß-catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50 ) reduced median EpCAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.

Development of a high-throughput cell-based reporter assay to identify stabilizers of tumor suppressor Pdcd4.

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain ("target") or an adjacent ("off-target") region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70(S6K1) overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z' averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers.

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.

Natural products active in aberrant c-Kit signaling.

Development of modulators of constitutively active, kinase domain mutants of c-Kit has proved to be very difficult. Therefore, a high-throughput differential cytotoxicity assay was developed to screen for compounds that preferentially kill cells expressing constitutively active c-Kit. The cells used in the assay, murine IC2 mast cells, express either the D814Y activating mutation (functionally equivalent to human D816Y) or wild type protein. This assay is robust and highly reproducible. When applied to libraries of natural product extracts (followed by assay-guided fractionation), two differentially active compounds were identified. To assess possible mechanisms of action, the active compounds were tested for inhibitory activity against a panel of signaling enzymes (including wild type and mutant c-Kit). Neither of the compounds significantly affected any of the 73 enzymes tested. The effects of commercially available modulators of known signaling components were also assessed using the screening assay. None of these inhibitors reproduced the differential activity seen with the natural products. Finally, both compounds were found to affect mitochondrial potential in cells expressing c-Kit(D814Y). These results suggest that the newly identified natural products may provide new avenues for intervention in aberrant c-Kit signaling pathways.

A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1.

The oncogenic transcription factor AP-1 (activator protein-1) is required for tumor promotion and progression. Identification of novel and specific AP-1 inhibitors would be beneficial for cancer prevention and therapy. The authors have developed a high-throughput assay to screen synthetic and natural product libraries for noncytotoxic inhibitors of mitogen-activated AP-1 activity. The cell-based high-throughput screen is conducted in a 384-well format using a fluorescent resonance energy transfer (FRET) substrate to quantify the activity of a beta-lactamase reporter under the control of an AP-1-dependent promoter. The ratiometric FRET readout makes this assay extremely robust and reproducible, particularly for use with natural product extracts. To eliminate false positives due to cell killing, a cytotoxicity assay was incorporated. The AP-1 beta-lactamase reporter was validated with inhibitors of kinases located upstream of AP-1 and with known natural product inhibitors of AP-1 (nordihydroguaiaretic acid and curcumin). The assay was able to identify other known AP-1 inhibitors and protein kinase C modulators, as well as a number of chemically diverse compounds with unknown mechanisms of action from natural products libraries. Application to natural product extracts identified hits from a range of taxonomic groups. Screening of synthetic compounds and natural products should identify novel AP-1 inhibitors that may be useful in the prevention and treatment of cancers.

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 microM 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 (> or = 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 microM) 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.