High-throughput Fluorescence Polarization Research Articles

Establishment of an at-line nanofractionation-based screening platform by coupling HPLC-MS/MS with high-throughput fluorescence polarization bioassay for natural SARS-CoV-2 fusion inhibitors

In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 μM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression.

Acute myeloid leukemia (AML) is a common type of acute leukemia in adults and relapse is one of the main reasons for treatment failure. FLT3‑ITD mutations are associated with poor prognosis, short disease‑free progression survival and high relapse rates in patients with AML. STAT5 is activated by FLT3‑ITD and drives the pathogenesis of AML. STAT5 activation is usually a hallmark of hematologic malignancies and occurs in ~70% of patients with AML. Moreover, STAT5 is a key molecule which regulates hematopoiesis, and its high expression is closely associated with drug resistance, thus direct targeting of STAT5 for AML is of great clinical value. The present study introduces a new small‑molecule inhibitor that targets STAT5, presenting a promising approach for AML therapy. A high throughput fluorescence polarization (FP) screening system for STAT5 was designed and established, and used to screen an existing compound library to obtain the highly active small molecule inhibitor, topotecan hydrochloride. Topotecan hydrochloride was demonstrated to be an effective inhibitor of STAT5 by molecular docking prediction and cellular thermal shift assay. Topotecan hydrochloride bound to STAT5, inhibiting its dimerization, phosphorylation and transcription of specific target genes. The compound exhibits cellular activity at the nanomolar level and significantly inhibits the proliferation of human AML cell lines and FLT3‑ITD+ AML cells. Furthermore, topotecan hydrochloride has the potential to exert an anti‑tumor effect invivo. Overall, topotecan hydrochloride offers a new opportunity for the treatment of AML and other hematologic malignancies by directly targeting STAT5.

Dual-Wavelength Fluorescence Polarization Immunoassay for Simultaneous Detection of Sulfonamides and Antibacterial Synergists in Milk

Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC50 values for 23 SAs below 100 μg L-1 and 5 ASGs below 50 μg L-1. The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.

Gambogic acid and juglone inhibit RNase P through distinct mechanisms

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 μM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.

Development of a sensitive and rapid fluorescence polarization immunoassay for high throughput screening eight glucocorticoids in beef

In this study, a sensitive, rapid, homogeneous, and high-throughput fluorescence polarization immunoassay (FPIA) for the rapid screening of eight glucocorticoids (GCs) in beef samples was successfully established. Two tracers including 5-aminofluorescein-labeled dexamethasone (5-AF-DMS) and fluorescein isothiocyanate-labeled dexamethasone (FITC-DMS) were studied to select appropriate antibody-tracer pairs using four previously produced broad-specific monoclonal antibodies. An optimal combination of the antibody 12D9 and the tracer FITC-DMS was selected. Under optimal detection conditions, the half inhibitory concentrations of dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) were 1.00, 2.17, 3.49, 12.45, 1.20, 5.66, 6.85 and 3.45 ng/mL, respectively. The average recoveries of the proposed method in beef samples ranged from 77.3–91.7% with the coefficient of variation less than 12%. The developed FPIA was time-saving that could be completed within 10 min. The FPIA was applied to beef samples and showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (R2 = 0.9894). Thus, the proposed method provides a rapid, reliable, sensitive, and high-throughput screening tool for the simultaneous screening of eight GCs in beef, which shows great potential in the food safety analysis.

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors

Signal transducer and activator of transcription 5B (STAT5B) is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated STAT5B and demonstrates suitability for high-throughput screening with a Z′ factor of 0.68 ± 0.07 and a signal to noise ratio of 6.7 ± 0.84.

High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction.

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein-protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion-associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z'-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT-paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy.

This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd , the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd , the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.

Probing the Ligand-Binding Pocket of BTN3A1.

Small-molecule phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate stimulate human Vγ9Vδ2 T cells after binding to the intracellular B30.2 domain of the immune receptor butyrophilin 3 isoform A1 (BTN3A1). To understand the ligand-target interaction in greater detail, we performed molecular docking. Based on the docking results, we synthesized the novel ligand (E)-(7-hydroxy-6-methylhept-5-en-1-yl)phosphonate and mutated proposed binding site residues. We evaluated the impact on butyrophilin binding of existing and novel ligands using a newly developed high-throughput fluorescence polarization assay. We also evaluated the ability of the compounds to stimulate proliferation and interferon-γ production of Vγ9Vδ2 T cells. Mutation of H381 fully blocked ligand binding, whereas mutations to charged surface residues impacted diphosphate interactions. Monophosphonate analogs bind similarly to BTN3A1, although they differ in their antigenicity, demonstrating that binding and efficacy are not linearly correlated. These results further define the structure-activity relationships underlying BTN3A1 ligand binding and antigenicity and support further structure-guided drug design.

High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer.

High-throughput fluorescence polarization immunoassays (FPIAs) for mycotoxin were conducted using a portable FP analyzer with a microdevice. Simultaneous FPIA measurements for 8 different deoxynivalenol (DON) concentrations in 12 chambers (total of 96 samples) and high-throughput FPIA measurements for single DON concentrations in more than 500 chambers were conducted. The results indicated that simultaneous FPIAs for 96 independent samples and for 500 samples were possible by FP imaging. The FP analyzer has a size of 65 cm (W 35 cm × D 15 cm × H 15 cm) and costs less than $5000. The sample volume was 1 nL. Furthermore, it is expected that sample reaction and FP detection can be automatically conducted with the analyzer by changing the microdevice and the software. Its features such as low cost and portability will contribute to on-site measurement and point-of-care testing. Additionally, the high-throughput feature will contribute to the study of molecular interactions based on FP measurements.

A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3).

Anti-cancer drug discovery efforts to directly inhibit the signal transducer and activator of transcription 3 (STAT3) have been active for over a decade following the discovery that 70% of cancers exhibit elevated STAT3 activity. The majority of research has focused on attenuating STAT3 activity through preventing homo-dimerization by targeting the SH2 or transcriptional activation domains. Such dimerization inhibitors have not yet reached the market. However, an alternative strategy focussed on preventing STAT3 DNA-binding through targeting the DNA-binding domain (DBD) offers new drug design opportunities. Currently, only EMSA and ELISA-based methods have been implemented with suitable reliability to characterize STAT3 DBD inhibitors. Herein, we present a new orthogonal, fluorescence polarization (FP) assay suitable for high-throughput screening of molecules. This assay, using a STAT3127-688 construct, was developed and optimized to screen molecules that attenuate the STAT3:DNA association with good reliability (Z’ value > 0.6) and a significant contrast (signal-to-noise ratio > 15.0) at equilibrium. The assay system was stable over a 48 hour period. Significantly, the assay is homogeneous and simple to implement for high-throughput screening compared to EMSA and ELISA. Overall, this FP assay offers a new way to identify and characterize novel molecules that inhibit STAT3:DNA association.

Abstract 4656: Development of an RNA-based cancer therapeutic targeting the let-7-LIN28 interaction

Abstract Based on the knowledge that microRNAs (miRNAs) are dysregulated in diseases such as cancer, various attempts have been explored to develop miRNA-based cancer therapeutics. Although many strategies have been used to restore the levels of therapeutically relevant microRNAs, clinical delivery of the processed or mature miRNA to cancer cells still remains a challenge. To overcome this challenge, innovative methods are being explored to increase the pools of tumor-suppressive miRNAs, such as enhancing miRNA biogenesis. This is especially true for the let-7 family of tumor-suppressive miRNAs, which has an additional layer of regulation over the canonical miRNA biogenesis pathway. In particular, the DROSHA and DICER cleavage steps are blocked when unprocessed let-7 is bound by the RNA-binding protein LIN28. LIN28 interacts specifically with unprocessed let-7 via a sequence-specific motif, GGAG, contained in most let-7 family members. Because previous attempts to restore mature let-7 levels through increasing let-7 pools have been quite successful at reducing tumor burden, identifying novel and clinically relevant ways to increase the level of this tumor-suppressive miRNA represents a critical need. In this study, we hypothesize that small-molecule inhibitors that disrupt the LIN28-let-7 interaction will lead to enhanced processing and increased levels of mature, tumor-suppressive let-7. Thus, an in vitro high-throughput fluorescence polarization screen has been conducted using His-tagged LIN28 and a Cy5 fluorophore-tagged let-7 RNA probe. The nine-nucleotide RNA probe was designed through in silico modeling to include the GGAG motif, which associates with LIN28 through the zinc knuckle domain of LIN28. Due to the size difference between unbound let-7 probe and LIN28-bound let-7 probe, a wide window of polarization values was achieved, resulting in a Z′ score of 0.61, indicating that the assay was robust to proceed with the screen. The screen was performed against 23,680 compounds, which consist of FDA-approved LOPAC and blood-brain barrier-permeable CNS library compounds. Eight compounds were identified as positive hits from the screen. These compounds are currently being characterized in secondary assays both in vitro and in cell culture. The significance of this research is that small-molecule inhibitors may provide novel therapeutic strategies to increase the pool of mature, tumor-suppressive let-7 in tumors with elevated LIN28. Citation Format: Sunghyun Myoung, Sergey Savinov, Lan Chen, Gaurav Chopra, Larisa Avramova, James Welch, Bradley Loren, David Thompson, Andrea L. Kasinski. Development of an RNA-based cancer therapeutic targeting the let-7-LIN28 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4656.

Development of a simple, rapid and high-throughput fluorescence polarization immunoassay for glycocholic acid in human urine

In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of glycocholic acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

Dual-wavelength fluorescence polarization immunoassay to increase information content per screen: Applications for simultaneous detection of total aflatoxins and family zearalenones in maize

To increase information content per screen, a dual-wavelength, homologous and high-throughput fluorescence polarization immunoassay (DWFPIA) for simultaneous detection of total aflatoxins (AFs) and family zearalenones (ZENs) was developed. Aflatoxin B1 (AFB1) and zearalanone (ZAN) were labeled with different fluoresceins, and then combined with corresponding broad-specific antibodies to develop the DWFPIA. Under optimal conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 2.68 μg/L and 4.08 μg/L for total four AFs (the weight ratio of AFB1: Aflatoxin B2: Aflatoxin G1: Aflatoxin G2 was 1: 1: 1: 1) and six ZEN analogs (the weight ratio of ZEN: ZAN: α-zearalenone: β-zearalenone: α-zearalanone: β-zearalanone was 1: 1: 1: 1: 1: 1) by mixing equal volumes (1: 1, v/v), respectively. The corresponding limit of detection values in maize flour samples were 4.98 μg/kg and 11.03 μg/kg, and the mean recoveries ranged from 78.6 to 103.6% with the coefficients of variation below 19.2%. The whole detection procedure, including sample preparation, took less than 30 min. Finally, the DWFPIA was applied to screen naturally contaminated maize flour samples, and the detection data were similar to those with HPLC-MS/MS.

Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors.

Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

ABSTRACTSecondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.