Fluorescence Polarization Assay Research Articles

The role of the size of affinity ligands in the detection and characterization of extracellular vesicles

Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.

Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

Synthetic modulators for plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Here, we describe a novel small-molecule inhibitor for 14-3-3 proteins ofArabidopsisthaliana. The inhibitor was identified fromunexpected productsin DMSO stock solution of an in-house chemical library. Mass spectroscopy, mutant-based analyses,fluorescence polarization assays, and thermal shift assaysrevealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor toArabidopsisleaves suppressed the stomatal aperture. The inhibitorshould provide a new insight into the design of potent and isoform-selective 14-3-3 modulators.

Discovery of a Plant 14‐3‐3 Inhibitor Possessing Isoform Selectivity and In Planta Activity

Synthetic modulators for plant 14‐3‐3s are promising chemical tools both for understanding the 14‐3‐3‐related signaling pathways and controlling plant physiology. Here, we describe a novel small‐molecule inhibitor for 14‐3‐3 proteins ofArabidopsis thaliana. The inhibitor was identified from unexpected products in DMSO stock solution of an in‐house chemical library. Mass spectroscopy, mutant‐based analyses, fluorescence polarization assays, and thermal shift assaysrevealed that the inhibitor covalently binds to an allosteric site of 14‐3‐3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide a new insight into the design of potent and isoform‐selective 14‐3‐3 modulators.

Human eukaryotic initiation factor 4G directly binds the 40S ribosomal subunit to promote efficient translation

Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Abstract 1815: Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth

Abstract Background: The pluripotency factor known as Lin28 plays a crucial role in the regulation of cancer cell stem-like characteristics, hence facilitating the formation of cancer and the evolution of therapy-resistant tumors. The protein exerts its function by utilizing its cold shock domain and zinc knuckle domain (ZKD) to engage with the Let-7 pre-microRNA and impede the process of Let-7 production. The implementation of chemical agents to hinder the interaction between Lin28 and Let-7 has been proven as a therapeutic approach for treating aggressive tumors. Methods: Computer-aided drug design techniques were employed in the development of a novel series of small molecule inhibitors, which are derivatives of our previously identified lead compounds Ln115, Ln15, and Ln7. Fluorescent Polarization Assay (FP), and Electrophoretic Mobility Shift Assay (EMSA) were used to screen the chemicals as Lin28-Let7 interaction disruptors using Lin28A/B ZKD domain. To further screen the chemicals, the Incucyte cell proliferation assay was performed using two Lin28A and Lin28B positive cells, IGROV-1, and DuNE cells, respectively. The inhibition of Lin28 activity was subsequently evaluated by measuring Let-7 micro-RNA and Lin28 protein levels using RT-PCR and Western Blot. Then, the metabolism profile was explored using Microsomal Stability Assay. Incucyte proliferation assay and MTS assay determined the IC50 of lead compound in Lin28 positive cells. The anti-stemness properties of the lead compound were subsequently evaluated through assays for stem gene expression levels and colony formation. To evaluate the potential of the lead compound to further move into animal studies, we tested it on a newly developed 3D Neuroendocrine Prostate Cancer (NEPC) model. Results: In this study, we provide a novel set of derivatives, within which, Ln268 was chosen as the lead compound. Ln268 shows higher metabolic stability with a half-life time of 462 min and clearance rate of 5 ul/min/mg, compared to its father compound at 347 min and 46 ul/min/mg, respectively. Ln268 treatment resulted in a time-dependent inhibition of cell growth with an IC50 of 2.5 uM at 72 hrs. Ln268 reduces mRNA levels of stemness and neurodifferentiation markers such as CHGA, CHG, SYP, SCGN, OCT3/4, NANOG, ID4, FOXC1, ALDH1A2, and FOXO3. In the 3D NEPC model, Ln268 suppresses spheroid growth in a dose- and time-dependent manner. Ln268 achieved 100% spheroid growth inhibition at the concentration of 100 uM, while the current commercially available C1632 only reached 50%. Conclusion: We discovered a novel Ln15 derivative named Ln268, which has a better metabolism profile and increased inhibitory activity to distrust Lin28-Let7 interaction and cancer cell proliferation. Ln268 has the potential to be improved and used as an anti-cancer treatment for a variety of malignancies, such as prostate cancer. Citation Format: Victor M. Matias-Barrios, Mariia Radaeva, Graciella Rosellinny, Artem Cherkasov, Xuesen Dong. Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1815.

Abstract 3051: JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models

Abstract The activator protein 1 (AP-1) transcription factor is a dimeric complex consisting of proteins from the JUN, FOS, MAF, and ATF families. AP-1 complexes have been identified as novel therapeutic targets in cancer due to their role in tumor growth, invasion, metastasis, angiogenesis and chemoresistance. Dimerization of components of the AP-1 complex via leucine zipper domain interactions is required for DNA binding and subsequent transcriptional activity. We designed a peptide (Jun antagonizing peptide, JunAP) targeting the basic leucine zipper motif of AP-1 to antagonize AP-1 complex formation and prevent associated activity. Fluorescence polarization and DNA ELISA assays demonstrate target binding and selectivity of JunAP towards AP-1 family members, co-immunoprecipitation experiments reveal that JunAP blocks protein-protein interactions between cJun and its key binding partners, and reporter assays confirm inhibition of AP-1 transcriptional activity in vitro. Pathway analysis of RNA sequencing data confirmed by qPCR analysis shows that JunAP disrupts key pathways required for cell survival and migration. Corresponding functional in vitro assays reveal that JunAP exhibits an inhibitory effect on invasion in Boyden chamber assays and demonstrates potent cytotoxicity in Jun-dependent cells. Further, in a BT549 triple negative breast cancer subcutaneous xenograft model, administration of JunAP results in tumor regression, demonstrating the anti-cancer potential of targeting the AP-1 complex. In summary, these data support JunAP as a potent peptide antagonist of the AP-1 transcription factor family that warrants further development as a potential therapeutic option for AP-1 driven tumors. Citation Format: Karen Mendelson, Zachary F. Mattes, Siok Leong, Ricardo Ramirez, Mark Koester, Claudio Scuoppo, Julia Diehl, Erin Gallagher, Jerel Gonzales, Franco Abbate, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3051.

A structural basis for the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a widespread environmental pollutant of the perfluoroalkyl substance (PFAS) class that is extremely resistant to environmental and metabolic degradation, leading to bioaccumulation. PFOA exposure has been linked to many health effects including endocrine disruption and metabolic dysregulation, but our understanding of the molecular mechanisms resulting in these outcomes remains incomplete. One target affected by PFOA is the ligand regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which plays a critical role in controlling metabolic homeostasis through regulating processes such as adipogenesis, glucose homeostasis, inflammation and osteogenesis. It has been previously established that PFOA activates PPARγ through binding to the PPARγ ligand binding domain (PPARγ LBD) leading to increased expression of PPARγ controlled target genes. However, the mechanism by which PFOA achieves this has remained elusive. Here, we employed a combination of X-ray crystallography and fluorescence polarization assays to provide a structural basis for PFOA mediated activation of PPARγ via binding to the PPARγ LBD. Using X-ray crystallography, the cocrystal structure of the PPARγ LBD:PFOA complex was solved. This revealed that PFOA occupies three distinct sites, two within the PPARγ LBD and one within the activation function 2 (AF2) on the protein surface. Structural comparison of PFOA binding with previously reported PPARγ:ligand complexes supports that PFOA activates PPARγ by a partial agonist mechanism at micromolar concentrations. Fluorescence polarization assays also revealed that PFOA binding to the AF2 is unlikely to occur in a cellular context and confirmed that PFOA behaves as a partial agonist in vitro, weakly recruiting a coactivator peptide to the AF2 of the PPARγ LBD. This discovery provides an advancement in understanding PFOA mediated regulation of PPARγ, giving new insight regarding regulation of PPARγ by PFAS and PFAS substitutes in general and can be applied to the design and assessment of safer PFAS.

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV β-scaffold in signal transduction.

Involvement of the Keap1-Nrf2-ARE pathway in the antioxidant activity of sinomenine

Sinomenine is a pure alkaloid isolated from Sinomenium acutum. This study is aimed to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2)-kelch-like ECH-associated protein-1(Keap1)-antioxidant response element (ARE) antioxidative signaling pathway in protecting sinomenine against H2O2-induced oxidative injury. Cytotoxicity and antioxidant experiments to initially determine the protective effects of sinomenine show that sinomenine has no effect on the decreased cell viability and presents similar potency in scavenging all three free radicals. The binding affinity between sinomenine and Keap1 was determined via fluorescence polarization assay, with IC50 of 13.52 μM. Quantum chemical calculation and theoretical simulation illustrated that sinomenine located into the Nrf2-binding site of Keap1 via hydrophobic and hydrogen interactions, showing high stability and binding affinity. On the basis of the stable binding of sinomenine with Keap1, sinomenine efficiently induced nuclear translocation of Nrf2, and increased in ARE activity in a concentration-dependent manner. Quantitative polymerase chain reaction provided further evidences that sinomenine-induced protection upregulated ARE-dependent genes, such as NAD(P)H quinone oxidoreductase 1, hemeoxygenase-1, and glutamate-cysteine ligase modifier subunit. Western blot confirmed that sinomenine increased the expressions of these antioxidative enzymes. Taken together, in vitro and in silico evaluations demonstrate that sinomenine inhibits the binding of Keap1 to Nrf2, promotes the nuclear accumulation of Nrf2 and thus leads to the upregulated expressions of Nrf2-dependent antioxidative genes. Our findings also highlight the use of sinomenine for pharmacological or therapeutic regulation of the Nrf2-Keap1-ARE system, which is a novel strategy to prevent the progression of oxidative injury.

Fluorescence Polarization Assays for Organic Compounds in Food Safety.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018-2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

Comparison of the Glucocorticoid Receptor Binding and Agonist Activities of Typical Glucocorticoids: Insights into Their Endocrine Disrupting Effects.

Over the past decades, the synthetic glucocorticoids (GCs) have been widely used in clinical practice and animal husbandry. Given the health hazard of these toxic residues in food, it is necessary to explore the detailed interaction mechanisms of typical GCs and their main target glucocorticoid receptor (GR). Hence, this work compared the GR binding and agonist activities of typical GCs. Fluorescence polarization assay showed that these GCs were potent ligands of GR. Their GR binding affinities were in the order of methylprednisolone>betamethasone≈prednisolone>dexamethasone, with IC50 values of 1.67, 2.94, 2.95, and 5.58 nM. Additionally, the limits of detection of dexamethasone, betamethasone, prednisolone, and methylprednisolone were 0.32, 0.14, 0.19, and 0.09 μg/kg in fluorescence polarization assay. Reporter gene assay showed that these GCs induced GR transactivation in a dose-dependent manner, confirming their GR agonist activities. Among which, dexamethasone at the concentration of 100 nM produced a maximal induction of more than 11-fold over the blank control. Molecular docking and molecular dynamics simulations suggested that hydrogen-bonding and hydrophobic interactions played an important role in stabilizing the GC-GR-LBD complexes. In summary, this work might help to understand the GR-mediated endocrine disrupting effects of typical GCs.

Evaluation of natural products from virtual screenings as SARS-CoV-2 main protease inhibitors using combinational experiments

Recently, andrographolide, kaempferol, maslinic acid, rutin, and schaftoside have been identified as potent SARS-CoV-2 main protease (Mpro) inhibitors via molecular docking studies. However, no comprehensive in vitro testing of these compounds against Mpro has been conducted. In this study, we rigorously evaluated the in vitro inhibition of Mpro by these compounds using combinational experiments, including fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays. Our data revealed that these compounds are not Mpro inhibitors based on the results from a set of in vitro assays. These results suggest that an efficient combination of a molecular docking approach and an experimental assay is essential for the discovery of Mpro inhibitors in the future.

Discovery of GPX4 inhibitors through FP-based high-throughput screening

Ferroptosis is a form of non-apoptotic cell death, regulated by phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), a selenoprotein with a selenocysteine residue (sec) in the active site. GPX4 is a promising target for cancer cells in therapy-resistant conditions via ferroptosis, which can reduce the level of lipid reactive oxygen species (ROS). So far, all existing GPX4 inhibitors covalently bind to GPX4 via a reactive alkyl chloride moiety or masked nitrile-oxide electrophiles with poor selectivity and pharmacokinetic properties and most were obtained by cell phenotype-based screening. Lacking of effective high-throughput screening methods for GPX4 protein limits the discovery of GPX4 inhibitors. Here, we report a fluorescence polarization (FP)-based high throughput screening (HTS) assay for GPX4-U46C-C10A-C66A in vitro, and found Metamizole sodium from our in-house compound library inhibits GPX4-U46C-C10A-C66A enzyme activity. Structure-activity relationships (SAR) demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. Our FP assay could be an effective tool for discovery of GPX4 inhibitors and Metamizole sodium was a potential inhibitor for GPX4 in vitro.

Design, synthesis, and biological evaluation of novel 8-substituted quercetin derivatives targeting the β‑catenin/B-cell lymphoma 9 interaction

The β-catenin/B-cell lymphoma 9 (BCL9) protein–protein interaction (PPI) is a potential target for aberrantly active Wnt/β-catenin signaling which actively participates in initiating and progressing of many cancers. Herein, we discovered novel 8-substituted quercetin derivatives with potential inhibitory activities targeting β-catenin/BCL9 PPI. Among all the derivatives, compound B4 displayed the most promising PPI inhibitory activity with an IC50 value of 2.25 μM in a competitive fluorescence polarization assay and a KD value of 1.44 μM for the β-catenin protein. Furthermore, B4 selectively inhibited the growth of colorectal cancer (CRC) cells, suppressed the transactivation of Wnt signaling, and downregulated the expression of oncogenic Wnt target gene. Especially, B4 showed potent anti-CRC activity in vivo with the tumor growth inhibition (TGI) of 75.99 % and regulated the tumor immune microenvironment.

Fluorescence Polarization Assay Based on a New Recognition Motif QepA for the One-Step Detection of Fluoroquinolones in Eggs.

A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g-1, with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4-12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.

Discovery of First-in-Class Small Molecule Inhibitors of the IRF4-PU.1/Spi-B Interaction

Introduction Many transcription factors (TF) are promising therapeutic targets in hematological malignancies but have been mislabeled as ‘undruggable’ due to their dynamic structures and lack of feasible paths for bioactive small molecules. Multiple myeloma (MM), the second most common hematological malignancy, is predominantly driven by oncogenic transcription factors such as c-Myc & interferon regulatory factor 4 (IRF4). Attempts at targeting IRF4 have relied on immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, which target upstream TFs of IRF4 - IKZF1 & IKZF3. Unfortunately, IMiDs are highly susceptible to resistance mechanisms in the clinic and fail to provide deep and sustained downregulation of IRF4 due to theirindirect mechanism of action. Currently, no known small molecules that directly bind and inhibit IRF4 exist. Moreover, no probes or screening based assays have been developed for IRF4 or any member of the IRF family.IRF4 is the top ranked dependency of MM and is a validated clinical target given the success of IMiD-based regimens. Thus, orally available small molecules that can directly target IRF4 will have significant benefit for patients with MM. Methods & Results To identify small molecules that inhibit or bind IRF4, we first cloned, expressed, and purified numerous constructs of the interferon accessory domain (IAD - residues 238-420) and full-length IRF4 (residues 1-451). To enable traditional high-throughput screening (HTS) campaigns, we first identified peptide ligands of IRF4 based on the IRF4 - PU.1/Spi-B interaction, an oncogenic signaling node in various hematological malignancies. We profiled peptides derived from PU.1 and Spi-B and confirmed binding to the IAD domain with K ds of 1 - 7 µM by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). Encouraged by the robust signal seen with our FP assay, we further developed a TR-FRET based method using a CoraFluor labeled IAD domain which significantly boosted our signal-to-noise ratio for our HTS efforts (Z' > 0.8). We screened this interaction against the Chembridge2020 drug library, which consists of 50,000 lead-like small molecules. The screen afforded 68 small molecules that had inhibition > 50% by TR-FRET (hit rate of ~0.1%). We prioritized our most potent compound, HIT-1, which has a TR-FRET IC 50 of 2 µM. Further binding validation including ITC, biotin-based pull-downs in human myeloma cell lines (HMCLs), and a BRET based target engagement assay confirm binding to both recombinant and full-length IRF4. Additionally, medicinal chemistry optimization led to the development of 1, with sub-micromolar affinity to IRF4, and an inactive control 2. Biological assessment of compounds 1 and inactive control 2 are underway in HMCLs; initial results show on-target modulation of IRF4 signaling processes. Compound 1 represents the first validated small molecule targeting the ‘undruggable’ IRF family and warrants its use in investigating the IRF4-PU.1/Spi-B interaction in oncology and immunology. Conclusions These small molecules are the first inhibitors of the IRF4-PU.1 interaction. We have developed novel biochemical assays that can be applied to other IRF family members, including IRF8. Additionally, we showcase translatable strategies to build novel biochemical assays for other transcription factors, leveraging known PPIs for probe development. These ligands represent starting points for hetero-bifunctional compounds and chemical probes for modulating IRF4 activity both in vitro and in vivo.

Co-Analysis of Genome-Wide DNA Methylation and Gene Expression Suggests Novel Regulatory Mechanism for RUNX1 in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by recurring chromosomal abnormalities that encode oncogenic fusion proteins. Approximately 10% of AML cases are associated with a chromosome 16 inversion [inv(16)(p13q22)] or translocation t(16;16)(p13q22). These chromosomal rearrangements results in the formation of the fusion oncogene CBFB-MYH11 and ultimately the fusion protein CBFβ-SMMHC. Although CBFβ-SMMHC was initially considered a dominant negative repressor of RUNX1, we have recently shown that it works together with RUNX1 to activate gene expression through direct target gene binding. RUNX1 is known to regulate gene expression at both transcriptional and epigenetic levels. Aberrant DNA methylation patterns have been reported across different cancer types including AML. Recent studies have shown that DNA methylation negatively influences RUNX1 DNA binding. We hypothesize that changes to DNA methylation and consequently dysregulated RUNX1 DNA binding contribute to the pathogenesis of inv(16) AML. In this study, we examined the epigenetic and transcriptomic landscapes of CBFB-MYH11 knockin mice and assessed the contribution of the resulting fusion protein CBFβ-SMMHC to RUNX1 DNA target binding. To assess the effects of DNA methylation on RUNX1 DNA binding in vitro, we initially performed fluorescence polarization and electrophoretic mobility shift assays using unmethylated and methylated RUNX1 DNA target sequences. RUNX1, RUNX1/CBFβ and RUNX1/CBFβ-SMMHC showed significantly lower binding affinity for methylated DNA compared to unmethylated DNA probes. To determine genome-wide DNA methylation changes, we performed enzymatic methyl sequencing on sorted Lin -Sca1 -c-Kit + bone marrow cells from control and pre-leukemic inv(16) mice. Inv(16) mice showed a significant genome-wide DNA hypermethylation compared to controls (13,478 hyper- vs 705 hypomethylated regions; q-value < 0.05), with 14% of the differentially methylated regions (DMRs) lying in promoters. Genes associated with these DMRs were enriched for gene ontology (GO) processes related to hematopoiesis, such as myeloid cell homeostasis and erythrocyte differentiation, and DNA replication, such as mitotic DNA replication initiation, among others. In addition, several RUNX1 target genes showed promoter hypermethylation, such as Klf1 or the thrombopoietin receptor Mpl. To understand the impact of aberrant DNA methylation on gene expression, we performed RNA-seq using the same cell population. Gene expression changes were observed in inv(16) mice compared to controls, with 741 genes being upregulated and 278 downregulated from a total of 1019 differentially expressed genes (DEG; absolute fold-change ≥ 2, q-value < 0.05). Enriched GO processes identified from the DEGs were associated with immune responses, such as response to interferon-alpha (and beta) and leukocyte activation involved in inflammatory response. We then intersected the DMR (focusing on promoters) and DEG lists to identify genes whose expression might be directly influenced by DNA methylation changes in inv(16) leukemia. Hypermethylation of promoters was associated with 102 genes, with 67% of them showing downregulation of gene expression in inv(16) mice, whereas hypomethylation was associated with 18 genes with 94% showing upregulation of gene expression. These results show that in inv(16) mice, promoter hypermethylation is associated with downregulation of gene expression, and hypomethylation with increased expression. We also confirmed that several RUNX1 target genes showing promoter hypermethylation also had reduced gene expression in inv(16) mice. The initial analysis suggests that CBFβ-SMMHC expression significantly affects the methylation landscape in mice, with important RUNX1 target genes exhibiting promoter hypermethylation and subsequent downregulation of gene expression. We are currently performing chromatin immunocleavage sequencing using the same cell population to determine RUNX1 binding in vivo. Overall, this study explores a novel regulatory mechanism of RUNX1 function through DNA methylation, and ultimately a new role for RUNX1 in the pathogenesis of inv(16) AML.