Fluorescence Polarization Assay Research Articles

Fluorescence Polarization Assay for Infection Diagnostics: A Review

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

High Affinity Inhibitors of the Macrophage Infectivity Potentiator Protein from Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila─A Comparison.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila, express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta-halogenated phenyl sulfonamide to be responsible for the high affinity.

Design, Synthesis, and Biological Evaluation of Lysine-Stapled Peptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor Activity In Vivo.

We introduce novel lysine-stapled peptide inhibitors targeting p53-MDM2/MDMX interactions. Leveraging the model peptides pDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a series of lysine-stapled analogues were designed and synthesized. Through in vitro cell assay screening, two lead compounds, SPDI-48-T1 and SPMI-48-T3, were identified for their excellent antiproliferation activity. Fluorescence polarization assays revealed that both compounds exhibited strong binding affinities against MDM2 and MDMX, ascertained by Kd values within the low micromolar spectrum. Further characterization of SPDI-48-T1 and SPMI-48-T3 demonstrated that SPDI-48-T1 possessed superior cell permeability and serum stability. Notably, SPDI-48-T1 displayed a dose-dependent suppression of tumor growth in an HCT116 xenograft mouse model. Our findings indicate that SPDI-48-T1 holds promise as a lead compound for further development as an anticancer agent by modulating p53-MDM2/MDMX interactions. Additionally, this study also proved that the lysine stapling strategy may serve as a robust approach for generating peptide ligands targeting other protein-protein interactions.

An engineered ubiquitin binding coiled coil peptide.

Recognition of ubiquitin (Ub) is often mediated by small Ub binding domains such as the Ubiquitin Interacting Motif (UIM). Most Ub binding events are low affinity interactions, and designing stronger binders for Ub can be challenging. We here report the design of a short crosslinked coiled coil (CC) which is conformationally and chemically stable, and which can act as a scaffold to present the key binding residues from known UIM sequences. Doing so gives rise to a hybrid CC peptide that reconciles the important features of both UIM and CC sequences. We show by fluorescence polarization assays that this crosslinked 'CC-UIM' peptide exhibits enhanced binding to Ub compared to the original UIM sequence. Furthermore, we report a crystal structure of this peptide in complex with Ub. These studies show that preorganization of a small number of important binding residues onto a stable helical scaffold can be a successful strategy for binder design.

Pore Engineering as a General Strategy to Improve Protein-Based Enzyme Nanoreactor Performance.

Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.

Discovery of potential inhibitors targeting SARS-CoV-2 Mpro.

The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has caused a global health emergency. The lack of a specific drug or treatment strategy against this disease makes it devastating. Given that the main protease (Mpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition prevents viral replication and constrains virus spread. Therefore, developing an effective SARS-CoV-2 Mpro inhibitor to treat COVID-19 is imperative. We employed a high-throughput screening (HTS) method based on fluorescence polarization (FP) assay and further confirmed by the fluorescence resonance energy transfer (FRET) method for the discovery of Mpro inhibitors. Then multiple approaches were taken to investigate the inhibition profiles of the hit compounds against Mpro, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay, surface plasmon resonance analysis (SPR), high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS), cytopathic effect (CPE) assay, molecule docking, and the drug-likeness analysis. In this study, four Mpro inhibitors with low toxicity were selected from HTS. According to SPR, all the hit compounds had medium binding affinities toward SARS-CoV-2 Mpro. HPLC-Q-TOF-MS results revealed the non-covalent linkage of each compound with SARS-CoV-2 Mpro. Molecule docking simulated the molecule interactions between each compound and the substrate binding pocket of SARS-CoV-2 Mpro. CPE assay was used to detect their inhibitory activities against coronaviruses HCoV-OC43 and HCoV-229E. In particular, the IMB63-8G compound demonstrated the highest antiviral potency [50% effective concentration (IC50) value of 1.71 μg/mL] and selectivity against HCoV-OC43 (SI = 39), which was more than 4-fold higher than that of ribavirin (RBV). Besides, the IMB63-8G compound possessed favorable drug-likeness characteristics. Our results will highlight the therapeutic potential of these compounds for the treatment of SARS-CoV-2 infection.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Applying a Fluorescence Polarization Assay for Detection of Brucellosis in Animals Using the Fluorescently Labeled Synthetic Oligosaccharides as Biosensing Tracer.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden's method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

Small Molecule Induces Time-Dependent Inhibition of Stat3 Dimerization and DNA-Binding Activity and Regresses Human Breast Tumor Xenografts.

Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 μM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 μM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, and Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1, pErk1/2MAPK, pShc, pJAK2, or pSrc induction, or on cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.

Structure-guided Development of Novel Benzothiophene Derivatives as PLK1-PBD Inhibitors

Background: Polo-like kinase 1 (PLK1), a validated target for tumor therapy, plays a key role in mitosis and is over-expressed in many tumors. In addition to its N-terminal kinase domain, PLk1 also harbors a C-terminal polo-box domain (PBD). Objective: A candidate based on PLK1-PBD was developed as a promising compound for future development. Methods: Seventeen small molecule PLK1-PBD inhibitors were designed, synthesized and evaluated for PLK1-PBD inhibitory activities by fluorescence polarization (FP) assay. The compounds with better inhibitory activities were further assessed for their anti-proliferative activities using a CCK-8 method. Results: The inhibitory rates of compounds 7a, 7d, 14a, 14d, 14e and 14f exceeded 98%. The IC50 values of compounds 7d, 14d, 14e, and 14f were 0.73 μM, 0.67 μM, 0.89 μM and 0.26 μM, proving better than MCC1019. Compound 14f showed the best inhibitory activity (IC50: 0.26 μM) and antiproliferative activities against three cancer cell lines (HeLa, HepG2 and MG63). Especially, compound 14f also exhibited acceptable safety profiles in the human ether-a-go-go related gene (hERG) and normal cell tests. The results of docking and prediction studies indicated that compound 14f had a high binding affinity to the target, with good drug-like absorption, distribution, metabolism, elimination, and toxicity (ADMET) properties. Conclusion: Compound 14f can be a promising compound for future development.

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 μM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

TracerDB: a crowdsourced fluorescent tracer database for target engagement analysis

Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery—it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket, and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

Erectile dysfunction in cardiovascular patients: A prospective study of the eNOS gene T‐786C, G894T, and INTRON variable number of the tandem repeat functional interaction

AbstractBackgroundCardiovascular disease induces erectile dysfunction modulated by endothelial nitric oxide synthase enzyme and an impaired ejection fraction that restricts penis vascular congestion. However, the mechanisms regulating endothelial dysfunction are not understood.ObjectivesExploring the functional impact of endothelial nitric oxide synthase genetic polymorphisms on erectile dysfunction and drug therapy optimization in high‐risk cardiovascular disease patients.Materials and methodsPatients with erectile dysfunction symptoms and candidates for andrology therapy were included (n = 112). Clinical data and endothelial nitric oxide synthase rs1799983 (G894T) and rs2070744 (T‐786C), genotyped by fluorescence polarization assays, were registered. The 27‐bp variable number of the tandem repeat polymorphism in intron 4 (intron4b/a) was analyzed by polymerase chain reaction‐restriction fragment length polymorphism. Association analyses were run with the R‐3.2.0 software.ResultsA significant association between endothelial nitric oxide synthase 786‐TT (p = 0.005) and the aa/ac of intron 4 variable number of the tandem repeat (p = 0.02) with higher erectile dysfunction susceptibility was observed in cardiovascular disease patients (60 ± 9 years, 66% severe erectile dysfunction, 56% ejection fraction). After 3‐months of phosphodiesterase type 5 inhibitors, erectile dysfunction (International Index of Erectile Function, 50 ± 16 scores, the International Index of Erectile Function‐Erectile Function 21 ± 10 scores, p < 0.001) and sexual quality of life (modified Sexual Life Quality Questionnaire 55 ± 23 scores, p < 0.001) had significantly improved. The cardiovascular ejection fraction was influenced positively with better sexual quality of life (0.1941), and also in the endothelial nitric oxide synthase G894‐T allele (p = 0.076) carriers, which could merit future analyses. Erectile dysfunction was present as the primary clinical manifestation in 62% of cases, with cardiovascular disease occurring concurrently. Only former smokers and obese subjects debuted prior to cardiovascular disease than to erectile dysfunction.ConclusionsOur study provides comprehensive insights into the functional interaction linking endothelial nitric oxide synthase gene polymorphisms, erectile function, and ejection fraction in high‐risk cardiovascular disease patients. Future therapeutic strategies could target endothelial nitric oxide synthase activity by including lifestyle changes and epigenetic modulations.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1 (AP1), we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of DMSO, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

Determining the Affinity and Kinetics of Small Molecule Inhibitors of Galectin-1 Using Surface Plasmon Resonance.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

Structure of the E. coli nucleoid-associated protein YejK reveals a novel DNA binding clamp.

Nucleoid-associated proteins (NAPs) play central roles in bacterial chromosome organization and DNA processes. The Escherichia coli YejK protein is a highly abundant, yet poorly understood NAP. YejK proteins are conserved among Gram-negative bacteria but show no homology to any previously characterized DNA-binding protein. Hence, how YejK binds DNA is unknown. To gain insight into YejK structure and its DNA binding mechanism we performed biochemical and structural analyses on the E. coli YejK protein. Biochemical assays demonstrate that, unlike many NAPs, YejK does not show a preference for AT-rich DNA and binds non-sequence specifically. A crystal structure revealed YejK adopts a novel fold comprised of two domains. Strikingly, each of the domains harbors an extended arm that mediates dimerization, creating an asymmetric clamp with a 30 Å diameter pore. The lining of the pore is electropositive and mutagenesis combined with fluorescence polarization assays support DNA binding within the pore. Finally, our biochemical analyses on truncated YejK proteins suggest a mechanism for YejK clamp loading. Thus, these data reveal YejK contains a newly described DNA-binding motif that functions as a novel clamp.

Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αIIbβ3 as Novel Antiplatelet Agents.

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin αIIbβ3, the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with αIIbβ3, we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with αIIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- αIIbβ3 interaction could carry a translational potential as antithrombotic agents.

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

Synthetic modulators of plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Herein, we describe a novel small-molecule inhibitor for 14-3-3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assays revealed 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 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

AbstractSynthetic modulators of plant 14‐3‐3s are promising chemical tools both for understanding the 14‐3‐3‐related signaling pathways and controlling plant physiology. Herein, we describe a novel small‐molecule inhibitor for 14‐3‐3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in‐house chemical library. Mass spectroscopy, mutant‐based analyses, fluorescence polarization assays, and thermal shift assays revealed 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 new insight into the design of potent and isoform‐selective 14‐3‐3 modulators.