Fluorescence Polarization Method Research Articles

Juglone suppresses vasculogenic mimicry in glioma through inhibition of HuR-mediated VEGF-A expression

Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)—an mRNA-binding protein—is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR.Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.

Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

Fluorescence polarization immunoassay for the determination and screening of medicines.

Modern clinical medicine with its achievements has helped a person to get rid of many infectious and non-communicable diseases, restore health, preserve and improve the quality of life of people with chronic dis- eases. Tens of thousands of medicines are used in medical practice. However, paradoxical as it sounds, having created medicines for almost all diseases, humanity has not become healthier and the need for medicines is only growing every year. More than 20 % of people receiving medications during therapy have various complications. Therefore, the search for medicines does not stop, but only increases at the present time. Another important problem is the detecting drugs in environmental objects and food products. Most drugs that get into wastewater from pharmaceutical factories and farms, even after treatment at wastewater treatment plants, are still diagnosed in the water. The method of fluorescence polarization is extremely widespread in clinical and biomedical fields. Thanks to the introduction into laboratory diagnostics of devices capable of measuring the signal of fluorescence polarization on microplates, polarization fluorescent analysis is used not only in the traditional format: the detection of drugs in human physiological fluids, environmental objects and food, but also in high-tech screening of drugs, significantly speeding up and facilitating the process of identifying new drugs.

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Microscale thermophoresis and fluorescence polarization assays of calcineurin-peptide interactions

Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It is very important to study the affinity between calcineurin and its substrate or other interacting proteins. Two conserved motifs have been reported on the interactive proteins of calcineurin, namely, the PxIxIT motif and the LxVP motif. Here, we used 5(6)-carboxyfluorescein to fluorescently label the N-terminus of the short peptides derived from the two motifs and then determined the affinity between the protein and polypeptides. Microscale thermophoresis (MST) is very suitable for determining calcineurin with peptides containing the LxVP motif. The Kd values of the binding of calcineurin with NFATc1-YLAVP, NHE1-YLTVP, and A238L-FLCVK peptides were 6.72 ± 0.19 μM, 17.14 ± 0.35 μM, and 15.57 ± 0.10 μM, respectively. The GST pull-down results further confirmed the binding trend of the three peptides to calcineurin. However, fluorescently labeled PxIxIT polypeptides are not suitable for MST due to their own aggregation. We determined the binding affinity of the RCAN1-PSVVVH polypeptide to calcineurin by the fluorescence polarization (FP) method. MST and FP assays are fast and accurate in determining the affinity between protein-peptide interactions. Our research laid the foundation for screening the molecules that affect the binding between calcineurin and its substrates in the future.

A simple and high-sensitive fluorescence polarization method for screening small molecule agonists of glucagon-like peptide-1 receptor

Glucagon-like peptide-1 receptor (GLP-1R) is an important therapeutic target for the treatment of type-2 diabetes. Currently-available GLP-1R agonists in clinic are peptides that require injection. It is necessary to develop oral small molecule drugs to provide conventional therapeutics for this chronic disease. At present, nuclear magnetic resonance, ELISA method and isotope tracing, have been used to screen small molecule drugs for anti-type 2 diabetes, but these methods have the disadvantages of either operational cumbersome or high risk.

Fluorescence polarization method for detection of lard mixed with olive oil

This study is based on fluorescence polarization as a potential alternative method for an investigation of contamination of lard in food. The purpose of this study was to obtain the characteristic of fluorescence polarization in olive oil samples mixed with lard. The characteristics of fluorescence polarization were obtained by measuring changes in the polarization of light using a linearly polarized green laser pointer and observed in the direction of the scattering angle of 90˚ for various direction of electric field of the laser. The results showed that the critical angle of direction of electric field of the laser and the average polarization change increase as the concentration of lard is raised. It could be due by additional asymmetric triglycerides and molecular orientation that play an important role in the sample. This result shows that additional mixture of lard in olive oil can be detected, and this method seems to be developed for the detection of low concentrations of lard contamination.

Neuroprotective Effect of N-Cyclohexylethyl-[A/G]-[D/E]-X-V Peptides on Ischemic Stroke by Blocking nNOS-CAPON Interaction.

The protein-protein interaction between neuronal nitric oxide syntheses (nNOS) and the carboxy-terminal PDZ ligand of nNOS (CAPON) is a potential target for the treatment of ischemic stroke. Our previous study had identified ZLc-002 as a promising lead compound for inhibiting nNOS-CAPON coupling. To find better neuroprotective agents disrupting the ischemia-induced nNOS-CAPON interaction, a series of N-cyclohexylethyl-[A/G]-[D/E]-X-V peptides based on the carboxy-terminal tetrapeptide of CAPON was designed, synthesized, and evaluated in this study. Herein, we reported an affinity-based fluorescence polarization (FP) method using 5-carboxyfluorescein (5-FAM) labeled CAPON (496-506) peptide as the probe for high-throughput screening of the small-molecule inhibitors of the PDZ domain of nNOS. N-Cyclohexylethyl-ADAV displayed the most potent affinity for the nNOS PDZ domain in the FP and isothermal titration calorimetry (ITC) (ΔH = -1670 ± 151.0 cal/mol) assays. To improve bioavailability, lipophilicity, and membrane permeability, the Asp methylation was employed to get N-cyclohexylethyl-AD(OMe)AV, which possesses good blood-brain barrier (BBB) permeability in vitro parallel artificial membrane permeability assay (PAMPA)-BBB (Pe = 6.07 cm/s) and in vivo assays. In addition, N-cyclohexylethyl-AD(OMe)AV (10 mg/kg body weight, i.v., immediately after reperfusion) substantially reduced infarct size in rats, which was measured 24 h after reperfusion and subjected to 120 min of middle cerebral artery occlusion (MCAO).

Binding Assays Using a Benzofurazan-Labeled Fluorescent Probe for Estrogen Receptor-Ligand Interactions.

Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (Kd) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

Abstract A43: Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction

Abstract K-Ras functionality is integral for cell growth and differentiation due its canonical role in signaling the PI3K and MAPK pathways. Genetic analysis on various cancers revealed that mutations in the K-Ras gene are responsible for 21% of all cases, which include lung, pancreatic and colorectal cancer. Prior efforts at directly targeting mutant K-Ras have been largely unsuccessful, thus creating a demand for novel therapeutic approaches. Common to the aforementioned cancers is expression of the Insulin-Like Growth Factor 2 mRNA Binding Protein 1 (IMP-1). IMP-1 is part of the VICKZ family of RNA-binding proteins known for post-transcriptional regulation, including its ability to shield its mRNA substrates from decay. One notable substrate of IMP-1 is the K-Ras mRNA, which was identified using UV-crosslinking experiments. Downregulation of IMP-1 through siRNA treatment led to a reduction of K-Ras protein by 60% in SW480 colorectal cancer cells and consequently reduced cell proliferation. The studies suggest a relationship between IMP-1 binding, mRNA turnover and the resultant protein expression of target substrates. To study the IMP-1-mRNA relationship and to help find inhibitors of such interaction, we developed a fluorescence polarization (FP) method that employed 45-nt K-Ras and 39-nt CD44 fluorescein-labeled RNA probes. Using full-length IMP1, truncated and point-mutated IMP1, we showed that the FP method faithfully recapitulated the relative affinity of recombinant proteins for both K-Ras and CD44 RNAs. The FP method was then used to find inhibitors of the IMP-1-K-Ras mRNA interaction and to elucidate potential domain target of inhibitors. We found that oligonucleotides SM6 and SM7, but not SM2, could significantly reduce the FP units, consistent with the results from electrophoretic mobility shift assay. In addition, the molecule UNBC143 was found to decrease FP units in a dose-dependent manner, suggesting its ability to inhibit IMP-1-K-Ras RNA interaction. The SM oligonucleotides and UNBC143 were also assessed on K-Ras expression in SW480 human colon cancer cells. These experiments and their results will be discussed in further detail upon presentation. Citation Format: Victor Liu, Chuyi Wang, Chow Lee. Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A43.

Five new 2-(2-phenylethyl)chromone derivatives from agarwood.

Agarwood has been used as an incense and in traditional medicines as aphrodisiac, sedative, cardiotonic, and carminative. In this study, five new 2-(2-phenylethyl)chromones (2, 13-16) and eleven known compounds (1, 3-12) were isolated from the agarwood. The structures of the new compounds were determined by 1H-, 13C-, and two-dimensional NMR together with electronic circular dichroism (ECD) spectroscopy. All isolated compounds were evaluated for the phosphodiesterase (PDE) 3A and 5A1 inhibitory activity by the fluorescence polarization method. Dimeric 2-(2-phenylehyl)chromones (13, 14, 16) had potent inhibitory activity to PDE 5A1 with IC50 values of micro molar range (13: 4.2μM, 14: 7.9μM, 16: 4.3μM), whereas they had weak activity to PDE 3A. In contrast, compound (15), which has a phenylpropionic acid moiety instead of the 2-(2-phenylethyl)chromone moiety in the dimers, showed moderate inhibition of both PDE 3A (IC50: 42.6μM) and PDE 5A1 (IC50: 15.1μM).

Fluorescence polarization assay for the identification and evaluation of inhibitors at YAP–TEAD protein–protein interface 3

The Hippo signaling pathway controls cell–cell contact, cell proliferation, as well as organ size by integrating changes in the cellular microenvironment. In recent years, the pivotal role of Hippo signaling in cancers has been well recognized. Inhibition of the pathway promotes the translocation of the major Hippo pathway effectors, the yes-associated protein (YAP) and its paralog TAZ, to the nucleus, where they interact with the transcription factor family transcriptional enhancer associate domain (TEAD), thus coactivating the expression of downstream genes, leading to cell transformation, tissue overgrowth, and tumor development. Therefore, the interruption of the YAP–TEAD transcriptional complex represents a novel opportunity for the treatment of cancer. Here, we established a fluorescence polarization (FP)-based assay for the identification and evaluation of YAP–TEAD protein–protein interface (PPI) inhibitors at the YAP Ω-loop binding region of TEAD, which is also called interface 3 at the YAP–TEAD binding surface. Furthermore, a patented small molecule (Patent-22) was evaluated by the FP assay, which confirmed that it was a YAP–TEAD PPI inhibitor at interface 3. Possessing great application value, this FP method is reliable, robust, and economical for inhibitor assessment and drug discovery.

Method of fluorescence polarization for a new alternative tool for investigation of cooking oil and lard

Testing the quality of cooking oil is very important, especially to ensure the quality and identification of other unexpected mixture of cooking oil. In this paper, the fluorescence polarisation method will be used to test the presence of lard contamination on cooking oil. Fluorescence polarization is similar to fluorescence intensity, in that it is based on the emission of light by an excited fluorophore. However, samples are excited by polarized light which is selected by specific polarizing filters. The angle of linearly polarized incoming light was adjusted by using a polarizer. The change of fluorescent polarization angle was measured by using a second polarizer (analyzer) to obtain a relation between θ-value and φ-value for all samples. The samples were various edible oils, i.e., chicken oil, lard, and canola oil. The test is carried out by measuring the change in the average polarization angle of the fluorescent light to the polarizing angle of each incoming light over the sample directly. The results of the critical angle showed that all three samples were characteristic. Chicken oil has φc = 20° with θAv = 11.28° to 13.63°, lard has φc = 40° with θAv = 7.13° to 9.51°, whereas canola oil has φc = 30° with θAv = 2.59°. These results show that the fluorescence polarization method has a good prospect for oil contamination test on cooking oil.

Multi-Residue Method for the Analysis of Stilbene Estrogens in Milk.

The rapid analysis of stilbene estrogens is crucially important in the environment, food and health sectors, but quantitation of lower detection limit for stilbene estrogens persists as a severe challenge. We herein described a homologous and sensitive fluorescence polarization (FP) assay based on estrogen receptor α ligand binding domain (ER-LBD) to monitor stilbene estrogens in milk. Under optimal conditions, the half maximal inhibitory concentrations (IC50) of the FP assay were 9.27 nM, 12.94 nM, and 22.38 nM for hexestrol, dienestrol and diethylstilbestrol, respectively. And the corresponding limits of detection (LOD) values were 2.94 nM, 2.89 nM, and 3.12 nM. Finally, the assay was applied to determine the stilbenes in milk samples where the mean recoveries ranged from 95.76% to 112.78% and the coefficients of variation (CV) below 12.00%. Furtherly, we have focused our study on high cross-reactivity phenomena by using two in silico approaches, including molecular docking analysis and topology analysis. Overall, docking results show that several residues in the hydrophobic pocket produce hydrophobic interactions with the tested drug molecules, which contribute to the stability of their binding. In this paper, we conclude that the FP method is suitable for the rapid detection of stilbenes in milk samples, requiring no expensive analytical equipment or time-consuming sample preparation. This work offers a practical approach that applies bioscience technology in food safety testing and improves analytical speed and laboratory efficiency.

Fluorescence Anisotropy Reloaded-Emerging Polarization Microscopy Methods for Assessing Chromophores' Organization and Excitation Energy Transfer in Single Molecules, Particles, Films, and Beyond.

Fluorescence polarization is widely used to assess the orientation/rotation of molecules, and the excitation energy transfer between closely located chromophores. Emerging since the 1990s, single molecule fluorescence spectroscopy and imaging stimulate the application of light polarization for studying molecular organization and energy transfer beyond ensemble averaging. Here, traditional fluorescence polarization and linear dichroism methods used for bulk samples are compared with techniques specially developed for, or inspired by, single molecule fluorescence spectroscopy. Techniques for assessing energy transfer in anisotropic samples, where the traditional fluorescence anisotropy framework is not readily applicable, are discussed in depth. It is shown that the concept of a polarization portrait and the single funnel approximation can lay the foundation for alternative energy transfer metrics. Examples ranging from fundamental studies of photoactive materials (conjugated polymers, light-harvesting aggregates, and perovskite semiconductors) to Förster resonant energy transfer (FRET)-based biomedical imaging are presented. Furthermore, novel uses of light polarization for super-resolution optical imaging are mentioned as well as strategies for avoiding artifacts in polarization microscopy.

EVALUASI KUALITAS MUTU MINYAK GORENG MENGGUNAKAN POLARISATOR TERPADU BERDASARKAN METODE FLUORESENSI ELEKTROOPTIK

Evaluation of quality of cooking oil using integrated polarizer based on fluorescence polarization method has been carried out. Integrated polarizer used a green laser with a wavelength of 532 ± 10 nm. Measurements were made by observing changes in fluorescence polarization angles that occurs when without an external electric field and with the provision of an external electric field produced from two copper plates given a voltage of 0-6 kV. The samples used were new cooking oil and expired cooking oil, palm cooking oil that has been contaminated by chicken oil and lard. The test results show that the change in polarization angle on cooking oil expires is greater than the new cooking oil, as well as changes in the polarization angle in palm cooking oil that has been contaminated by lard is greater than pure palm cooking oil and palm cooking oil. This indicates that the saturated fatty acid content in lard is greater than that of pure palm cooking oil and chicken oil. With these results, the fluorescence polarization method is expected to be a method for evaluating halal cooking oil.

Design of Integrated Polarizer to Evaluate Quality of Cooking Oil Based on the Fluorescence Polarization Method

An integrated polarizer was designed as an alternative test tool to evaluate quality of cooking oils. Integrated Polarizer was composed of light source with wavelength of 532 ± 10 nm, polarizer, analyser, cuvette, electrodes, and high voltage source. In this research, the tool works based on the fluorescence polarization method. Measurements were made by observing the fluorescence polarization angle changes that occur and by applying an external electric field at the samples using high voltage of 0-9 kV in the parallel plate. The results show that the expired cooking oil has a greater polarization angle than the edible cooking oil. The results also show that the change in the polarization angle will increase in proportion to the increase of the heating time. The condition of saturated fatty acids in each sample has an effect on the change of polarization angle.

Formulation of critical micellar concentration of non- phosphate detergents by surface tension measurement

Non-phosphate builders which are environmentally friendly are required for use in detergent formulation to replace the sodium tri-poly phosphate (STPP). Phosphates are excellent fertilizer for algae, bacteria, and other flora and fauna in rivers, lakes and oceans, making them bloom at very rapid rates. This exhausts the oxygen supply both in the surface and in the bottom layers of water bodies, and leading the death of fishes. Sodium metasilicate a non-phosphate builder from Lokoja Quartzite was used to formulate a non-phosphate biodegradable synthetic detergent using 25%, 50% and 75% sodium hydroxide concentrations. The critical micellar concentration (CMC) of a non-phosphate detergent was determined via measurement of surface tension. This method is rapid, reliable and cheap compared to the fluorescence polarization method which required a sophiscated materials such as 1,6-diphenyl-1,3,5-hexatriene as fluorescence probe. The detergents formulated with 75% and 50% sodium hydroxide concentrations have CMC values of 8.6685 mM and 6.9348 mM respectively. From this study, a non phosphate builder synthesized from Lokoja Quartzite has potential of protecting aquatic animals when compared with the conventional STPP currently used in our detergent formulation. Also, the simple and economical approach for determination of detergent’s CMC has demonstrated it’s potentials. These values agree well with the literature value ranges between 7 to 10 mM. Keywords : Detergent; CMC; Surfactants; Non-phosphate; 1,6-diphenyl-1,3,5-hexatriene

Highly Simple and Sensitive Molecular Amplification-Integrated Fluorescence Anisotropy for Rapid and On-Site Identification of Adulterated Beef.

Fluorescence polarization (FP) signal is a self-referencing fluorescence signal, and it is less dependent on dye concentration and environmental interferences, which makes FP measurement a highly attractive alternative sensing technology to conventional fluorescent detection methods. Here we adopted a strategy for rapid increase of molecular weight to increase the FP signal for the detection of meat adulteration. The molecular weight of fluorescent labeled primers increased rapidly by slight preamplification, and the FP values were varied accordingly. We found a positive correlation between adulteration ratio and the FP signals. Detection limit for adulterated beef can be reached as low as 0.1% (wt %), meeting or better than the most detection requirements. On the basis of this proposed amplification-integrated FP method, both the standard samples and the commercial processed beef samples were successfully authenticated with satisfied results.

Three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone derivatives enantiomeric to agarotetrol from agarwood.

Agarwood (jinkoh in Japanese) is a resinous wood from Aquilaria species of the family Thymelaeaceae and has been used as incense and in traditional medicines. Characteristic chromone derivatives such as agarotetrol have been isolated from agarwood. In previous study, we isolated two new 2-(2-phenylethyl)chromones together with six known compounds from MeOH extract of agarwood. Further chemical investigation of the MeOH extract led to isolation of eighteen 2-(2-phenylethyl)chromones, including three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones with stereochemistry enantiomeric to agarotetrol-type, viz. (5R,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (2), (5R,6S,7S,8R)-2-[2-(4'-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (6), and (5R,6S,7S,8R)-2-[2-(4'-hydroxy-3'- methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (13). The absolute configurations of the new compounds were determined by exciton chirality method. All isolated compounds were tested for their phosphodiesterase (PDE)3A inhibitory activity by fluorescence polarization method. Compounds 8, 12-15, 21-24 showed moderate PDE3A inhibitory activity.