Seco-cyclic phorbol derivatives and their anti-HIV-1 activities.

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (Kd) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 µmol·L-1 and a CC50 of 153.92 µmol·L-1, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Insertion Depth Modulates Protein Kinase C-δ-C1b Domain Interactions with Membrane Cholesterol as Revealed by MD Simulations.

Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

Tigliane-Type Diterpene Esters from the Fruits of Shirakiopsis indica and Their Anti-HIV Activity.

Four new diterpene esters, shirakindicans A-D (1-4), along with eight related known diterpene esters (5-12), were isolated from the fruits of the Bangladeshi medicinal plant Shirakiopsis indica. The structures of 1-4 were elucidated by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Shirakindican A (1) was assigned as a tigliane-type diterpene ester possessing an unusual 6ß-hydroxy-1,7-dien-3-one structure, while shirakindican B (2) exhibits a tiglia-1,5-dien-3,7-dione structure. The anti-HIV activities of the isolated diterpene esters were evaluated and showed significant activities for sapintoxins A (5) and D (11), with EC50 values of 0.0074 and 0.044 µM, respectively, and TI values of 1 100 and 5 290. Sapatoxin A (12) also exhibited anti-HIV activity with an EC50 value of 0.13 µM and a TI value of 161.

Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane.

Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 µs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.

New phorbol ester derivatives as potent anti-HIV agents.

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.

Phorbol Diesters and 12-Deoxy-16-hydroxyphorbol 13,16-Diesters Induce TGFα Release and Adult Mouse Neurogenesis.

A small library of phorbol 12,13-diesters bearing low lipophilicity ester chains was prepared as potential neurogenic agents in the adult brain. They were also used in a targeted UHPLC-HRMS screening of the latex of Euphorbia resinifera. Two new 12-deoxy-16-hydroxyphorbol 13,16-diesters were isolated, and their structures were deduced using two-dimensional NMR spectroscopy and NOE experiments. The ability of natural and synthetic compounds to stimulate transforming growth factor alpha (TFGα) release, to increase neural progenitor cell proliferation, and to stimulate neurogenesis was evaluated. All compounds that facilitated TGFα release promoted neural progenitor cell proliferation. The presence of two acyloxy moieties on the tigliane skeleton led to higher levels of activity, which decreased when a free hydroxyl group was at C-12. Remarkably, the compound bearing isobutyryloxy groups was the most potent on the TGFα assay and at inducing neural progenitor cell proliferation in vitro, also leading to enhanced neurogenesis in vivo when administered intranasally to mice.

The allylic oxidation of tigliane esters.

As part of a study on the structure-activity relationships of the anticancer agent tigilanol tiglate (EBC-46, 2), the allylic oxidation of phorbol triacetate (1c) and of the acetonide of its 3αH-dihydroderivative (5) was investigated. The aim was to introduce an oxygen function at C-5 en route to point-like analogues of 2, but functionalization of C-10 was instead observed. This was followed by oxidative fragmentation of ring B to the 9,10-secotigliane derivative 6 and oxidation of the endocyclic Δ6 double bond to the C-6/C-10 oxygen bridged 7-oxotigliane 7. Despite the over-functionalization of ring B, these observations suggest the possibility to modify positions overlooked in the oxidase phase of tigliane biosynthesis and explore novel areas of the phorbol chemical space.

12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway.

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

Prodrugs of PKC modulators show enhanced HIV latency reversal and an expanded therapeutic window.

AIDS is a pandemic disease caused by HIV that affects 37 million people worldwide. Current antiretroviral therapy slows disease progression but does not eliminate latently infected cells, which resupply active virus, thus necessitating lifelong treatment with associated compliance, cost, and chemoexposure issues. Latency-reversing agents (LRAs) activate these cells, allowing for their potential clearance, thus presenting a strategy to eradicate the infection. Protein kinase C (PKC) modulators-including prostratin, ingenol esters, bryostatin, and their analogs-are potent LRAs in various stages of development for several clinical indications. While LRAs are promising, a major challenge associated with their clinical use is sustaining therapeutically meaningful levels of the active agent while minimizing side effects. Here we describe a strategy to address this problem based on LRA prodrugs, designed for controllable release of the active LRA after a single injection. As intended, these prodrugs exhibit comparable or superior in vitro activity relative to the parent compounds. Selected compounds induced higher in vivo expression of CD69, an activation biomarker, and, by releasing free agent over time, significantly improved tolerability when compared to the parent LRAs. More generally, selected prodrugs of PKC modulators avoid the bolus toxicities of the parent drug and exhibit greater efficacy and expanded tolerability, thereby addressing a longstanding objective for many clinical applications.

Hydroxy-octadecenoic acids instead of phorbol esters are responsible for the Jatropha curcas kernel cake's toxicity.

The toxic kernel cake of Jatropha curcas (KCakeJ) is an emerging health and environmental concern. Although phorbol esters are widely recognized as the major toxin of KCakeJ, convincing evidence is absent. Here, we show that rather than phorbol esters an isomeric mixture of 11-hydroxy-9E-octadecenoic acid, 12-hydroxy-10E-octadecenoic acid and 12-hydroxy-10Z-octadecenoic acid (hydroxy-octadecenoic acids, molecular formula C18H34O3) is the major toxic component. The toxicities of hydroxy-octadecenoic acids on experimental animals, e.g. acute lethality, causing inflammation, pulmonary hemorrhage and thrombi, allergies, diarrhea and abortion, are consistent with those on human/animals caused by Jatropha seed and/or KCakeJ. The hydroxyl group and the double bond are essential for hydroxy-octadecenoic acids' toxicity. The main pathway of the toxicity mechanism includes down-regulating UCP3 gene expression, promoting ROS production, thus activating CD62P expression (platelet activation) and mast cell degranulation. The identification of the major toxin of KCakeJ lays a foundation for establishing an environmentally friendly Jatropha biofuel industry.

Identification, characterization, and comparison of n-alkanols and anesthetics binding to the C1b subdomain of protein kinase cα: similar function with different binding sites.

Protein kinase C (PKC) is a family of lipid-activated enzymes involved in anesthetic preconditioning signaling pathways. Previously, n-alkanols and general anesthetics have been found to activate PKC by binding to the kinase C1B subdomain. In the present study, we attempt to ascertain the molecular mechanism and interaction mode of human PKCα C1B subdomain with a variety of exogenous n-alkanols and volatile general anesthetics as well as endogenous activator phorbol ester (PE) and co-activator diacylglycerol (DG). Systematic bioinformatics analysis identifies three spatially vicinal sites on the subdomain surface to potentially accommodate small-molecule ligands, where the site 1 is a narrow, amphipathic pocket, the site 2 is a wide, flat and hydrophobic pocket, and the site 3 is a rugged, polar pocket. Further interaction modeling reveals that site 1 is the cognate binding region of natural PE activator, which can moderately simulate the kinase activity in an independent manner. The short-chain n-alkanols are speculated to also bind at the site to competitively inhibit PE-induced kinase activation. The long-chain n-alkanols and co-activator DG are found to target site 2 in a nonspecific manner, while the volatile anesthetics prefer to interact with site 3 in a specific manner. Since the site 1 is composed of two protein loops that are also shared by sites 2 and 3, binding of n-alkanols, DG and anesthetics to sites 2 and 3 can trigger a conformational displacement on the two loops, which enlarges the pocket size and changes the pocket configuration of site 1 through an allosteric mechanism, consequently enhancing kinase activation by improving PE affinity to the site.

A new highly oxygenated abietane diterpenoid and a new lysosome generating phorbol ester from the roots of Euphorbia fischeriana Steud.

Twenty-two diterpenoids (1-22), including two new ones (1, a tigliane-type diterpenoid and 8, an abietane-type diterpenoid) were isolated from the roots of Euphorbia fischeriana Steud. Among them, compounds 4, 7, 12, 14-16, 19-21 are isolated from this plant for the first time. Their structures were elucidated through extensive 1D, 2D NMR and the HRESIMS data. The 13C data of 4 is hereby presented for the first time. The macrocyclic diterpenes 1 and 2 showed marked enhancement of lysosomal biosynthesis after evaluation using lysoTracker staining method.

Isolation of phenanthrenes and identification of phorbol ester derivatives as potential anti-CHIKV agents using FBMN and NAP from Sagotia racemosa.

In an effort to identify inhibitors of Chikungunya virus (CHIKV) replication, a systematic study of 594 extracts of plant species originating from the French Guiana plateau region was performed in a virus-cell-based assay for CHIKV assay. The extract obtained from the stem bark of Sagotia racemosa was selected for its potent antiviral activity. Using a classical bioassay-guided procedure, three undescribed degraded diterpenoids, i.e. trigohowilols C and D and trigoflavidol D, as well as trigoxyphin K, stictic acid, hyperhomosekikaic acid and five known flavonoids were isolated. The structures of these compounds were elucidated by extensive NMR spectroscopic data analysis. Although trigohowilols C and D were isolated from the most active fraction they didn't show any antiviral activity. By using the Feature-Based Molecular Networking (FBMN) and Network Annotation Propagation (NAP) workflows, it has been shown that the strong anti-CHIKV activity found for this fraction might be due to the presence of analogues of 12-O-tetradecanoylphorbol-13-acetate (TPA), one of the most potent inhibitors of CHIKV replication identified to date.

Molecularly Imprinted Polymer-Coated Probe Electrospray Ionization Mass Spectrometry Determines Phorbol Esters and Deoxyphorbol Metabolites in Jatropha curcas Leaves.

In this study, a molecularly imprinted polymer-coated probe electrospray ionization mass spectrometry (MIPCPESI-MS) method was developed for detection of phorbol esters (PEs) and deoxyphorbol metabolites in Jatropha curcas leaves. Such an approach was established by sticking on a metallic needle a molecularly imprinted polymer to particularly design a MIP-coated probe for selective sampling and ionization of PEs and deoxyphorbol metabolites. By a subsequent application of a high voltage and methanol, as spray solvent, ESI was generated for direct and rapid analysis under ambient and open-air conditions. MIP-coated probe exhibited a high sampling capacity of the PEs and its metabolites in methanolic extracts of J. curcas leaves compared with the non-imprinted polymer (NIP)-coated probe. MIPCPESI-MS allowed the detection of phorbol 12,13-diacetate (PDA) from J. curcas leaves with minimal sample preparation, and with detection limit and quantification reaching 0.28 µg/mL and 0.92 µg/mL, respectively. Also, good linearity was obtained with R2 > 0.99 and precision and accuracy values between 4.06-13.49% and - 1.60 to - 15.26%, respectively. The current method was successfully applied to screening methanolic extracts of six different J. curcas leaf genotypes (three toxic and three non-toxic). PDA and three PE deoxyphorbol metabolites were identified only from toxic genotypes, in which PDA was determined with concentration ranging from 222.19 ± 23.55 to 528.23 ± 19.72 µg/g. All these findings support that the MIPCPESI-MS method developed here has a high potential for the analysis of PEs in plant extracts enabling differentiation of toxic and non-toxic genotypes earlier in the leaves.

Structural Identification and Systematic Comparison of Phorbol Ester, Dioleoylglycerol, Alcohol and Sevoflurane Binding Sites in PKCδ C1A Domain.

Protein kinase C (PKC) is a family of signal transducing enzymes that have been implicated in anesthetic preconditioning signaling cascade. Evidences are emerging that certain exogenous neuromodulators such as n-alkanols and general anesthetics can stimulate PKC activity by binding to regulatory C1A domain of the enzyme. However, the accurate binding sites in C1A domain as well as the molecular mechanism underlying binding-stimulated PKC activation still remain unelucidated. Here, we report a systematic investigation of the intermolecular interaction of human PKCδ C1A domain with its natural activator phorbol ester (PE) and co-activator dioleoylglycerol (DOG) as well as exogenous stimulators butanol, octanol and sevoflurane. The domain is computationally identified to potentially have three spatially vicinal ligand-binding pockets 1, 2 and 3, in which the pockets 1 and 2 have previously been determined as the binding sites of PE and DOG, respectively. Systematic cross-binding analysis reveals that long-chain octanol and DOG are well compatible with the flat, nonpolar pocket 2, where the nonspecific hydrophobic contacts and van der Waals packing are primarily responsible for the binding, while the general anesthetic sevoflurane prefer to interact with the rugged, polar pocket 3 through specific hydrogen bonds and electrostatic forces. Short-chain butanol appears to bind effectively none of the three pockets. In addition, the pocket 1 consists of two angled arms 1 and 2 that are also involved in pockets 2 and 3, respectively. Dynamics characterization imparts that binding of long-chain octanol and DOG to pocket 2 or binding of sevoflurane to pocket 3 can induce a conformational displacement in arm 1 or 2, thus further opening the included angle and enlarging pocket 1, which can improve the pocket 1-PE affinity via an allosteric mechanism, consequently stimulating the PE-induced PKCδ activation.

Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues.

To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.

The C1 domain of Vav3, a novel potential therapeutic target.

Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.

Anti-HIV Activities and Mechanism of 12-O-Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae.

APOBEC3G is a member of the human cytidine deaminase family that restricts Vif-deficient viruses by being packaged with progeny virions and inducing the G to A mutation during the synthesis of HIV-1 viral DNA when the progeny virus infects new cells. HIV-1 Vif protein resists the activity of A3G by mediating A3G degradation. Phorbol esters are plant-derived organic compounds belonging to the tigliane family of diterpenes and could activate the PKC pathway. In this study, we identified an inhibitor 12-O-tricosanoylphorbol-20-acetate (hop-8), a novel ester of phorbol which was isolated from Ostodes katharinae of the family Euphorbiaceae, that inhibited the replication of wild-type HIV-1 and HIV-2 strains and drug-resistant strains broadly both in C8166 cells and PBMCs with low cytotoxicity and the EC50 values ranged from 0.106 µM to 7.987 µM. One of the main mechanisms of hop-8 is to stimulate A3G expressing in HIV-1 producing cells and upregulate the A3G level in progeny virions, which results in reducing the infectivity of the progeny virus. This novel mechanism of hop-8 inhibition of HIV replication might represents a promising approach for developing new therapeutics for HIV infection.

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

BACKGROUND: Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. METHODS: To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. RESULTS: Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. CONCLUSIONS AND SIGNIFICANCE: This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.