The Natural Product Magnolol as a Lead Structure for the Development of Potent Cannabinoid Receptor Agonists

Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenyl)phenol), the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB) receptors. We now investigated the structure-activity relationships of (tetrahydro)magnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB₁/CB₂ full agonist 2-(2-methoxy-5-propyl-phenyl)-4-hexylphenol (61a, K _i CB₁∶0.00957 µM; K _i CB₂∶0.0238 µM), and the CB₂-selective partial agonist 2-(2-hydroxy-5-propylphenyl)-4-pentylphenol (60, K _i CB₁∶0.362 µM; K _i CB₂∶0.0371 µM), which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC₅₀ value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies.     

Synthesis of Okicamelliaside,a Glucoside of Ellagic Acid with Potent Anti-Degranulation Activity

During our scrutiny of GC-EI-MS date for C₁₅ alcohols as putative intermediates on the ABA biosynthetic pathway in Cercospora cruenta, a trace amount of 5-[2',2'-dimenthyl-6'-methylene-1'-cyclohexyl]-3-methyl-4-penten-1-ol (2,3-dihydro-γ-ionylideneethanol) was identified. Feeding experiments indicated that this compound was not an intermediate to ABA, but a catabolite that originated from γ-ionylideneacetaldehyde. The stereochemistry of 2,3-dihydro-γ-ionylideneethanol was deduced to be (3R,1'S) from a comparison with an authentic specimen prepared via baker’s yeast asymmetric reduction.     

Synthesis of New Furanone Derivatives with Potent Anticancer Activity

Russian Journal of Bioorganic Chemistry - New compounds based on Furanone derivatives were synthesized by reaction of 3-(4-nitrobezylidine)-5-phenylfuran-2(3H)-one with various reagents as...     

A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity

Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.     

Plants from Brazilian Cerrado with Potent Tyrosinase Inhibitory Activity

The increased amount of melanin leads to skin disorders such as age spots, freckles, melasma and malignant melanoma. Tyrosinase is known to be the key enzyme in melanin production. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase as well as can be used for the treatment of dermatological disorders associated with melanin hyperpigmentation. Using in vitro tyrosinase inhibitory activity assay, extracts from 13 plant species from Brazilian Cerrado were evaluated. The results showed that Pouteria torta and Eugenia dysenterica extracts presented potent in vitro tyrosinase inhibition compared to positive control kojic acid. Ethanol extract of Eugenia dysenterica leaves showed significant (p<0.05) tyrosinase inhibitory activity exhibiting the IC₅₀ value of 11.88 µg/mL, compared to kojic acid (IC₅₀ value of 13.14 µg/mL). Pouteria torta aqueous extract leaves also showed significant inhibitory activity with IC₅₀ value of 30.01 µg/mL. These results indicate that Pouteria torta and Eugenia dysenterica extracts and their isolated constituents are promising agents for skin-whitening or antimelanogenesis formulations.     

Preparation,Structure, and Potent Antifouling Activity of Sclerotioramine Derivatives

A series of 30 sclerotioramine derivatives (2–31) of the natural compound, (+)-sclerotiorin (1), has been successfully semi-synthesized by a one-step reaction with high yields (up to 80%). The structures of these new derivatives were established by extensive spectroscopic methods and single-crystal X-ray diffraction analysis for 3, 6, and 10. (+)-Sclerotiorin (1) and its semisynthetic derivatives (2–31) were evaluated for their antifouling activity. Most of them except 6, 7, 8, 12, and 28 showed potent antifouling activity against the larval settlement of the barnacle Balanus amphitrite. More interestingly, most of the aromatic amino-derivatives (13–17, 19–21, 23, 25–27, and 29–31) showed strong antifouling activity; however, only two aliphatic amino-derivatives (5 and 10) had the activity.     

Zebra Mussel Antifouling Activity of the Marine Natural Product Aaptamine and Analogs

Several aaptamine derivatives were selected as potential zebra mussel (Dreissena polymorpha) antifoulants because of the noteworthy absence of fouling observed on Aaptos sponges. Sponges of the genus Aaptos collected in Manado, Indonesia consistently produce aaptamine-type alkaloids. To date, aaptamine and its derivatives have not been carefully evaluated for their antifoulant properties. Structure–activity relationship studies were conducted using several aaptamine derivatives in a zebra mussel antifouling assay. From these data, three analogs have shown significant antifouling activity against zebra mussel attachment. Aaptamine, isoaaptamine, and the demethylated aaptamine compounds used in the zebra mussel assay produced EC₅₀ values of 24.2, 11.6, and 18.6 μM, respectively. In addition, neither aaptamine nor isoaaptamine produced a phytotoxic response (as high as 300 μM) toward a nontarget organism, Lemna pausicostata, in a 7-day exposure. The use of these aaptamine derivatives from Aaptos sp. as potential environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a possible control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses.     

Potent Lipolytic Activity of Lactoferrin in Mature Adipocytes

Momilactone A, a major rice diterpene phytoalexin, could be synthesized by dehydrogenation at the 3-position of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in rice leaves. The presence of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in UV-irradiated rice leaves was confirmed by comparing the mass spectra and retention times after a GC/MS analysis of the natural and synthetic compounds. The soluble protein fraction from UV-irradiated rice leaves showed dehydrogenase activity to convert 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide into momilactone A. The enzyme required NAD⁺ or NADP⁺ as a hydrogen acceptor. The optimum pH for the reaction was 8. The K _m value to 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide was 36 μM when NAD⁺ was supplied as a cofactor at a concentration of 1 mM. 3β-Hydroxy-9β-pimara-7,15-dien-19,6β-olide and its dehydrogenase activity were induced in a time-dependent manner by UV irradiation.     

Resistance to Pseudorabies Virus with Enhanced Interferon Production and Natural Killer Cell Activity in Mice Treated with Serum Thymic Factor

Susceptibility to pseudorabies virus (PRV) infection in mice, which were continuously depleted of natural killer (NK) cell activity by injection of anti-asialo GM1, was examined. Effects of serum thymic factor (FTS) on susceptibility of mice to PRV were also investigated. In mice with depleted NK cell activity, the mortality of PRV-infected mice was markedly high, whereas that of FTS-pretreated mice was significantly lower than the controls. Reduced susceptibility to PRV was demonstrated in mice treated with anti-asialo GM1 antisera before the PRV infection. Such a reduced susceptibility was not observed in mice inoculated with the antisera on day 1 post-infection (PI). To analyze the FTS-induced resistance to PRV infection, NK cell activity, macrophage activity, and interferon (IFN) productions were studied. Interferon production and NK cell activity were enhanced in the FTS-pretreated mice, suggesting that interferon may play an important role in this FTS-induced resistance to PRV infection.     

Northeast Red Beans Produce a Thermostable and pH-Stable Defensin-Like Peptide with Potent Antifungal Activity

A 5.4-kDa antifungal peptide was purified from Phaseolus vulgaris L. cv. “northeast red bean” using a protocol that entailed affinity chromatography, ion exchange chromatography, and gel filtration. The molecular mass was determined by matrix-assisted laser desorption ionization time-of-flight. The N-terminal amino acid sequence of the peptide was highly homologous to defensins and defensin-like peptides from several plant species. The peptide impeded the growth of a number of pathogenic fungi, including Mycosphaerella arachidicola Khokhr. (IC₅₀ = 1.7 μM), Setosphaeria turcica Luttr., Fusarium oxysporum Schltdl., and Valsa mali Miyabe & G. Yamada. Antifungal activity of the peptide was fully preserved at temperatures up to 100 °C and pH values from 0 to 12. Congo red deposition at the hyphal tip of M. arachidicola was detected after exposure to the peptide, signifying that the peptide had suppressed hyphal growth. The antifungal peptide did not manifest antiproliferative activity toward human breast cancer MCF7 cells and hepatoma HepG2 cells, in contradiction to the bulk of previously reported plant defensins. The data suggest distinct structural requirements for antifungal and antiproliferative activities.     

Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds.     

Antiviral Activity in Tissue Culture Systems of bis-Benzimidazoles, Potent Inhibitors of Rhinoviruses

(S,S)-1,2-bis(5-methoxy-2-benzimidazolyl)-1,2-ethanediol showed antiviral activity in monolayer tissue culture systems against 55 strains of rhinovirus, three types of poliovirus, and strains of type A and B coxsackieviruses. Neither the compound nor any of the analogues tested showed virucidal activity. Its antiviral activity was not associated with interference with viral attachment to or penetration into the cell. At a concentration of 0.1 mg/ml, this group of compounds was generally nontoxic to WI-38, primary bovine kidney, and African green monkey kidney cells and had antiviral activity with 100% inhibition of virus-induced cytopathic effects (CPE). At antiviral levels, these compounds prevented CPE of up to 10(6) median tissue culture infective dose units of virus and completely inhibited formation of new infective virions. The compounds showed antiviral activity both prophylactically and therapeutically against rhinoviruses. Infected cultures could be cleared of CPE up to 90 hr after infection.     

Cyclotraxin-B,the First Highly Potent and Selective TrkB Inhibitor,Has Anxiolytic Properties in Mice

In the last decades, few mechanistically novel therapeutic agents have been developed to treat mental and neurodegenerative disorders. Numerous studies suggest that targeting BDNF and its TrkB receptor could be a promising therapeutic strategy for the treatment of brain disorders. However, the development of potent small ligands for the TrkB receptor has proven to be difficult. By using a peptidomimetic approach, we developed a highly potent and selective TrkB inhibitor, cyclotraxin-B, capable of altering TrkB-dependent molecular and physiological processes such as synaptic plasticity, neuronal differentiation and BDNF-induced neurotoxicity. Cyclotraxin-B allosterically alters the conformation of TrkB, which leads to the inhibition of both BDNF-dependent and -independent (basal) activities. Finally, systemic administration of cyclotraxin-B to mice results in TrkB inhibition in the brain with specific anxiolytic-like behavioral effects and no antidepressant-like activity. This study demonstrates that cyclotraxin-B might not only be a powerful tool to investigate the role of BDNF and TrkB in physiology and pathology, but also represents a lead compound for the development of new therapeutic strategies to treat brain disorders.     

GMP-Compliant,Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3⁺ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3⁻CD16⁺CD56⁺ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.     

Type II Fatty Acid Biosynthesis, a New Approach in Antimalarial Natural Product Discovery

Malaria, one of the most problematic infectious diseases worldwide, is on the rise. The absence of an effective vaccine and the spread of drug-resistant strains of Plasmodium clearly indicate the necessity for the development of new chemotherapeutic agents and the identification of novel targets. The recent discovery of a relict, non-photosynthetic plastid-like organelle, the so-called apicoplast, in Plasmodium has opened up new avenues in malaria research. It also initiated the Plasmodium falciparum genome sequencing project, which revealed a number of biochemical pathways previously unknown to Plasmodium, i.e. cytosolic shikimate pathway, apicoplastic type II fatty acid, non-mevalonate isoprene and haem biosyntheses. Since these vital biosynthetic processes are absent in humans or fundamentally different from those found in humans, they represent excellent targets for pharmaceutical interventions. We are interested in the type II fatty acid synthase (FAS II) system of malaria parasite and focus on the FabI enzyme, the only known enoyl-ACP reductase in Plasmodium involved in the final reduction step of the fatty acid chain elongation cycle. Here we describe the general aspects of fatty acid biosynthesis, its essentiality to the malaria parasite and our continuing efforts to discover in Turkish medicinal plants natural antimalarial agents, which specifically target the plasmodial FabI enzyme.Phytochemical Society of Europe (PSE)-Pierre Fabre Prize 2004 Lecture     

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.