Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies.

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(ß-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(ß-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Aspirin Affects MDA-MB-231 Vesicle Production and Their Capacity to Induce Fibroblasts towards a Pro-Invasive State.

Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.

An NMR-Based Chemometric Strategy to Identify Leishmania donovani Nucleoside Hydrolase Inhibitors from the Brazilian Tree Ormosia arborea.

Nucleoside hydrolases are a strategic target for the development of drugs to treat leishmaniasis, a neglected disease that affects 700 thousand to one million people annually. The present study aimed to identify Leishmania donovani nucleoside hydrolase (LdNH) inhibitors from the leaves of Ormosia arborea, a tree endemic to Brazilian ecosystems, through a strategy based on 1H NMR analyses and chemometrics. The aqueous EtOH extract of O. arborea leaves inhibited LdNH activity by 95%. The extract was fractionated in triplicate (13 in each step, making a total of 39 fractions). Partial least squares discriminant analysis (PLS-DA) was used to correlate the 1H NMR spectra of the fractions with their LdNH inhibitory activity and thus to identify the spectral regions associated with the bioactivity. The strategy aimed at isolating the probable bioactive substances and led to two new A-type proanthocyanidins, linked to a p-coumaroyl unit (1 and 2), which appeared as noncompetitive inhibitors of LdNH (IC50: 28.2 ± 3.0 µM and 25.6 ± 4.1 µM, respectively). This study confirms the usefulness of the NMR-based chemometric methods to accelerate the discovery of drugs from natural products.

Geissoschizoline, a promising alkaloid for Alzheimer's disease: Inhibition of human cholinesterases, anti-inflammatory effects and molecular docking.

Due to the lack of effective pharmacotherapy options to treats Alzheimer's disease, new strategies have been approached in the search for multi-target molecules as therapeutic options. In this work, four indole alkaloids, geissoschizoline, geissoschizone, geissospermine, and 3',4',5',6'-tetradehydrogeissospermine were isolated from Geissospermum vellosii (Pao pereira) and evaluated for their anticholinesterase activities. While geissospermine inhibited only butyrylcholinesterase (BChE), the other alkaloids behaved as non-selective inhibitors of acetylcholinesterase (AChE) and BChE. In cell viability tests, only geissoschizoline was not cytotoxic. Therefore, geissoschizoline actions were also evaluated in human cholinesterases, where it was twice as potent inhibitor of hBChE (IC50 = 10.21 ± 0.01 µM) than hAChE (IC50 = 20.40 ± 0.93 µM). On enzyme kinetic studies, geissoschizoline presented a mixed-type inhibition mechanism for both enzymes. Molecular docking studies pointed interactions of geissoschizoline with active site and peripheral anionic site of hAChE and hBChE, indicating a dual site inhibitor profile. Moreover, geissoschizoline also played a promising anti-inflammatory role, reducing microglial release of NO and TNF-α at a concentration (1 µM) ten and twenty times lower than the IC50 values of hBChE and hAChE inhibition, respectively. These actions give geissoschizoline a strong neuroprotective character. In addition, the ability to inhibit hAChE and hBChE, with approximate inhibitory potencies, accredits this alkaloid for therapeutic use in the moderate to severe phase of AD. Thus, geissoschizoline emerges as a possible multi-target prototype that can be very useful in preventing neurodegeneration and restore neurotransmission.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Phytochemical Study of Tapirira guianensis Leaves Guided by Vasodilatory and Antioxidant Activities.

The aim of this research was to perform a phytochemical study of the methanol leaves extract of T. guianensis (MET) guided by vasodilatory and antioxidant activities. The chemical profile of MET and the ethyl acetate fraction (EA fraction) was determined by HPLC-UV-MS and EA fraction guided fractionation by reverse-phase chromatography. The vasorelaxant effects of MET, fractions, sub-fractions and constituents were assessed on rat aorta pre-contracted with phenylephrine. Antioxidant activity was evaluated by using a DPPH assay. The results show that MET-induced vasodilation was dependent on NO/cGMP; and that the PI3K/Akt pathway seems to be the main route involved in eNOS activation. The EA fraction showed greater vasodilatory and antioxidant potency and was submitted to further fractionation. This allowed the isolation and characterization of quercetin, quercetin 3-O-(6″-O-galloyl)-ß-d-galactopyranoside and 1,4,6-tri-O-galloyl-ß-d-glucose. Also, galloyl-HHDP-hexoside and myricetin deoxyhexoside were identified by HPLC-UV-MS. These compounds are being described for the first time for T. guianensis. 1,4,6-tri-O-galloyl-ß-d-glucose and quercetin 3-O-(6″-O-galloyl)-ß-d-galactopyranoside showed no vasodilatory activity. Quercetin and myricetin glycoside seems to contribute to the MET activity, since they have been reported as vasodilatory flavonoids. MET-induced vasodilation could contribute to the hypotensive effect of T. guianensis previously reported.

Characterization of a Ternary System Based on Hybrid Nanoparticles.

Hybrid nanoparticles (NPs) are emerging as an important family of multifunctional nanoscale materials. Due to its unique properties cyclodextrins (CDs) present the possibility to be employed in nanostructured devices for multiple applications. Therefore the study of hybrids NPs containing CDs, metal nanoparticles and an organic molecular guest is promising for applications in separation science, sensing, optics, catalysis and medical therapy. To investigate the hybrid nanoparticles properties in light of technological and medical science interfaces we chose tramadol hydrochloride as the model molecular guest. Using 2D 1H NMR and FTIR spectroscopies and ESI-MS we studied the topology and host-guest stoichiometry of inclusion compound between 2-hydroxypropyl-α-cyclodextrin and tramadol. These techniques confirmed that the inclusion compounds present 1:1 stoichiometry with the tramadol aromatic moiety inserted in the CD cavity and the amine part interacting with the CD superficial OHs. Performing capillary electrophoresis experiments, the stability constants of inclusion compounds were obtained and showed that the increase of temperature during the synthesis of these NPs can decrease their stabilities. Precipitated palladium nanoparticles were determined to have 80-200 nm of size distribution and the incorporation of tramadol was evidenced by additional 1H NMR experiments.

The Use of NMR Metabolite Profiling and in vivo Hypoglycemic Assay for Comparison of Unfractionated Aqueous Leaf Extracts of Two Ocimum Species.

Ocimum basilicum and Ocimum gratissimum (Lamiaceae) are used to treat diabetes mellitus in Africa. In a previous work, we identified chicoric acid as a hypoglycemic substance in O. gratissimum. This study aims to compare the chemical metabolite profile and the hypoglycemic activity of unfractionated aqueous extracts from leaves of both Lamiaceae species. The metabolite composition of OB and OG decoctions (10% w/v) was analyzed using HPLC-DAD and NMR tools. Chicoric acid showed to be the major phenolic in both extracts, besides caftaric, caffeic, and rosmarinic acids; nevertheless, there is approximately three times more of this substance in OG. From 1D- and 2D-NMR analyses, 19 substances were identified in OB, while 12 in OG. The in vivo acute hypoglycemic activity of the extracts was assessed intraperitoneally in streptozotocin (STZ)-induced diabetic mice. The doses of 100 and 200 mg/kg of both extracts significantly reduced their glycemia, compared to controls (P < 0.05). OB was a little more effective than OG, despite the lower content of chicoric acid in OB. This result strongly suggests that components other than chicoric acid contribute to the hypoglycemic activity of the two extracts. Despite the abundance of caffeic and rosmarinic acids in OB, their hypoglycemic activity observed at 8.3 µmol/kg was low. This is the first chemical profile of crude extracts from Ocimum species by NMR. Our findings confirmed the potential of both species in DM treatment in spite of marked differences in their chemical composition. However, long-term studies are necessary in order to identify the most promising of the two species for the development of an herbal medicine.

Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.

Antimycobacterial and nitric oxide production inhibitory activities of Ocotea notata from Brazilian restinga.

The genus Ocotea (Lauraceae) is distributed mainly in tropical and subtropical regions. Some species of this genus as O. puberula and O. quixos have been described in the literature, showing antibacterial activity. And Ocotea macrophylla showed anti-inflammatory activity with inhibition of COX-1, COX-2, and LOX-5. The purpose of this study was the phytochemical investigation of the plant species Ocotea notata from Restinga Jurubatiba National Park, Macaé, RJ, Brazil, and the search for antimycobacterial fractions and compounds. The crude extract was evaluated for antimycobacterial activity and presented 95.75 ± 2.53% of growth inhibition at 100 µg/mL. Then, it was subjected to a liquid-liquid partition and subsequently was chemically investigated by HPLC, revealing the major presence of flavonoids. In this process the partition fractions hexane, ethyl acetate, and butanol are shown to be promising in the antimycobacterial assay. In addition, ethyl acetate fraction was chromatographed and afforded two flavonoids identified by MS and NMR as afzelin and isoquercitrin. The isolated flavonoids afzelin and isoquercitrin were evaluated for their antimycobacterial activity and for their ability to inhibit NO production by macrophages stimulated by LPS; both flavonoids isoquercitrin (Acet22) and afzelin (Acet32) were able to inhibit the production of NO by macrophages. The calculated IC50 of Acet22 and Acet32 was 1.03 and 0.85 µg/mL, respectively.

Resveratrol is active against Leishmania amazonensis: in vitro effect of its association with Amphotericin B.

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC50s) of 27 and 42 µM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G0/G1 phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH2-to-CH3 ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo.

The beneficial effect of fluoxetine on behavioral and cognitive changes in chronic experimental Chagas disease unveils the role of serotonin fueling astrocyte infection by Trypanosoma cruzi.

BACKGROUND: In Chagas disease (CD), a neglected tropical disease caused by the parasite Trypanosoma cruzi, the development of mental disorders such as anxiety, depression, and memory loss may be underpinned by social, psychological, and biological stressors. Here, we investigated biological factors underlying behavioral changes in a preclinical model of CD. METHODOLOGY/PRINCIPAL FINDINGS: In T. cruzi-infected C57BL/6 mice, a kinetic study (5 to 150 days postinfection, dpi) using standardized methods revealed a sequential onset of behavioral changes: reduced innate compulsive behavior, followed by anxiety and depressive-like behavior, ending with progressive memory impairments. Hence, T. cruzi-infected mice were treated (120 to 150 dpi) with 10 mg/Kg/day of the selective serotonin reuptake inhibitor fluoxetine (Fx), an antidepressant that favors neuroplasticity. Fx therapy reversed the innate compulsive behavior loss, anxiety, and depressive-like behavior while preventing or reversing memory deficits. Biochemical, histological, and parasitological analyses of the brain tissue showed increased levels of the neurotransmitters GABA/glutamate and lipid peroxidation products and decreased expression of brain-derived neurotrophic factor in the absence of neuroinflammation at 150 dpi. Fx therapy ameliorated the neurochemical changes and reduced parasite load in the brain tissue. Next, using the human U-87 MG astroglioma cell line, we found no direct effect of Fx on parasite load. Crucially, serotonin/5-HT (Ser/5-HT) promoted parasite uptake, an effect increased by prior stimulation with IFNγ and TNF but abrogated by Fx. Also, Fx blocked the cytokine-driven Ser/5-HT-promoted increase of nitric oxide and glutamate levels in infected cells. CONCLUSION/SIGNIFICANCE: We bring the first evidence of a sequential onset of behavioral changes in T. cruzi-infected mice. Fx therapy improves behavioral and biological changes and parasite control in the brain tissue. Moreover, in the central nervous system, cytokine-driven Ser/5-HT consumption may favor parasite persistence, disrupting neurotransmitter balance and promoting a neurotoxic environment likely contributing to behavioral and cognitive disorders.

1,2,4-thiadiazol-5(4H)-ones: a new class of selective inhibitors of Trypanosoma cruzi triosephosphate isomerase. Study of the mechanism of inhibition.

CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of small molecular weight compounds with potential use against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified a new selective inhibitor chemotype of TIM from T. cruzi (TcTIM), 1,2,4-thiadiazol-5(4H)-one. OBJECTIVE: Study the mechanism of TcTIM inhibition by a 1,2,4-thiadiazol derivative. METHODS: We performed the biochemical characterization of the interaction of the 1,2,4-thiadiazol derivative with the wild-type and mutant TcTIMs, using DOSY-NMR and MS experiments. Studies of T. cruzi growth inhibition were additionally carried out. RESULTS AND CONCLUSION: At low micromolar concentrations, the compound induces highly selective irreversible inactivation of TcTIM through non-covalent binding. Our studies indicate that it interferes with the association of the two monomers of the dimeric enzyme. We also show that it inhibits T. cruzi growth in culture.

Two new triterpenoid saponins from the flowers of Albizia lebbeck.

The chemical investigation of the fresh flowers of Albizia lebbeck (L.) Benth. (Fabaceae, Mimosoideae) led to the isolation of two new echinocystic acid saponins. They were isolated by using chromatographic methods and their structures were elucidated by detailed 1H and 13C NMR spectral data including 2 D-NMR (COSY, HSQC, HMBC and APT) spectroscopic techniques, high-resolution electrospray ionization mass spectrometry (HRESIMS) and acid hydrolysis. Their structures were established as 16-hydroxy-3-[[O-ß-D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-2-(acetylamino)-2-deoxy-ß-D-glucopyranosyl]oxy]-(3ß,16α)-olean-12-en-28-oic acid O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-6-deoxy-α-L-mannopyranosyl-(1→2)-ß-D-glucopyranosyl ester (1) and 16-hydroxy-3-[[O-ß-D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-2-(acetylamino)-2-deoxy-ß-D-glucopyranosyl]oxy]-(3ß,16α)-olean-12-en-28-oic acid 6-O-[(2S,3R,4R)-tetrahydro-3-hydroxy-4-(hydroxymethyl)-2-furanyl]-ß-D-glucopyranosyl ester (2). Additionally, the permeability property and the capacity of interaction with biological membranes of compounds 1 and 2 were investigated.

Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors.

Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L-1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L-1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.

Haemolytic activity and immunological adjuvant effect of a new steroidal saponin from Allium ampeloprasum var. porrum.

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known compounds, its structure was established as (3ß,5α,6ß,25R)-3-{(O-ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→2)-O-[O-ß-D-glucopyranosyl-(1→3)]-O-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranosyl)oxy}-6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune response against ovalbumin antigen.

High-resolution inhibition profiling and ligand fishing for screening of nucleoside hydrolase ligands in Moringa oleifera Lamarck.

In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.

Integration of NMR studies, computational predictions, and in vitro assays in the search of marine diterpenes with antitumor activity.

Among the compounds of natural origin, diterpenes have proved useful as drugs for the treatment of cancer. Marine organisms, such as soft corals and algae, are a promising source of diterpenes, being a rich and unexplored source of cytotoxic agents. This study evaluated a library of 32 natural and semisynthetic marine diterpenes, including briarane, cembrane, and dolabellane nuclei, with the aim of determining their cytotoxicity against three human cancer cell lines (A549, MCF7, and PC3). The three most active compounds were submitted to a flow cytometry analysis in order to determine induction of apoptosis against the A549 cell line. An NMR analysis was conducted to determine and evaluate the interactions between active diterpenes and tubulin. These interactions were characterized by a computational study using molecular docking and MD simulations. With these results, two cembrane and one chlorinated briarane diterpenes were active against the three human cancer cell lines, induced apoptosis in the A549 cell line, and showed interactions with tubulin preferably at the taxane-binding site. This study is a starting point for the identification and optimization of the marine diterpenes selected for better antitumor activities. It also highlights the power of integrating NMR studies, computational predictions, and in vitro assays in the search for compounds with antitumor activity.

Isolation and characterization of flavonoids from Tapirira guianensis leaves with vasodilatory and myeloperoxidase-inhibitory activities.

The aim of this study was to perform the isolation and characterization of vasodilatory flavonoids from Tapirira guianensis Aubl. (Annacardiaceae) leaves. In this context, ethyl acetate fraction (EA fraction) was obtained and subjected to fractionation batches by HSCCC affording: myricetin 3-O-α-L-rhamnopyranoside (myricitrin, 1); quercetin 3-O-(6"-O-galloyl)-ß-D-galactopyranoside (2); quercetin 3-O-α-L-arabinofuranoside (avicularin, 3); and quercetin 3-O-α-L-rhamnopyranoside (quercitrin, 4). Myricitrin (1) induced a relaxation of 56.07 ± 13.04% at 300 µM (P < 0.05; n = 5), indicating that this flavonoid contributes to the vasodilatory activity of EA fraction. In addition, all EA fraction flavonoids were evaluated for their capacity of inhibiting myeloperoxidase activity and flavonoid (2) (IC50 1.0 ± 0.3 µM) was the strongest peroxidase inhibitor. In conclusion, it was possible to verify that myricitrin together with quercetin are mainly responsible for vasodilatory potential, besides flavonoid 2 for myeloperoxidase inhibition. Together these flavonoids seem to be responsible for Tapirira guianensis cardiovascular effects.

New Leishmania donovani nucleoside hydrolase inhibitors from Brazilian flora.

This study presents new inhibitors of the nucleoside hydrolase from Leishmania donovani (LdNH) with in vitro leishmanicidal activity. Biological screening of 214 Brazilian plant extracts was performed to select plants with enzyme inhibitory activity. Two plants were selected for their results, and for their lack of prior phytochemical description: Leandra amplexicaulis DC. (Melastomataceae) and Urvillea rufescens Cambess (Sapindaceae). Three flavonoids were isolated by bioguided fractionation of the hydroethanolic extracts: kaempferol 3-O-α-l-rhamnopyranoside (1) and kaempferol 3-O-ß-d-xylopyranosyl-(1→2)-α-l-rhamnopyranoside (2) from L. amplexicaulis, as well as tricetin-4'-O-methyl flavone (3) from U. rufescens. These flavonoids showed inhibitory activities (IC50) of 197.4 µM (1), 74.7 µM (2) and 1.1 µM (3) on the LdNH. Their binding mode was proposed based on molecular docking with LdNH and by NMR Saturation Transfer Difference studies. Kinetic studies demonstrate that the most potent inhibitor (3) acts by uncompetitive inhibition. This study reports for the first time the inhibition of LdNH by naturally sourced flavonoids.