Anti-Trypanosoma cruzi Potential of Vestitol Isolated from Lyophilized Red Propolis.

Chagas disease (CD) is a worldwide public health problem, and the drugs available for its treatment have severe limitations. Red propolis is a natural extract known for its high content of phenolic compounds and for having activity against T. cruzi. The aim of this study was to investigate the trypanocidal potential of red propolis to isolate, identify, and indicate the mode of action of the bioactive compounds. The results revealed that the total phenolic content was 15.4 mg GAE/g, and flavonoids were 7.2 mg QE/g. The extract was fractionated through liquid-liquid partitioning, and the trypanocidal potential of the samples was evaluated using the epimastigote forms of the Y strain of T. cruzi. In this process, one compound was characterized by MS, 1H, and 13C NMR and identified as vestitol. Cytotoxicity was evaluated employing MRC-5 fibroblasts and H9C2 cardiomyocytes, showing cytotoxic concentrations above 15.62 µg/mL and 31.25 µg/mL, respectively. In silico analyses were applied, and the data suggested that the substance had a membrane-permeation-enhancing effect, which was confirmed through an in vitro assay. Finally, a molecular docking analysis revealed a higher affinity of vestitol with farnesyl diphosphate synthase (FPPS). The identified isoflavan appears to be a promising lead compound for further development to treat Chagas disease.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Understanding the Antilymphoma Activity of Annona macroprophyllata Donn and Its Acyclic Terpenoids: In Vivo, In Vitro, and In Silico Studies.

Annona macroprophyllata Donn (A. macroprophyllata) is used in traditional Mexican medicine for the treatment of cancer, diabetes, inflammation, and pain. In this work, we evaluated the antitumor activity of three acyclic terpenoids obtained from A. macroprophyllata to assess their potential as antilymphoma agents. We identified the terpenoids farnesyl acetate (FA), phytol (PT) and geranylgeraniol (Gg) using gas chromatography-mass spectroscopy (GC-MS) and spectroscopic (1H, and 13C NMR) methods applied to petroleum ether extract of leaves from A. macroprophyllata (PEAm). We investigated antitumor potential in Balb/c mice inoculated with U-937 cells by assessing brine shrimp lethality (BSL), and cytotoxic activity in these cells. In addition, to assess the potential toxicity of PEAm, FA, PT and Gg in humans, we tested their acute oral toxicity in mice. Our results showed that the three terpenoids exhibited considerable antilymphoma and cytotoxic activity. In terms of lethality, we determined a median lethal dose (LD50) for thirteen isolated products of PEAm. Gg, PT and AF all exhibited a higher lethality with values of 1.41 ± 0.42, 3.03 ± 0.33 and 5.82 ± 0.58 µg mL-1, respectively. To assess cytotoxic activity against U-937 cells, we calculated the mean cytotoxic concentration (CC50) and found that FA and PT were closer in respect to the control drug methotrexate (MTX, 0.243 ± 0.007 µM). In terms of antilymphoma activity, we found that FA, PT and Gg considerably inhibited lymph node growth, with median effective doses (ED50) of 5.89 ± 0.39, 6.71 ± 0.31 and 7.22 ± 0.51 mg kg-1 in females and 5.09 ± 0.66, 5.83 ± 0.50 and 6.98 ± 0.57mg kg -1 in males, respectively. Regarding acute oral toxicity, we classified all three terpenoids as category IV, indicating a high safety margin for human administration. Finally, in a molecular docking study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, we found binding of terpenoids to some amino acids of the catalytic site, suggesting an effect upon activity with a resulting decrease in the synthesis of intermediates involved in the prenylation of proteins involved in cancer progression. Our findings suggest that the acyclic terpenoids FA, PT, and Gg may serve as scaffolds for the development of new treatments for non-Hodgkin's lymphoma.

Structural insights into the cyclization of unusual brasilane-type sesquiterpenes.

The biosynthesis of brasilane-type sesquiterpenoids (BTSs) attracts much attention owing to their unique skeleton of 5/6 bicyclic structure that contains five Me groups. Here, the crystal structures of a BTS cyclase TaTC6 from Trichoderma atroviride FKI-3849 and its complexes with farnesyl pyrophosphate (FPP) and analogue were reported. These structural information reveal that TaTC6 exploits a hydrophobic pocket to constrain the hydrocarbon region of FPP in a "U-shape" to facilitate the initial C1-C11 bond formation after pyrophosphate ionization. Following, four carbocations of reaction intermediates were molecularly docked into the hydrophobic pocket to reveal critical residues involved in the cyclization cascade. Finally, an S239-stabilized water molecule that is 3.9 Å away from the C8 of the last allyl cation may conduct hydration to quench the reaction cascade. Mutating S239 to alanine led to ca. 40% reduction in activity compared with the wild-type enzyme. The conservation of the residues that constitute the hydrophobic pocket is also discussed. Overall, this study will give an insight into the mechanism of how the active site of STCs constrain the conformation of the flexible FPP and series allylic carbocations for the complicated-ring formation and unusual carbon rearrangement in the biosynthesis of BTSs.

Bisphosphonate-Based Molecules as Potential New Antiparasitic Drugs.

Neglected tropical diseases such as Chagas disease and leishmaniasis affect millions of people around the world. Both diseases affect various parts of the globe and drugs traditionally used in therapy against these diseases have limitations, especially with regard to low efficacy and high toxicity. In this context, the class of bisphosphonate-based compounds has made significant advances regarding the chemical synthesis process as well as the pharmacological properties attributed to these compounds. Among this spectrum of pharmacological activity, bisphosphonate compounds with antiparasitic activity stand out, especially in the treatment of Chagas disease and leishmaniasis caused by Trypanosoma cruzi and Leishmania spp., respectively. Some bisphosphonate compounds can inhibit the mevalonate pathway, an essential metabolic pathway, by interfering with the synthesis of ergosterol, a sterol responsible for the growth and viability of these parasites. Therefore, this review aims to present the information about the importance of these compounds as antiparasitic agents and as potential new drugs to treat Chagas disease and leishmaniasis.

Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia.

BACKGROUND: The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. METHODS: We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. RESULTS: The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. CONCLUSIONS: Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

Pharmacophore modeling, docking and molecular dynamics to identify Leishmania major farnesyl pyrophosphate synthase inhibitors.

Leishmaniasis is caused by protozoa of the genus Leishmania spp. and is considered the second most important protozoa in the world due to the number of cases and mortality. Despite its importance in terms of public health, the treatment of patients is limited and has mostly low levels of efficacy and safety. Farnesyl pyrophosphate synthase (FPPS) acts in the early stages of isoprenoid synthesis, and is important for maintaining the integrity of the lipid bilayer of the parasite that causes the disease. The aim of this work was to identify one potential inhibitor of the FPPS of Leishmania major through virtual screening by pharmacophore modeling and docking. A total of 85,000 compounds from a natural products database (ZINC15) was submitted for virtual hierarchical screening, and the top ranked molecule in both methods was analyzed by intermolecular interaction profile and 20 ns molecular dynamics simulations. These results showed a promising compound from natural products that mimic the major interactions present in the substrate/inhibitor.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production.

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Toxic effects of phytol and retinol on human glioblastoma cells are associated with modulation of cholesterol and fatty acid biosynthetic pathways.

Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.

Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites.

Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates-Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Approaches for Designing new Potent Inhibitors of Farnesyl Pyrophosphate Synthase.

INTRODUCTION: Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give rise to one molecule of geranyl diphosphate, which on a further reaction with another molecule of isopentenyl diphosphate forms the 15-carbon isoprenoid farnesyl diphosphate. This molecule is the obliged precursor for the biosynthesis of sterols, ubiquinones, dolichols, heme A, and prenylated proteins. The blockade of FPPS prevents the synthesis of farnesyl diphosphate and the downstream essential products. Due to its crucial role in isoprenoid biosynthesis, this enzyme has been winnowed as a molecular target for the treatment of different bone disorders and to control parasitic diseases, particularly, those produced by trypanosomatids and Apicomplexan parasites. AREAS COVERED: This article discusses some relevant structural features of farnesyl pyrophosphate synthase. It also discusses the precise mode of action of relevant modulators, including both bisphosphonate and non-bisphosphonate inhibitors and the recent advances made in the development of effective inhibitors of the enzymatic activity of this target enzyme. EXPERT OPINION: Notwithstanding their lack of drug-like character, bisphosphonates are still the most advantageous class of inhibitors of the enzymatic activity of farnesyl pyrophosphate synthase. The poor drug-like character is largely compensated by the high affinity of the bisphosphonate moiety by bone mineral hydroxyapatite in humans. Several bisphosphonates are currently in use for the treatment of a variety of bone disorders. Currently, the great prospects that bisphosphonates behave as antiparasitic agents is due to their accumulation in acidocalcisomes, organelles with equivalent composition to bone mineral, hence facilitating their antiparasitic action.

New insights into molecular recognition of 1,1-bisphosphonic acids by farnesyl diphosphate synthase.

As part of our project pointed at the search of new antiparasitic agents against American trypanosomiasis (Chagas disease) and toxoplasmosis a series of 2-alkylaminoethyl-1-hydroxy-1,1-bisphosphonic acids has been designed, synthesized and biologically evaluated against the etiologic agents of these parasitic diseases, Trypanosoma cruzi and Toxoplasma gondii, respectively, and also towards their target enzymes, T. cruzi and T. gondii farnesyl pyrophosphate synthase (FPPS), respectively. Surprisingly, while most pharmacologically active bisphosphonates have a hydroxyl group at the C-1 position, the additional presence of an amino group at C-3 resulted in decreased activity towards either T. cruzi cells or TcFPPS. Density functional theory calculations justify this unexpected behavior. Although these compounds were devoid of activity against T. cruzi cells and TcFPPS, they were efficient growth inhibitors of tachyzoites of T. gondii. This activity was associated with a potent inhibition of the enzymatic activity of TgFPPS. Compound 28 arises as a main example of this family of compounds exhibiting an ED50 value of 4.7 µM against tachyzoites of T. gondii and an IC50 of 0.051 µM against TgFPPS.

Impact of an intragenic retrotransposon on the structural integrity and evolution of a major isoprenoid biosynthesis pathway gene in Hevea brasiliensis.

Isoprenoids belong to a large family of structurally and functionally different natural compounds found universally from prokaryotes to higher animals and plants. In Hevea brasiliensis, the commercially important cis-polyisoprene (rubber) is synthesised as part of its defence mechanism in addition to other common isoprenoids like phytosterols, growth hormones etc. Farnesyl diphosphate synthase (FDPS) is a key enzyme in this process which catalyses the conversion of isoprene units into polyisoprene. Although prior sequence information is available, the structural variants of the FDPS gene presently existing in Hevea population are largely unknown. Since gene structure has a major role in gene regulation, extensive sequence analysis of this gene from different genotypes was carried out to identify the prevailing structural variants. We identified several SNPs and large indels which were associated with a partial transposable element (TE). Modification of key regulatory motifs and splice sites induced by the retroelement was also identified in the first intron. Screening of popular rubber clones, wild germplasm accessions and Hevea species revealed that the retroelement is responsible for the generation of new alleles with varying degrees of sequence homology. Segregation analysis of a progeny population confirmed that the alleles are not paralogs and are inherited in a Mendelian mode. Our findings suggest that the first intron of the FDPS gene has been subjected to various chromosomal rearrangements due to the interaction of a retrotransposon, resulting in novel alleles which may substantially contribute towards the evolution of this major gene in rubber. Moreover, the results indicate the possible existence of a retrotransposon-mediated epigenetic gene regulatory mechanism in Hevea.

Nuevas estrategias en el tratamiento de la leucemia mieloide crónica / New strategies in the treatment of chronic myeloid leukemia

En los últimos años se han propuesto nuevos modelos terapéuticos para ser utilizados en pacientes con leucemia mieloide crónica (LMC) resistentes o intolerantes al Imatinib. La mayor parte de estos modelos están basados en intervenciones sobre sitios específicos del sistema de transmisión de señales que la célula utiliza para hacer llegar información al núcleo. Una de las estrategias incorporadas es el uso de inhibidores de la farnesiltransferasa y ya se han identificado productos terapéuticos factibles de ser utilizados por vía oral, debido al pequeño tamaño de sus moléculas. Actualmente hay 4 inhibidores en fases más avanzadas, fundamentalmente el R115777 (zarnestra, tipifarnib). La búsqueda de otros modelos de tratamiento dio lugar a la aparición de nuevas moléculas que pudieran ser utilizadas para tratar los casos de resistencia o intolerancia al Imatinib, entre estas el Desatinib y el Dilotinib. Otra molécula en fase de experimentación en casos de resistencia al Imatinib es la conocida actualmente como .PKC 412 (N benzil-estauroporina), CGP41251 que es un potente inhibidor selectivo de las isoformas de la proteincinasa C. El desarrollo de la terapéutica molecular ha avanzado rápidamente y su aplicación en la LMC ha logrado resultados muy positivos que se deben incrementar con la incorporación de los nuevos medicamentos en estudio y de aquellos que seguramente deben aparecer en un futuro.

New therapeutic models have been recommended in the last years to be used in patients with chronic myeloid leukemia (CML) resistant or intolerant to Imatinib. Most of the models are based on interventions on specific sites of the signal transmission system that the cells used to send information to the nucleus. One of the strategies that has been incorporated is the use of farnesyl transferase inhibitors. Therapeutic products that may be administered by oral route, due to the small size of their molecules, have already been identified. At present, there are 4 inhibitors in more advanced stages, mainly the R115777 (zarnestra, tipifarnib). The search for other tretament models gave rise to the appearance of new molecules that may be used to treat the cases of resistance or intolerance to Imatinib, such as Desatinib and Dilotinib. Other molecule under experimentation phase is that currently known as PKC 412 (N-benzoylstaurosporine), CGP41251 that is a powerful selective inhibitor of the protein kinase C isoforms . The development of the molecular therapeutics has advanced rapidly and its application to CML has attained very positive results that should increase with the incorporation of the new drugs under study and of those that will certainly emerge in the future.