Total Synthesis and Antimalarial Activity of 2-(p-Hydroxybenzyl)-prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine MYP1 Isolated from Marine Bacteria.

Highly efficient and straightforward synthetic routes toward the first total synthesis of 2-(p-hydroxybenzyl)-prodigiosins (2-5), isoheptylprodigiosin (6), and geometric isomers of tambjamine MYP1 ((E/Z)-7) have been developed. The crucial steps involved in these synthetic routes are the construction of methoxy-bipyrrole-carboxaldehydes (MBCs) and a 20-membered macrocyclic core and a regioselective demethylation of MBC analogues. These new synthetic routes enabled us to generate several natural prodiginines 24-27 in larger quantity. All of the synthesized natural products exhibited potent asexual blood-stage antiplasmodial activity at low nanomolar concentrations against a panel of Plasmodium falciparum parasites, with a great therapeutic index. Notably, prodiginines 6 and 24-27 provided curative in vivo efficacy against erythrocytic Plasmodium yoelii at 25 mg/kg × 4 days via oral route in a murine model. No overt clinical toxicity or behavioral change was observed in any mice treated with prodiginines and tambjamines.

Simultaneous Microcystis algicidal and microcystin synthesis inhibition by a red pigment prodigiosin.

Microcystis blooms and their secondary metabolites microcystins (MCs) occurred all over the world, which have damaged aquatic ecosystems and threatened public health. Techniques to reduce the Microcystis blooms and MCs are urgently needed. This study aimed to investigate the algicidal and inhibitory mechanisms of a red pigment prodigiosin (PG) against the growth and MC-producing abilities of Microcystis aeruginosa (M. aeruginosa). The numbers of Microcystis cells were counted under microscope. The expression of microcystin synthase B gene (mcyB) and concentrations of MCs were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) methods, respectively. The inhibitory effects of PG against M. aeruginosa strain FACHB 905 with 50% algicidal concentration (LC50) at 120 h was 0.12 µg/mL. When M. aeruginosa cells exposed to 0.08 µg/mL, 0.16 µg/mL, 0.32 µg/mL PG, the expression of mcyB of M. aeruginosa was down-regulated 4.36, 8.16 and 18.51 times lower than that of the control at 120 h. The concentrations of total MC (TMC) also were 1.66, 1.72 and 5.75 times lower than that of the control at 120 h. PG had high algicidal effects against M. aeruginosa, with the activities of superoxide dismutase (SOD) initially increased and then decreased after 72 h, the contents of malondialdehyde (MDA) increase, the expression of mcyB gene down-regulation, and MCs synthesis inhibition. This study was first to report the PG can simultaneously lyse Microcystis cells, down-regulate of mcyB expression and inhibit MCs production effectively probably due to oxidative stress, which indicated PG poses a great potential for regulating Microcystis blooms and MCs pollution in the environment.

Physiological response and morphological changes of Heterosigma akashiwo to an algicidal compound prodigiosin.

Harmful algal blooms (HABs) occur all over the world, producing severely negative effects on human life as well as on marine ecosystems. The algicidal compound, prodigiosin, secreted by algicidal bacteria Hahella sp. KA22 can lyse the harmful alga Heterosigma akashiwo. This study is aimed to investigate the algicidal mechanism of prodigiosin against H. akashiwo by detecting physiological and morphological responses of H. akashiwo to presence of prodigiosin. The results indicated that prodigiosin showed strong algicidal effects on H. akashiwo at the concentration of 3 µg/mL. Chlorophyll a and protein levels of the microalgae decreased significantly while malonaldehyde levels increased at this concentration. Contents of ascorbic acid and activities of superoxide dismutase and peroxidase increased fast with the quick decrease of the reactive oxygen species (ROS). For the 3 µg/mL prodigiosin treatment group, transcription of genes related to photosynthesis and respiration were significantly inhibited at 12 h while respiration related genes increased at 24 h. Collectively, the results indicated that prodigiosin could kill the microalgae by inducing ROS overproduction which could destroy the cell integrity and change the antioxidant system levels and functional gene expression. Our results demonstrated that prodigiosin is an effective algicide for the control of harmful algae.

The impact of concentration and administration time on the radiomodulating properties of undecylprodigiosin in vitro.

Undecylprodigiosin pigment (UPP) is reported to display cytotoxic activity towards various types of tumours. Nevertheless, its efficacy in modifying the cellular response to ionising radiation is still unknown. In this study, the radiomodulating effects of UPP were investigated. The effects of UPP were assessed in vitro by treating cultures of human peripheral blood with UPP and ionising radiation using two treatment regimens, the UPP pre-irradiation treatment and UPP post-irradiation treatment. The activity of UPP was investigated evaluating its effects on the radiation-induced micronuclei formation, cell proliferation, and induction of apoptosis. The redox modulating effects of UPP were examined measuring the catalase activity and the level of malondialdehyde, as a measure of oxidative stress. The results showed that UPP effects on cellular response to ionising radiation depend on its concentration and the timing of its administration. At low concentration, the UPP displayed radioprotective effects in γ-irradiated human lymphocytes while at higher concentrations, it acted as a radiosensitiser enhancing either mitotic catastrophe or apoptosis depending on the treatment regimen. The UPP modified redox processes in cells, particularly when it was employed prior to γ-irradiation. Our data highlight the importance of further research of the potential of UPP to sensitize tumour cells to radiation therapy by inhibiting pathways that lead to treatment resistance.

[Bacterial pigment prodigiosin and its genotoxic effects].

The prodigiosin preparation was isolated and purified from Serratia marcescens ATCC 9986, using chromatographic methods. The analysis of the preparation by TLC, NMR-spectrometry and mass-spectrometry allowed to confirm the red pigment fraction as the prodigiosin and detect its purity. Originally, the specific features of the toxic and genotoxic effects of prodigiosin and the possibility of induction of mutations by pigment in the cells of Salmonella typhimurium TA 100 (Ames test) and chromosome damage of mammalian erythroblasts have been determined.

Small-molecule lipid-bilayer anion transporters for biological applications.

The development of small-molecule lipid-bilayer anion transporters for potential future use in channel replacement therapy for the treatment of diseases caused by dysregulation of anion transport (such as cystic fibrosis), and in treating cancer by perturbing chemical gradients within cells, thus triggering apoptosis, is an area of intense current interest. This Minireview looks at recent developments in the design of small-molecule transmembrane anion transporters and focuses on the progress so far in employing these compounds in biological systems.

Synthetic prodiginine obatoclax (GX15-070) and related analogues: anion binding, transmembrane transport, and cytotoxicity properties.

Synthetic prodiginine obatoclax shows promise as a potential anticancer drug. This compound promotes apoptosis of cancer cells, although the mechanism of action is unclear. To date, only the inhibition of BCL-2 proteins has been proposed as a mechanism of action. To gain insight into other possible modes of action, we have studied the anion-binding properties of obatoclax and related analogues in solution, in the solid state, and by means of density functional theory calculations. These compounds are well suited to interact with anions such as chloride and bicarbonate. The anion-transport properties of the compounds synthesized were assayed in model phospholipid liposomes by using a chloride-selective-electrode technique and (13)C NMR spectroscopy. The results demonstrated that these compounds are efficient anion exchangers that promote chloride, bicarbonate, and nitrate transport through lipid bilayers at very low concentrations. In vitro studies on small-cell lung carcinoma cell line GLC4 showed that active ionophores are able to discharge pH gradients in living cells and the cytotoxicity of these compounds correlates well with ionophoric activity.

Mitochondrial dysfunction in Trypanosoma cruzi: the role of Serratia marcescens prodigiosin in the alternative treatment of Chagas disease.

BACKGROUND: Chagas disease is a health threat for many people, mostly those living in Latin America. One of the most important problems in treatment is the limitation of existing drugs. Prodigiosin, produced by Serratia marcescens (Rhodnius prolixus endosymbiont), belongs to the red-pigmented bacterial prodiginine family, which displays numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive, and anticancer properties. Here we describe its effects on Trypanosoma cruzi mitochondria belonging to Tc I and Tc II. RESULTS: Parasites exposed to prodigiosin altered the mitochondrial function and oxidative phosphorylation could not have a normal course, probably by inhibition of complex III. Prodigiosin did not produce cytotoxic effects in lymphocytes and Vero cells and has better effects than benznidazole. Our data suggest that the action of prodigiosin on the parasites is mediated by mitochondrial structural and functional disruptions that could lead the parasites to an apoptotic-like cell death process. CONCLUSIONS: Here, we propose a potentially useful trypanocidal agent derived from knowledge of an important aspect of the natural life cycle of the parasite: the vector-parasite interaction. Our results indicate that prodigiosin could be a good candidate for the treatment of Chagas disease.

New insights on the antitumoral properties of prodiginines.

Apoptosis is involved in the action of several (and perhaps all) cancer-chemotherapeutic agents. Prodiginines are a family of natural red pigmented secondary metabolites, produced by different bacteria and most of them are characterized by a common pyrrolylpyrromethene skeleton. The biosynthesis of prodigiosin and derivatives has been extensively studied in Serratia marcescens. S. marcescens is a Gramnegative bacterium belonging to Enterobacteriaceae. Prodiginines show numerous biological activities pointing out immunosuppressive and anticancer properties. Some prodiginines displayed apoptotic effects in vitro and antitumor activity in vivo. Their cytotoxic effect is attributed to the presence of the C- 6 methoxy substituent. The A-pyrrole ring plays a key role in both the copper nuclease activity and the cytotoxicity of prodiginines. Here we review the main characteristics of prodigiosin and their derivatives as well as the most prominent pharmacological activity of prodiginines and related compounds, including novel synthetic PG-derivatives with lower toxicity like GX15-070 (Obatoclax). The molecular targets of prodiginines are discussed and the mechanism of action for these molecules is a current topic in biomedicine with a real therapeutica potential in the clinic.

Apoptosis-mediated cytotoxicity of prodigiosin-like red pigment produced by gamma-Proteobacterium and its multiple bioactivities.

Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment (PG-L-1). Among the cell lines tested, U937 cells showed the highest susceptibility to PG-L-1 toxicity. PG-L-1 induced typical apoptotic nuclear morphological changes, and single cell gel electrophoresis revealed that PG-L-1 caused DNA fragmentation in U937 cells. In PG-L-1 treated U937 cells, the acidic compartment such as lysosomes disappeared, suggesting that PG-L-1-induced disorder of intracellular pH compartmentalization might trigger apoptotic signal. Since p38 MAP kinase inhibitor specifically prevented the PG-L-1 mediated cell death, p38 MAP kinase may be involved in the cytotoxic mechanism. In fact, immunoblot analysis of p38 MAP kinase revealed that phosphorylation of p38 MAP kinase occurred in PG-L-1-treated U937 cells. In addition to the activity to induce apoptotic cell death as reported in several PG family members, our chemiluminescence analysis suggested that PG-L-1 inhibited superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated U937 cells in a dose-dependent manner. Since PG-L-1 had no effect on the chemiluminescence response caused by xanthine oxidase/hypoxanthine system, PG-L-1 acts on the enzyme system responsible for O(2)(-) generation rather than direct scavenging toward O(2)(-). Our results suggest that PG-L-1 causes multiple biochemical effects on the target cells such as increase in pH in acidic intracellular compartment, activation of p38 MAP kinase, inhibition of O(2)(-) generation, and eventually induces apoptotic cell death.

Copper-nuclease efficiency correlates with cytotoxicity for the 4-methoxypyrrolic natural products.

The DNA-targeting activities of the 4-methoxypyrrolic natural products, that include prodigiosin (1), tambjamine E (2), and the blue pigment (3), have been compared using fluorescence spectroscopy to study DNA binding and agarose gel electrophoresis to assess their ability to facilitate oxidative copper-promoted DNA cleavage. Fluorescence emission titration of 3 with calf-thymus DNA (CT-DNA) shows that the natural product occupies a site size (n) of ca. two base pairs and possesses an affinity constant (K) of approximately 6x10(5) x M(-1). Similar to prodigiosin (1), the blue pigment 3 was found to facilitate oxidative double-strand DNA (dsDNA) cleavage without the aid of an external reducing agent. Quantitation of ds- (n2) and ss- (n1) breaks provided n1:n2 ratios of approximately 8-12, which were significantly greater than the number expected from the accumulation of ss-breaks (approximately 120). This was contrasted by the nicking activity of tambjamine E (2), which only generates ss-breaks in the presence of copper. The superior copper-nuclease activity of 1 and 3 also correlated with their superior anticancer properties against leukemia (HL-60) cells. These results are discussed with respect to the mode of cytotoxicity by the 4-methoxypyrrolic natural products.

Cytotoxicity of prodigiosin and benznidazole on V79 cells.

The cytotoxicity of prodigiosin, an antibiotic and potential trypanocide produced by Serratia marcescens, and Benznidazole, a trypanocidal drug, were assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), MTT reduction and neutral red uptake (NRU). IC(50) values of 1-20 microM were obtained for prodigiosin in the NRU, MTT and NAC tests. Prodigiosin had greater trypanocidal activity (IC(50)=5 microM) than Nifurtimox (IC(50)=150 microM) a known trypanocide drug used in Chagas' disease therapy. Benznidazole was less toxic (IC(50)=2000 microM) than prodigiosin (IC(50)=1-20 microM) in V79 cells based on the MTT and NAC assays. Benznidazole stimulated the NRU until 2 mM. Indeed, the cell viability measured with the NRU was higher at all concentrations of benznidazole tested than that measured by MTT reduction and NAC assays.

Synthesis and immunosuppressive activity of novel prodigiosin derivatives.

Prodigiosins (Ps) represent a family of naturally occurring red pigments characterized by a common pyrrolylpyrromethene skeleton. Some members of this family have been shown to possess interesting immunosuppressive properties exerted with a novel mechanism of action, different from that of currently used drugs. In fact, Ps inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with a cell surface receptor component called common gamma-chain, which is exclusive of all IL-2 cytokine family receptors. Blocking common gamma-chain transduction activity results in a potent and specific immunosuppressive activity. With respect to the interesting and unexploited immunomodulating properties of this family of compounds we initiated a medicinal chemistry program aimed at finding novel prodigiosin derivatives with improved immunosuppressive activity and lower toxicity. Utilizing an unprecedented and flexible way of assembling the prodigiosin frame, a number of new derivatives have been prepared and tested leading to the choice of 4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2, 2'-bi-1H-pyrrole (PNU-156804, 16) as a lead immunosuppressant.

Toxigenic studies with the antibiotic pigments from Serratia marcescens.

Prodigiosin, obtained from the bacterium, Serratia marcescens, was extracted in five organic solvents, petroleum ether, chloroform, acetone, ethanol, and methanol, and the fractions were labeled PE-1, C-2, A-3, E-4, and M-5 respectively. The effects of prodigiosin and its fractions on embryogenesis showed the whole pigment and C-2 fraction to be highly toxigenic while other fractions demonstrated toxicities approaching LD50 values of 26-30 mug/egg when dissolved in 100% dimethyl sulfoxide. The E-4 fraction in DMSO was least toxic. Ninety-five percent ethanol proved to be highly toxic at a dose level of 0.1 ml/egg indicating that it was an unsuitable solvent for studies of this nature. Disc-agar diffusion sensitivity studies were performed against E. coli, E. aerogenes, S. aureus, B. subtilis, and P. aeruginosa with prodigiosin and fractions dissolved in 100% DMSO. The solvent was found to have no diffusible bacteriostatic activity in vitro. However, prodigiosin and the ethanol (E-4) and methanol (M-5) fractions produced inhibition zones with every organism tested. Data presented below indicate that prodigiosin extracts have toxigenic effects on chick embryos and inhibit the growth of several species of bacteria.