Miriam S. Moraes

Synthesis and antiplasmodial activity of betulinic acid and ursolic acid analogues.

More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC50 = 220 and 175 nM, respectively) while 1a and b demonstrated good activity (IC50 = 4 and 5 μM, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC50 of 4 μM and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

The low molecular weight S-nitrosothiol, S -nitroso-N -acetylpenicillamine, promotes cell cycle progression in rabbit aortic endothelial cells

S-Nitrosylation reactions are considered to be a major mechanism by which NO-related bioactivities are regulated in vivo. In the present study, we show the effects of the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), on cell cycle progression of rabbit aortic endothelial cells (RAEC). SNAP at low concentrations (0.1mM) stimulated the p21Ras-ERK1/2 MAP kinase signaling pathway. Activation of this signaling pathway was strongly inhibited in cells stably transfected with S-nitrosylation insensitive p21Ras (p21(Ras (C118S))). Furthermore, the SNAP-induced effects on cell cycle progression were eliminated in RAEC expressing N17Ras, a negative dominant mutant of p21Ras. Upon stimulation with SNAP, ERK1/2 MAP kinases become phosphorylated and translocate to the nucleus promoting the phosphorylation of the transcription factor Elk1. Synthesis of Cyclin D1 and stimulation of the cyclin-dependent kinases cdk4 and cdk6 resulted in the phosphorylation of the nuclear protein Rb and its dissociation from the E2F family of transcription factors. Cells then pass the restriction point in the late G1 phase. Cyclins E and A were expressed as the cell cycle progressed through the S phase upon stimulation with SNAP. Further transition in the cell cycle from the G2 to M phase was evidenced by the G2/M peak found in a histogram of the cell-phase distribution in SNAP-treated RAEC. These observations suggest that low molecular weight S-nitrosothiols may promote cell cycle progression possibly through the transnitrosation of p21Ras, and activation of the Ras-ERK1/2 MAP kinases signaling pathway.

S-Nitrosoglutathione and Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Regulate Compartmentalized Ras S-Nitrosylation and Stimulate Cell Proliferation

AIMS S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS Ras signaling pathway stimulation by 50 and/or 100 μM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 μM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.

Signal Transduction in G0/G1-Arrested Mouse Y1 Adrenocortical Cells Stimulated by Acth and FGF2

In G0/G1 cell cycle arrested mouse Y1 adrenocortical tumor cells ACTH39, a weak mitogen and strong anti-mitogenic agent, blocks FGF2 mitogenic activity at G1 phase, keeping untouched ERK-MAPK activation and c-Fos protein induction. Here we report two anti-mitogenic mechanisms initiated in ACTH receptors and mediated by cAMP/PKA: a) post-transcriptional down regulation of c-Myc protein; b) dephosphorylation of AKT/PKB. In Y-1 cells the activity of the Mad/Max/Myc network of transcription factors seems to be regulated by c-Myc levels. FGF2 induces c-myc gene and stabilizes c-Myc protein by a process dependent on ERK-MAPK (PD98059 sensitive), but not on PI3K (Wortmannin resistant). ACTH39, on the other hand, causes rapid decrease in c-Myc levels induced by FGF2 in wild type Y1 cells, but not in PKA-deficient Y1 clones. The ACTH inhibition of DNA synthesis stimulated by FGF2 is reversed by transient transfection and induction of the MycER chimera (fusion of c-Myc and estrogen-receptor), suggesting that c-Myc down regulation is an efficient anti-mitogenic mechanism activated by ACTH. Y1 cells display high constitutive levels of AKT/PKB, that is dependent on elevated RasGTP. FGF2 up regulates RasGTP, PI3K and AKT/PKB. ACTH antagonizes this mitogenic effect of FGF2, promoting rapid dephosphorylation of AKT/PKB.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

BackgroundThe discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation.ResultsAmong the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series.ConclusionsThree new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

The PfNF-YB transcription factor is a downstream target of melatonin and cAMP signalling in the human malaria parasite Plasmodium falciparum.

Abstract:  Plasmodium falciparum causes the most severe form of malaria and is responsible for the majority of deaths worldwide. The mechanism of cell cycle control within intra‐erythrocytic stages has been examined as a potential means of a promising way to identifying how to stop parasite development in red blood cells. Our group determined that melatonin increases parasitemia in P. falciparum and P. chabaudi through a complex signalling cascade. In vertebrates, melatonin controls the expression of transcription factors, leading us to postulate rather that the indoleamine would affect PfNF‐YB expression in human malaria parasites. We show here that PfNF‐YB transcription factor is highly expressed and colocalized in the nucleus in mature parasites during intra‐erythrocytic stages, thus suggesting an important role in cell division. Moreover, we demonstrate for the first time that melatonin and cAMP modulate the PfNF‐YB transcription factor expression in P. falciparum at erythrocytic stages. In addition, PfNF‐YB is found to be more ubiquitinated in the presence of melatonin. Finally, the proteasome inhibitor bortezomib is able to modulate PfNF‐YB expression as well. Taken together, our dada reinforce the role played by melatonin in the cell cycle control of P. falciparum and point this indolamine as a target to develop new antimalarial drugs.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Protein tyrosine phosphatase alpha regulates cell detachment and cell death profiles induced by nitric oxide donors in the A431 human carcinoma cell line.

Abstract We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27BPTPα) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27BPTPα) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27BPTPα) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.