Wnt Drug Discovery: Weaving Through the Screens, Patents and Clinical Trials.

The Wnt signaling pathway is intricately involved in many aspects of development and is the root cause of an increasing number of diseases. For example, colorectal cancer is the second leading cause of death in the industrialized world and aberration of Wnt signaling within the colonic stem cell is the cause of more than 90% of these cancers. Despite our advances in successfully targeting other pathways, such as Human Epidermal Growth Factor Receptor 2 (HER2), there are no clinically relevant therapies available for Wnt-related diseases. Here, we investigated where research activities are focused with respect to Wnt signaling modulators by searching the United States Patent and Trade Office (USPTO) for patents and patent applications related to Wnt modulators and compared this to clinical trials focusing on Wnt modulation. We found that while the transition of intellectual property surrounding the Wnt ligand-receptor interface to clinical trials is robust, this is not true for specific inhibitors of ß-catenin, which is constitutively active in many cancers. Considering the ubiquitous use of the synthetic T-cell Factor/Lymphoid Enhancer Factor (TCF/Lef) reporter system and its success in identifying novel modulators in vitro, we speculate that this model of drug discovery does not capture the complexity of in vivo Wnt signaling that may be required if we are to successfully target the Wnt pathway in the clinic. Notwithstanding, increasingly more complex models are being developed, which may not be high throughput, but more pragmatic in our pursuit to control Wnt signaling.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Immunostimulatory and cytotoxic activities of Indigofera suffruticosa (Fabaceae).

One of the major disadvantages of the current cancer therapy is the suppression of the immune system. Brazilian flora is considered one of the most diverse in the world and many plants were found to contain active constituents that can be valuable sources of new drugs. The plant Indigofera suffruticosa was studied to determine its potential to stimulate the immune system and also to be effective against tumour cells. We investigated the effects of the alkaloidal fraction and the pure alkaloid indigo obtained from I. suffruticosa on macrophage activation by measuring nitric oxide (NO) and tumour necrosis factor-α (TNF-α) production. Cytotoxic activity was also evaluated against the two tumour murine cells lines, LM2 (breast adenocarcinoma) and LP07 (lung adenocarcinoma). The alkaloidal fraction induced a high NO production and a moderated TNF-α release. The pure indigo demonstrated an elevated NO and TNF-α production. The fraction and the pure compound also exhibited cytotoxic activity against both adenocarcinoma cell lines and indigo showed the strongest cytotoxic activity with IC50 value of 0.89 µg mL⁻¹ against LM2 and 1.44 µg mL⁻¹ against LP07. Our results presented the immunostimulatory and cytotoxic activity of I. suffruticosa, enhancing macrophage function and therefore contributing to the host defence against tumours.

Antimycobacterial activity of Indigofera suffruticosa with activation potential of the innate immune system.

Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The antimycobacterial activity and the innate immune response of methanol (METH) and dichloromethane (DCM) extracts of Indigofera suffruticosa Miller (Fabaceae) were evaluated. We observed that the minimum inhibitory concentrations (MICs) for METH and DCM extracts were 125 and 1000 microg/mL, respectively. However, they were able to induce the innate immune response through the production of high levels of NO and TNF-alpha (p < 0.001) by peritoneal exudate cells (PECs). These results suggest that I. suffruticosa extracts may have an important immunological role in the control of TB once macrophage activity is induced by them.

Immunostimulatory effects of the phenolic compounds from lichens on nitric oxide and hydrogen peroxide production / Efeito imunoestimulante de compostos fenólicos de líquens na produção de peróxido de hidrogênio e óxido nítrico

The effects of isolated compounds from Brazilian lichens and their derivatives on H2O2 and NO production were studied using murine macrophages as a part of an attempt to understand their possible immunomodulatory properties. The compound cytotoxicity was studied using MTT assay. Macrophage stimulation was evaluated by the determination of NO (Griess assay) and H2O2 (horseradish peroxidase/phenol red) in supernatants of peritoneal macrophage cultures of Swiss mice. This research demonstrated stimulatory activities of some phenolic compounds isolated from lichens and their derivatives on H2O2 and NO production. Structure-activity relationships suggest several synthetic directions for further improvement of immunological activity.

Os efeitos dos compostos isolados de líquens brasileiros e seus derivados na produção de NO e H2O2 foram estudados utilizando macrófagos murinos na tentativa de desvendar suas possíveis propriedades imunomodulatórias. A citotoxicidade dos compostos foi estudada utilizando o ensaio de MTT. A estimulação dos macrófagos foi avaliada através da determinação de NO (metodologia de Griess) e H2O2 (peroxidase de raíz forte/vermelho de fenol) no sobrenadante de culturas de macrófagos peritoneais de camundongos Swiss. Este estudo demonstrou atividade estimulante de alguns compostos fenólicos e seus derivados na produção de NO e H2O2. A relação estrutura atividade sugere inúmeras direções sintéticas para futuros melhoramentos da atividade imunológica.

Anti-angiogenic effects of pterogynidine alkaloid isolated from Alchornea glandulosa.

BACKGROUND: Angiogenesis, a complex multistep process that comprehends proliferation, migration and anastomosis of endothelial cells (EC), has a major role in the development of pathologic conditions such as inflammatory diseases, tumor growth and metastasis. Brazilian flora, the most diverse in the world, is an interesting spot to prospect for new chemical leads, being an important source of new anticancer drugs. Plant-derived alkaloids have traditionally been of interest due to their pronounced physiological activities. We investigated the anti-angiogenic potential of the naturally occurring guanidine alkaloid pterogynidine (Pt) isolated from the Brazilian plant Alchornea glandulosa. The purpose of this study was to examine which features of the angiogenic process could be disturbed by Pt. METHODS: Human umbilical vein endothelial cells (HUVEC) were incubated with 8 muM Pt and cell viability, proliferation, apoptosis, invasion and capillary-like structures formation were addressed. Nuclear factor kappaB (NFkappaB), a transcription factor implicated in these processes, was also evaluated in HUVEC incubated with Pt. Quantifications were expressed as mean +/- SD of five independent experiments and one-way analysis of variance (ANOVA) followed by the Dunnet test was used. RESULTS: A significant decrease in proliferation and invasion capacity and an effective increase in apoptosis as assessed by bromodeoxyuridine (BrdU), double-chamber and terminal transferase dUTP nick end labeling (TUNEL) assay, respectively, have been found. Pt also led to a drastic reduction in the number of capillary-like structures formation when HUVEC were cultured on growth factor reduced-Matrigel (GFR-Matrigel) coated plates. In addition, incubation of HUVEC with Pt resulted in reduced NFkappaB activity. CONCLUSION: These findings emphasize the potential use of Pt against pathological situations where angiogenesis is stimulated as tumor development.

Isolated flavonoids against mammary tumour cells LM2.

The purpose of the present study was to investigate antitumour and anti-inflammatory activities of flavonoids isolated from Byrsonima crassa, Davilla elliptica and Mouriri pusa. The antitumour activity was measured by the MTT assay in murine mammary tumour cells (LM2) and the IC50 values of the flavonoids tested ranged from (31.5 +/- 2.97) to (203.1 +/- 5.9) microg/ml. The flavonoids 1 (myricetin-3-O-alpha-L-rhamnopyranoside) and 3 (quercetin-3-O-galactopyranoside) from D. elliptica were the most active ones against the tumour cells. The same samples were tested to determine the inhibition of the release of nitric oxide (NO) and of the tumour necrosis factor-alpha (TNF-alpha) in murine macrophages by the Griess and ELISA sandwich assay, respectively. Almost all the samples showed inhibitory activity to the release of NO but not of TNF-alpha. Of all substances tested, flavonoids 2 (quercetin) and 6 (myricetin) may show promising activity in the treatment of murine breast cancer by immunomodulatory and antiproliferative activities.