Host cell cAMP-Epac-Rap1b pathway inhibition by hawthorn extract as a potential target against Trypanosoma cruzi infection.

Although the two drugs currently available for the treatment of Chagas disease, Benznidazole and Nifurtimox, have proven to be effective in the acute phase of the disease, the 60-90-day treatment leads to high toxicity and unwanted side effects, presenting, in addition, a low efficacy in the chronic phase of the disease. For this reason, new therapies that are more effective are needed. In this regard, we have recently shown that the inhibition of the Epac-Rap1b pathway suppressed the cAMP-mediated host cell invasion by Trypanosoma cruzi. Interestingly, it has been described that vitexin, a natural flavone that protects against ischemia-reperfusion damage, acts by inhibiting the expression of Epac and Rap1 proteins. Vitexin can be found in plants of the genus Crataegus spp., traditionally known as hawthorn, which are of great interest considering their highly documented use as cardio-protectors. Pre-treating cells with an extract of Crataegus oxyacantha produced levels of T. cruzi invasion comparable to the ones observed for the commercially available Epac1-specific inhibitor, ESI-09. In addition, extract-treated cells exhibited a decrease in the activation of Rap1b, suggesting that the effects of the extract would be mediated by the inhibition of the cAMP-Epac-Rap1 signaling pathway. Using HPLC-HRMS2, we could confirm the presence of vitexin, and other flavones that could act as inhibitors of Epac/Rap1b, in the extracts of C. oxyacantha. Most significantly, when cells were treated with the extract of C. oxyacantha in conjunction with Nifurtimox, an increased modulation of invasion was observed.

Screening of Protein-Protein Interaction Modulators Using BRET-Based Technology.

Protein-protein interactions (PPIs) play central roles in most molecular mechanisms underlying cellular and biological processes. Within the methods developed to study PPIs is bioluminescence resonance energy transfer (BRET). Taking advantage of this technique, we have set a BRET-based assay that enables the screening of modulators of essential PPIs for Trypanosoma cruzi survival. Considering the complexity of the evaluated mixture, pure chemical compounds or natural extracts, two approaches are described, BRET in living cells or from lysates.

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors.

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein-protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein-protein interactions were set in living cells and lysates.

Synthesis and antifungal activity of bile acid-derived oxazoles.

Peracetylated bile acids (1a-g) were used as starting materials for the preparation of fourteen new derivatives bearing an oxazole moiety in their side chain (6a-g, 8a-g). The key step for the synthetic path was a Dakin-West reaction followed by a Robinson-Gabriel cyclodehydration. A simpler model oxazole (12) was also synthesized. The antifungal activity of the new compounds (6a-g) as well as their starting bile acids (1a-g) was tested against Candida albicans. Compounds 6e and 6g showed the highest percentages of inhibition (63.84% and 61.40% at 250 µg/mL respectively). Deacetylation of compounds 6a-g, led to compounds 8a-g which showed lower activities than the acetylated derivatives.

Antifungal terpenoids from Hyalis argentea var. latisquama.

A detailed chemical study of the aerial parts and rhizomes of Hyalis argentea var. latisquama yielded a variety of sesqui- and diterpenes. In total, 26 compounds were isolated and identified, of which four are new, namely, two ent-kaurenes (1 and 2), a diterpene lactone (3), and a lindenanolide (4). The previously reported compounds included a series of lindenanolides, guaianolides, elemanolides, and additional diterpenes. The antifungal activity of the isolated compounds was tested against Cryptococcus neoformans and Candida albicans. Among the isolated compounds, the lindenanolides were the only structural class that showed strong antifungal activity, and onoseriolide acetate (5) was the most active. On the other hand, the isolated guaianolides were only moderately active, while the diterpenes did not show significant antifungal activity.

Antioxidant neolignans from Cordia americana.

Five new neolignans with a bicyclo[2.2.2]octene framework were isolated from an ethanolic extract of the bark of Cordia americana. The structures and relative configurations of the compounds were elucidated by a combination of spectroscopic methods. All the isolated compounds showed good antioxidant activities in the DPPH radical scavenging (0.5-100 µg/mL) and Ferric-reducing antioxidant power (FRAP, 1-100 µg/mL) assays. One of the compounds displayed mild fungistatic activity at 0.1 µmol/spot against Fusarium virguliforme while, at the same time, all compounds were inactive against several strains of Gram (+) and Gram (-) bacteria at all assayed concentrations (10-1,000 µg/mL).

Cytotoxic terpenoids from Nardophyllum bryoides.

The investigation of the ethanol extract of fresh aerial parts of the Patagonian shrub Nardophyllum bryoides collected in the province of Chubut, Argentina, yielded eleven terpenoids. These include: three seco-ent-halimane diterpenoids (1-3), two ent-halimanes (4-5) and six pentacyclic oleanane and ursane triterpenoids (6-11). Four of these compounds (2, 6, 8 and 11) are hitherto unknown, while two others (1 and 4) have been previously reported but only as synthetic products. Several of these compounds showed moderate cytotoxicity against a human pancreatic adenocarcinoma cell line while compounds 4 and 5 were active at micromolar concentrations. The main component, seco-chiliolidic acid (1), could be isolated from this extract in large amounts, turning N. bryoides into a sustainable source of this bioactive compound.