Mycotic infection is becoming a serious health problem since effective antifungal agents for control of pathogenic fungi, especially drug-resistant pathogens, is often very limited. Fungal resistance to antimycotic agents frequently involves mutations caused by environmental stressors. In fungal pathogens, stress signals resulting from oxidative, cell wall stress, etc., are integrated into the upstream mitogen-activated protein kinase (MAPK) pathways that regulate genes countering the stress. Noteworthy is that mutations in MAPK signaling system result in fungal tolerance to cell wall disrupting agents or phenylpyrrole. In a chemo-biological platform to achieve targeted antifungal intervention, the model yeast Saccharomyces cerevisiae served as a tool for identifying mechanisms of actions of redox-active or cell wall disrupting agents. This also enabled the identification of new utility of known compounds or the utilization of natural products/derivatives as chemosensitizing agents to intensify the efficacy of conventional antimycotic agents. Compounds targeting cellular antioxidant, mitochondrial or cell wall integrity systems effectively inhibited the growth of pathogens and/or overcame fungal tolerance to antimycotic agents. Therefore, chemo-biological approaches lead to the development of novel intervention strategies, such as antifungal chemosensitization, which enhance the drug susceptibility of targeted fungi, and ensure the maintenance of healthy microbiome dynamics.

Hepatitis C virus is an enveloped, ssRNA virus, which infects 3% of the world population. No fully efficient therapy for treating hepatitis C exists. This is mainly due to the quasispecies structure of the RNA genome population, which favors the emergence of resistant viral variants. Despite the high variability rate, significant sequence and, more importantly, structure conservation can be found in the so-called functional genomic RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are potential therapeutic targets. This study validates the use of RNA-based inhibitors (aptamers) as molecular tools to control the functionality of the cis-acting replication element (CRE) within the HCV genome. The CRE is an essential partner for viral replication. Also this structural domain is involved in the regulation of the protein synthesis. A set of forty-four RNA aptamers was assayed for the ability to interfere with the viral RNA synthesis in a subgenomic replicon system. Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with specific and well-defined functional RNA domains of the CRE, yielding a decrease in the HCV genomic RNA levels higher than 90%. Concomitantly, one of them also promoted a significant increase in viral translation (>50%), likely by its interaction with the nucleotides surrounding the viral stop translation codon. The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE, thus explaining their antiviral activity. Present findings confirm the potential of the CRE as an anti-HCV drugs target and support the use of aptamers as molecular tools for challenging the functionality of RNA domains in viral genomes.

 

Flavonoids represent an interesting class of naturally occurring compounds that have been attracting attention of the scientific community because of their wide range of biological properties, being the antitumor activity one of the most studied. The antitumor activity of flavonoids is associated with, at least in part, their ability to induce apoptosis by affecting the expression or activity of a wide variety of molecules involved in apoptosis pathways, namely the caspase family proteins [1]. As a result of the search for new bioactive compounds with antitumor activity by our research group, two flavonoids have been identified as procaspase-7 activators [2]. Inspired by the potential of these flavonoids as caspase modulators, we have synthesized several analogues using natural flavonoids, as the starting material. The synthetic approach was based on the reaction with alkyl halides in alkaline medium under microwave irradiation. The structure elucidation of synthesized compounds was established on the basis of NMR techniques (¹H NMR, ¹³C NMR, HSQC and HMBC). Some of the synthesized compounds were evaluated for their ability to modulate procaspases-3 and 7 activity using yeast cell based assays [2]. Molecular docking studies with procaspases-3, 6 and 7 were also performed.

References:

[1] Ravishankar, D., Rajora, A. K., Greco, F., Osborn, H. M. (2013), Flavonoids as Prospective Compounds for Anti-cancer Therapy, The International Journal of Biochemistry & Cell Biology, 45(12), 2821-2831.

[2] Pereira, C., Lopes-Rodrigues, V., Coutinho, I., Neves, M. P., Lima, R. T., Pinto, M., Saraiva, L. (2014), Potential Small-molecule Activators of Caspase-7 Identified Using Yeast-based Caspase-3 and-7 Screening Assays, European Journal of Pharmaceutical Sciences, 54, 8-16.

Acknowledgments:

This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and the projects TDC/DTP-FTO/1981/2014 and REQUIMTE-Pest-C/EQB/LA0006/2013.

The negatively charged polysaccharide Hyaluronic acid (HA) has diverse physiological and pathophysiological functions depending on its chain size. Space‑filling, anti‑inflammatory and antiangiogenic effects are triggered by high molecular weight HA (HMW HA) (>20 kDa). Hydrolyzation of HMW HA by Hyal‑1 results in low molecular weight HA (LMW HA) (<20 kDa) which leads to inflammatory and angiogenic effects.^[1] For this reason Hyal‑1 is an interesting target for drug discovery. The surface display of active Hyal‑1 on Escherichia coli, via Autodisplay, enables the screening for potential inhibitors in a whole cell system. Based on this technique we determined the inhibitory effect of different natural substances on human Hyal-1. The IC₅₀ values of the plant extracts Malvae sylvestris flos, Equiseti herba and Ononidis radix were determined to be between 1.4 and 1.7 mg/mL. Furthermore, the IC₅₀ values of four triterpenoid saponines were determined. The obtained IC₅₀ value for glycyrrhizinic acid, a known Hyal-1 inhibitor, was 177 µM. The IC₅₀ values for the newly identified inhibitors gypsophila saponin 2, SA1641, and SA1657 were 108 µM, 296 µM and 371 µM, respectively.^[2] For the synthesis of new small molecule inhibitors targeting human Hyal-1 these extracts and natural compounds could be used as a starting point.

References

[1]   Stern R, Semin Cancer Biol, 2008, 18, 275-280.

[2]   Orlando Z, et al. Molecules, 2015, 20, 15449-15498.

Myeloperoxidase (MPO) is an important target for drug design because of its contributing role in many inflammatory syndromes such as atherosclerosis, rheumatoid arthritis, end-stage renal disease or neurodegeneration. Rational drug design assisted by virtual screening is an interesting tool to design new chemical entities that could inhibit MPO. After a high throughput virtual screening of a database, bis-2,2′-[(dihydro-1,3(2H,4H)-pyrimidinediyl)bis(methylene)]phenol was chosen as a starting hit and we used different strategies of chemical synthesis to perform pharmacomodulation described by the three following approaches: I) changing the position of the two nitrogen atoms in the hexahydropyrimidine cycle leading to piperazine derivatives, II) omitting one nitrogen atom in the hexahydropyrimidine leading to piperidine derivatives, III) opening the cycle of hexahydropyrimidine and keeping one nitrogen atom in the aliphatic chain leading to alkylamine derivatives. This led to 30 compounds that have been assessed in an in vitro inhibition MPO test. We found that the alkylamine compounds were active but to a lesser extent than the starting hit. Exception for propylamine derivatives with a phenyl cycle should be noticed. As indolic compounds have demonstrated interesting inhibiting properties, we combined indole ring with thephenolhydropyrimidine structure which led to compounds more active than the hit. Among them, propylamine derivatives were new MPO inhibitors with a nanomolar IC50. Kinetics studies for the most potent inhibitors were conducted and reflected a fast reaction with compound I (MPO-Porphyrin•+-Fe(IV)=O) resulting in the accumulation of compound II (MPO-Porphyrin-Fe(IV)=O). Structure-activity relationship will be discussed to highlight the chemical group of interest in the interaction with MPO.

Ischemia-reperfusion or traumatic brain injury induces the accumulation of reactive oxygen species (ROS) in brain. ROS causes oxidative stress to astrocytes as well as neurons and oxidative stress induces the damage to organelles, proteins or lipids. The removal of damaged cellular cytosolic components is indispensable for the cell to keep the homeostasis. Macroautophagy (hereafter referred to as autophagy) is the process to degrade defective proteins and damaged organelles. In the process of autophagy, damaged cellular cytosolic components are isolated by autophagosomes. Microtubule-associated protein 1 light chain 3B (LC3B) plays a significant role in the autophagosome formation, and the conversion from unconjugated-LC3B (LC3B-I) to phosphatidylethanolamine (PE) conjugated-LC3B (LC3B-II) is the index of the activitation of autophagy. GABARAPL1 is the paralogue of LC3B and the function of GABARAPL1 is not fully understood.

In this study, we demonstrated GABARAPL1 mRNA and protein expression are up-regulated by H₂O₂ in rat C6 glioma cells and the induction of GABARAPL1 by H₂O₂ was accompanied with the conversion from LC3B-I to LC3B-II, indicating the formation of autophagosomes. Thus, GABARAPL1 may play a role in autophagic process, which is induced by H₂O₂. However, elucidation of the function of GABARAPL1 in autophagy will require further studies.

Research and development of drugs with combined pharmacological effects – those that affect the different receptor types – constitute an urgent problem of modern pharmacology. In this work we are presenting the synthesis and pharmacological investigation of some new menthol, thymol and salicylic acid derivatives designed as GABA- and TRP allosteric modulators. Our findings identified menthyl ester of GABA (2-isopropyl-5-methylcyclohexyl 4-aminobutyrate) as a compound with anticonvulsant activity over a wide range of doses: 87-1350 mg/kg, whereas menthyl ester of glycine (2-isopropyl-5-methylcyclohexyl 2-aminoacetate) shows significant sedative effect over 6 hours after oral administration at 175 mg/kg. Complex compounds obtained by SnCl₄ interaction with salicyloylhydrazones have demonstrated anti-inflammatory, anxiolytic, antidepressant and analgesic activities in different models in vivo. All aforementioned compounds can be considered as those that exhibit a combined pharmacological activity due to their simultaneous binding to different receptor types.

According to World Health Organization, cardiovascular diseases are the first cause of death worldwide. Although health improved in the last decades, lifestyle changes led to an increased incidence of cardiovascular diseases. Currently, the available antithrombotic drugs are associated with significant drawbacks that limit their use and the development of more advantageous drugs with less secondary effects is necessary. A new class of polysulfated small-molecules with anticoagulant and antiplatelet activities was discovered in our group.^{1, 2} However, these polysulfated derivatives showed poor antithrombotic efficacy by in vivo oral administration in mice, predicted to be due to poor absorption in the gastrointestinal (GI) tract.^{2, 3} The main aim of this work was to improve the oral bioavailability of these compounds. In order to get new optimized analogues two strategies were considered: i) obtaining conjugates with bile acids and ii) introduction of a triazole ring.

Naringin-deoxycholic acid conjugate was obtained through a crosslinking reaction using 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro borate (TBTU) as coupling reagent. Triazole linked xanthone glycoside was obtained through a copper(I)-catalyzed alkyne-azide cycloaddition following by O- and N-deacetylation. Sulfation was successfully achieved with triethylamine-sulfur trioxide adduct under microwave irradiation.

The three sulfated derivatives were screened for anticoagulant activity using the three classic clotting times: activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). All the sulfated compounds prolonged the clotting times and the most active compound was the persulfated naringin-deoxycholic acid conjugate, exhibiting a double concentration value on the APTT (APTT₂) in the micromolar range (around 44 µM). These new optimized analogues with anticoagulant activity are expected to cross the GI tract membranes after oral administration.

 

References

1. Correia-da-Silva, M.; Sousa, E.; Duarte, B.; Marques, F.; Cunha-Ribeiro, L. M.; Pinto, M. M. M. Eur. J. Med. Chem. 2011, 46, 2347-2358.
2. Correia-da-Silva, M.; Sousa, E.; Duarte, B.; Marques, F.; Carvalho, F.; Cunha-Ribeiro, L. M.; Pinto, M. M. J. Med. Chem. 2011, 54, 5373-84.
3. Correia-da-Silva, M.; Sousa, E.; Duarte, B.; Marques, F.; Carvalho, F.; Cunha-Ribeiro, L. M.; Pinto, M. M. J. Med. Chem. 2011, 54, 95-106.

 

 Acknowledgments: This research was partially supported by ERDF through the COMPETE and national funds through FCT, under the project PEst-C/MAR/LA0015/2013.

Phytohaemagglutinin (PHA, or phytohemagglutinin) is a lectin found commonly in plants, especially legumes. It has some physiological effects on cell metabolism; it induces mitosis and affects the cell membrane regarding transport and permeability to proteins. It agglutinates most mammalian red blood cell types and have the mitogenic effect. This is the reason that PHA is extensively used in the laboratory as well as clinical set up for karyotyping analysis. The downside of PHA use is its cost and storage (-20^oC) resulting into increased cost. We have synthesised acetylated pyrazolines  as anticancer agents and during their evaluation for anticancer potential in normal control cells, we were surprised by their cell proliferation activity. We thought of relating our compound to PHA (PHA mimetic) and performed the basis karyotyping experiment keeping PHA as standard and found our compounds to be PHA mimics. The compounds are thus being evaluated for their further PHA mimetic potential using B/T cell specific cell cycle analysis and karyotyping experiment keeping PHA as standard and found our compounds outstanding PHA in every aspect. The compounds are thus being evaluated for their further PHA mimetic potential.

 Human CK2 is a heterotetrameric constitutively active serine / threonine protein kinase and plays an important role in current cancer research [1]. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. Most protein-protein interaction (PPI) studies or screening assays are based on fluorescence detection and require the labeling of the target enzyme by a fluorophore. Unfortunately, through labeling by commercial applications the catalytic subunit CK2α loses activity. Furthermore, the labeling ratio of the protein sample differs and is not exactly reproducible.

The solution for this problem was a bioorthogonal click reaction of the protein kinase. By expanding the genetic code, the unnatural amino acid para‑acidophenylalanine (pAzF) could be incorporated into CK2 [2]. Performing the SPAAC click reaction (Strain-Promoted Alkyne-Azide Cycloaddition) by the use of DBCO 545 (dibenzylcyclooctyne‑fluor 545) led to a specifically labeled human protein kinase CK2 [3].

This site specific labeling does not impair the phosphorylation activity of the kinase, which was evaluated by capillary electrophoresis. The innovatively labeled kinase in combination with the Autodisplay technology could be a significant advancement for inhibitor screening assays by flow cytometry and for CK2α/CK2β interaction studies [4].

 

References:

1. Trembley, J.H. et al.: Biofactors 2010, 36(3): 187-95.
2. Chin, J.W. et al.: J. Am. Chem. Soc. 2002, 124(31): 9026-7.
3. Mbua, N.E. et al.: Chembiochem. 2011, 12(12): 1912-21.
4. Jose, J., Meyer, T.F.: Microbiol. Mol. Biol. Rev. 2007, 71(4): 600-19.