Flavonoid Derivatives as New Potent Inhibitors of Cysteine Proteases: An Important Step toward the Design of New Compounds for the Treatment of Leishmaniasis.

Leishmaniasis is a neglected tropical disease, affecting more than 350 million people globally. However, there is currently no vaccine available against human leishmaniasis, and current treatment is hampered by high cost, side-effects, and painful administration routes. It has become a United Nations goal to end leishmaniasis epidemics by 2030, and multitarget drug strategy emerges as a promising alternative. Among the multitarget compounds, flavonoids are a renowned class of natural products, and a structurally diverse library can be prepared through organic synthesis, which can be tested for biological effectiveness. In this study, we synthesised 17 flavonoid analogues using a scalable, easy-to-reproduce, and inexpensive method. All synthesised compounds presented an impressive inhibition capacity against rCPB2.8, rCPB3, and rH84Y enzymes, which are highly expressed in the amastigote stage, the target form of the parasite. Compounds 3c, f12a, and f12b were found to be effective against all isoforms. Furthermore, their intermolecular interactions were also investigated through a molecular modelling study. These compounds were highly potent against the parasite and demonstrated low cytotoxic action against mammalian cells. These results are pioneering, representing an advance in the investigation of the mechanisms behind the antileishmanial action of flavonoid derivatives. Moreover, compounds have been shown to be promising leads for the design of other cysteine protease inhibitors for the treatment of leishmaniasis diseases.

Low biological phosphorus removal from effluents treated by slow sand filters.

The legislation for environment protection requires strict controls of the wastewater releasing in water bodies. The wastewater treatment plants (WWTP) have been used for organic matter degradation; however, the residual total phosphorus (TP) removal has not been efficient. TP and nitrogen present in wastewater are associated to eutrophication of water bodies and algae growth. Therefore, this study discusses the efficiency of phosphorus removal by a slow filter (SF), complementary to a WWTP and the microbial community involved. The results showed that the use of SF, with or without macrophytes, is not suitable to remove TP. Spatial variation in microbial communities distributed in three distinct zones was identified in the SF. Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes covered the hydrolytic and fermentative bacteria. The acetogenesis, nitrification, and denitrification, as well as the removal of phosphorus from the effluent, were performed by representatives affiliated to different groups. Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria among these, Dokdonella sp., Frateuria sp., Comamonas sp., Diaphorobacter sp., Nitrosospira sp., Ferruginibacter sp., Flavobacterium sp., and the uncultured OD1 were the most abundant bacteria in the SF. The low efficiency for TP removing from SF effluents can be explained by the low abundance of phosphorus accumulating organisms (PAOs), with the association of the low concentration of biodegradable organic matter in the inlet effluent. Therefore, the alternative to using SF as a complement to WWTPs, as recommended by some Brazilian environmental agencies, did not prove to be viable and new approaches must be evaluated. KEY POINTS: • The phosphorus removal was performed by a slow filter system in a WWTP but obtained a low efficiency. • Microbial spatial variation was distributed into distinct zones from slow filter. • Low abundance of PAOs was observed due to the low availability of organic matter.

Leishmanicidal, antiproteolytic, and mutagenic evaluation of alkyltriazoles and alkylphosphocholines.

A series of 16 simple long-chain alkyltriazoles and two novel alkylphosphocholine derivatives containing an azide moiety were evaluated in vitro for their leishmanicidal activity against. Among the 18 compounds tested, the eight most active compounds against promastigote forms were selected for further evaluation against amastigote forms. These compounds were also evaluated for their cytotoxicity against murine macrophages and tested as inhibitors of cysteine protease rCPB2.8, an important target for development of antileishmanial drugs. The mutagenicity of some of these compounds was also evaluated in prokaryotic and eukaryotic cells to assess any genetic effects of the leishmanicidal candidates. The compound 4, an alkylphosphocholine derivative, was found to be the most potent against amastigote forms with an IC50 of 3.81 µM, comparable to that of pentamidine (IC50 = 6.62 µM) and amphotericin B (IC50 = 6.10 µM), two established leishmanicidal drugs. Compound 4 also exhibited the best selectivity index (SI) values of the series, demonstrating low toxicity against macrophages and a cLogP value higher than 5. Among the alkyltriazoles, compounds 13 and 14 were the most active against promastigote and amastigote forms. They were then evaluated for their mutagenicity in vitro; the mutagenicity index (MI) values were lower than 2, suggesting that these compounds are not mutagenic.

In vitro and in vivo activity of an organic tellurium compound on Leishmania (Leishmania) chagasi.

Tellurium compounds have shown several biological properties and recently the leishmanicidal effect of one organotellurane was demonstrated. These findings led us to test the effect of the organotellurium compound RF07 on Leishmania (Leishmania) chagasi, the agent of visceral leishmaniasis in Latin America. In vitro assays were performed in L. (L.) chagasi-infected bone marrow derived macrophages treated with different concentrations of RF07. In in vivo experiments Golden hamsters were infected with L. (L.) chagasi and injected intraperitoneally with RF07 whereas control animals received either Glucantime or PBS. The effect of RF07 on cathepsin B activity of L. (L.) chagasi amastigotes was assayed spectrofluorometrically using fluorogenic substrates. The main findings were: 1) RF07 showed significant leishmanicidal activity against intracellular parasites at submicromolar concentrations (IC50 of 529.7±26.5 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 of 5,426±272.8 nM); 2) kinetics assays showed an increasing leishmanicidal action of RF07 at longer periods of treatment; 3) one month after intraperitoneal injection of RF07 L. (L.) chagasi-infected hamsters showed a reduction of 99.6% of parasite burden when compared to controls that received PBS; 4) RF07 inhibited the cathepsin B activity of L. (L.) chagasi amastigotes. The present results demonstrated that the tellurium compound RF07 is able to destroy L. (L.) chagasi in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support further study of the potential of RF07 as a possible alternative for the chemotherapy of visceral leishmaniasis.

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B.

We have developed positional scanning synthetic combinatorial libraries to define the substrate specificity of carboxydipeptidases. The library Abz-GXXZXK(Dnp)-OH, where Abz is ortho-aminobenzoic acid, K(Dnp) is N(epsilon)-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively occupied with 1 of 19 amino acids (cysteine was omitted), and X represents randomly incorporated residues, was assayed initially with human cathepsin B, and arginine was defined as one of the best residues at the P(1) position. To examine the selectivity of S(1)('), S(2), and S(3) subsites, the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH were then synthesized. The peptide Abz-GIVRAK(Dnp)-OH, which contains the most favorable residues in the P(3)-P(1)(') positions identified by screening of the libraries with cathepsin B, was hydrolyzed by this enzyme with k(cat)/K(m)=7288 mM(-1)s(-1). This peptide is the most efficient substrate described for cathepsin B to this point, and it is highly selective for the enzyme among the lysosomal cysteine proteases.