Binding at the interface: We tested the inhibitory activity of a set of peptide sequences derived from an α-helix of the dimeric trypanothione reductase from Leishmania infantum. Replacement of a glutamic acid residue with a lysine promoted monomer dissociation and enzyme inhibition.

A series of 9-mer and 13-mer amide-bridged cyclic peptides derived from the linear prototype Ac-PKIIQSVGIS-Nle-K-Nle-NH (Toro et al. ChemBioChem2013) has been designed and synthesized by introduction of the lactam between amino acid side chains that are separated by one helical turn (i, i+4). All of these compounds were tested in vitro as both dimerization and enzyme inhibitors of Leishmania infantum trypanothione reductase (Li-TryR). Three of the 13-mer cyclic peptide derivatives (3, 4 and 6) inhibited the oxidoreductase activity of Li-TryR in the low micromolar range and they also disrupted enzyme dimerization. Cyclic analogues 3 and 4 were more resistant to proteases than was the linear prototype. Furthermore, the most potent TryR inhibitors in the linear and cyclic series displayed potent in vitro activity against Leishmania infantum upon conjugation with cationic cell-penetrating peptides. To date, these conjugated peptides can be considered the first example of TryR dimerization inhibitors that are active in cell culture.

A series of new selenocyanates and diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole, etc.) was synthesized, and their in vitro leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells were determined. Interestingly, most tested compounds were active in the low micromolar range and led us to identify four lead compounds (1h, 2d, 2e, and 2f) with 50% effective dose (ED 50 ) values ranging from 0.45 to 1.27 M and selectivity indexes of >25 for all of them, much higher than those observed for the reference drugs. These active derivatives were evaluated against infected macrophages, and in order to gain preliminary knowledge about their possible mechanism of action, the inhibition of trypanothione reductase (TryR) was measured. Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl selenocyanate) and 2d [3,3=-(diselenodiyldimethanediyl)bis(2-bromothiophene)] exhibited good association between TryR inhibitory activity and antileishmanial potency, pointing to 1h, for its excellent theoretical ADME (absorption, distribution, metabolism, and excretion) properties, as the most promising lead molecule for leishmancidal drug design.

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein )1 min )1 ) followed by silver carp (1.084 ± 0.061 U mg protein )1 min )1 ), and catla (0.193 ± 0.006 U mg protein )1 min )1 ). Trypsin activity of silver carp and rohu was 89-91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell-shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N-a-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanychloromethane (TPCK) indicated the presence of trypsin-like and chymotrypsin-like enzymes in all these three carps. SDS-PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS-PAGE evidenced the presence of various protease activity bands ranging from 21. 6-93.7, 21.6-63.8 and 26.7-98.5 kDa for catla, rohu and silver carp respectively. In pH-stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others. KEY WORDS

The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β-peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β-peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes.

A novel series of thirty-one N-substituted urea, thiourea, and selenourea derivatives containing diphenyldiselenide entities were synthesized, fully characterized by spectroscopic and analytical methods, and screened for their in vitro leishmanicidal activities. The cytotoxic activity of these derivatives was tested against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1 cells. Thirteen of the synthesized compounds showed a significant antileishmanial activity, with 50% effective concentration (EC 50 ) values lower than that for the reference drug miltefosine (EC 50 , 2.84 M). In addition, the derivatives 9, 11, 42, and 47, with EC 50 between 1.1 and 1.95 M, also displayed excellent selectivity (selectivity index ranged from 12.4 to 22.7) and were tested against infected macrophages. Compound 11, a derivative with a cyclohexyl chain, exhibited the highest activity against intracellular amastigotes, with EC 50 values similar to those observed for the standard drug edelfosine. Structure-activity relationship analyses revealed that N-aliphatic substitution in urea and selenourea is recommended for the leishmanicidal activity of these analogs. Preliminary studies of the mechanism of action for the hit compounds was carried out by measuring their ability to inhibit trypanothione reductase. Even though the obtained results suggest that this enzyme is not the target for most of these derivatives, their activity comparable to that of the standards and lack of toxicity in THP-1 cells highlight the potential of these compounds to be optimized for leishmaniasis treatment.

Seed extracts from indigenous and introduced legumes were prepared and used to search for inhibitors of fish muscle proteinases. Fish flesh extracts were prepared from samples of Merluccius productus (Pacific whiting or merluza) obtained off the Oregon coast and in the Gulf of California, respectively. The proteinase activity in the fish muscle for the Pacific whiting was the highest, followed by parasitized merluza. The lowest proteinase activity was for the nonparasitized merluza. Six out of 12 seed extracts reduced the proteinase activity from the fish flesh by more than 50%. The reduction of enzyme activity was higher for samples of fish flesh extracts from the Gulf of California than for the Oregon samples. Seed extracts also reduced the proteinase activity of commercial serine and cysteine proteinases such as trypsin, chymotrypsin, and papain. This inhibitory capacity was maintained even after heating the seed extracts to 90 degrees C for 15 min. Several seed extracts show promise for use as proteinase inhibitors during production of surimi, the intended commercial product of massive fisheries such as Pacific whiting or merluza.

A table of contents entryHelical peptides stabilized via all-hydrocarbon or lactam side-chain bridging were investigated as disruptors of Leishmania infantum trypanothione reductase and the biological results were rationalized by NMR spectroscopy studies and molecular dynamics simulations.Abstract-All-hydrocarbon and lactam-bridged staples linking amino acid side-chains have been used to stabilize the α-helical motif in short 13-mer peptides that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR). The design of the best positions for covalent hydrocarbon closure relied on a theoretical prediction of the degree of helicity of the corresponding cyclic peptides in water. Selected (i, i+4) and (i, i+7) hydrocarbon-stapled peptides were prepared by using solid-phase synthesis protocols and optimized ring-closing metathesis reactions under microwave conditions. Structural analysis by NMR spectroscopy confirmed high helical contents in aqueous TFE solutions for both types of helix-constrained cyclic peptides. Remarkably, the ability to prevent Li-TryR dimerization was reduced in both (i, i+4) and (i, i+7) hydrocarbon stapled peptides but was retained in the corresponding (i, i+4) Glu-Lys lactam-bridged analogue, which also showed a higher resistance to proteolytic degradation by proteinase K relative to the linear peptide prototype. In silico studies indicated that the introduction of a hydrocarbon staple vs a lactam bridge likely perturbs critical interactions required for proper binding of the peptide to the Li-TryR monomer.corresponding N-Fmoc-protected amino acid (1.2 equiv), HCTU (1.2 equiv) and DIEA (2.4 equiv) in anhydrous DMF (0.5-1.0 mL) was added over the swollen peptidil-resin (1.0 equiv) in anhydrous DMF in a microwave vial (5-10 mL). The coupling reaction was heated at 40 ºC using microwave radiation for 10 min. Each coupling step was repeated 3 times using freshly prepared Fmoc-amino acid/coupling reagent mixtures. After complete couplings, the resin was drained and washed with DMF/CH 2 Cl 2 /DMF/CH 2 Cl 2 (4 x 0.5 min).Coupling reactions to primary and secondary amines were monitored by the ninhydrin and the chloranil tests, respectively. Ring closing metathesis (RCM) reaction.To the peptidyl-resins 12 and 13 (0.05-0.07 mmol) swollen in anhydrous CH 2 Cl 2 (5 mL) in a microwave vial (5-10 mL) second generation Grubb's catalyst (0.2 equiv) was added and the vial was sealed and gently bubbled with argon. Then, the reaction was heated at 40 ºC using microwave radiation for 30-150 min. The resin was then filtered and washed successively with CH 2 Cl 2 /MeOH.Residual ruthenium impurities were removed by stirring the resin bound peptide with a solution of DMSO (50 equiv relative to the catalyst) in DMF for 12 h. 37