Exploring oxidovanadium(IV) complexes as YopH inhibitors: mechanism of action and modeling studies

YopH tyrosine phosphatase, a virulence factor produced by pathogenic species of Yersinia, is an attractive drug target. In this work, three oxidovanadium(IV) complexes were assayed against recombinant YopH and showed strong inhibition of the enzyme in the nanomolar range. Molecular modeling indicated that their binding is reinforced by H-bond, cation−π, and π–π interactions conferring specificity toward YopH. These complexes are thus interesting lead molecules for phosphatase inhibitor drug discovery

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases

Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: <i>In Vitro</i> and <i>In Silico</i> Approaches

<div><p>The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed <i>in vivo</i> due to poor pharmacokinetic profiles. In this work we propose a combined <i>in vitro</i>/<i>in silico</i> approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted <i>in silico</i>, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined <i>in vitro</i>/<i>in silico</i> approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.</p></div

Energies and stoichiometry ratios for binding of recombinant murine PrP (1AG2) to compounds from D, G, J, L, R and Y series obtained from molecular docking.

<p>Energies and stoichiometry ratios for binding of recombinant murine PrP (1AG2) to compounds from D, G, J, L, R and Y series obtained from molecular docking.</p

Representative dot-blot showing PK-resistant PrP (PrP^Res) accumulated in ScN2a cells grown in the presence of compounds.

<p>ScN2a cells were grown for 4-well plates in the presence of compounds from the R series at 1, 5, and 10 μM final concentrations. Cell lysates were subjected to PK treatment and dot-blotting with antiserum R30. Control wells (C) show untreated cells.</p

<i>In silico</i> prediction of physicochemical and pharmacokinetic properties.

<p><i>In silico</i> prediction of physicochemical and pharmacokinetic properties.</p

Evaluation of the compounds' capacity to delay PrP^Sen conversion into PrP^Res by RT-QuIC assay seeded with 263 K scrapie.

<p>RT-QuIC reactions were seeded with 10fg of 263 K infected hamster brain or the equivalent amount of normal brain homogenates (NBH). The substrate for the reaction was recombinant hamster PrP^90–231. NaCl was used at 300 mM final concentration. Compounds Y13 and Y17 were assayed at 25 and 50 μM. The assay was followed by ThT fluorescence (excitation 450 nm; emission 480 nm) emission over time (average of 4 replicate wells).</p

Decrease of PrP^Res levels in ScN2a cells.

<p>ScN2a cells were treated with the compounds belonging to the L (panels A and B), G, R', R and Y (panel C) series at 10 µM. After four days of incubation with the compounds, cells were lysed, treated with PK and PrP^Res was detected in the dot-blot assay with anti-PrP antibody (R30). Control bar (medium) represents intensity density of the blot from wells without compound addition, corresponding to 100% of PrP^Res content. Quantification of the assay was done by integration of the density of each dot using ImageJ software. All bars had <b>*</b><i>P</i><0.05 in relation to control.</p

Dose-response curves for compounds from the J and Y series.

<p>Lysates of ScN2a cells grown in the presence of the compounds for 4-blotted as described in the Methods section. Quantification of the relative dot-blot signal intensities were done with ImageJ considering as the control the final DMSO concentration in each well. The curves were fitted by a sigmoidal curve with SigmaPlot software v. 10.0.</p