Protection of PtpB proteolytic cleavage by KuwE.

<p>Schematic representation of the region protected by KuwE in PtpB structure. The amino acid sequence VVTLLAAGRPVLTHCFAGKDR (<i>m/z</i> 2224) identified by mass spectrometry is highlighted in blue sticks and cartoon. KuwE is showed as orange sticks while PtpB is represented as green (alpha-helix) and magenta (beta-chain) cartoon.</p

Kinetics measurements of PtpB inhibitors.

<p>Lineweaver-Burk double-reciprocal plots representing inhibitory profiles of compounds KuwE, PirIII, Ega1, 6016, Ac3 and ∆3 against PtpB. Kinetic experiments were conducted in the presence of increasing concentrations of inhibitors: 0 µM (), 1 µM (), 2 µM (), 3 µM (), 6 µM (), 10 µM (), 20 µM (), 25 µM (), 30 µM (), 35 µM (), 40 µM (), 45 µM (); <i>p</i>NPP was used as substrate in all experiments. For KuwE, Ac3 and 6016, all lines converged at the <i>y</i>-axis (1/<i>V</i>_max), whereas the slope (K_Mapp/<i>V</i>_max) and <i>x</i>-axis interception (1/<i>K</i>_Mapp) varies according to the inhibitor concentration; the constant value of <i>V</i>_max and the increased values of <i>K</i>_Mapp are consistent with a competitive inhibition mechanism. For PirIII, Ega1 and Δ3, all lines converge at the <i>x</i>-axis (1/<i>K</i>_Mapp) and the <i>y</i>-axis interception (1/<i>V</i>_max) varies as a function of the inhibitor concentration; the constant value of <i>K</i>_Mapp and the increased values of <i>V</i>_max indicate that these compounds are noncompetitive inhibitors. </p

Differences between PtpB and PTP1B.

<p>Sequence alignment of Mtb PtpB (UniProtKB code: P96830, 276 aa complete sequence) and human PTP1B (UniProtKB code: P18031, 435 aa complete sequence). Not conserved amino acids of the PtpB active site motif, which may be exploited to design selective Mtb PtpB inhibitors, are highlighted by a red box. Sequence alignment was performed with ClustalX. Sequence numbering corresponds to human PTP1B. Bars below the sequence alignment correspond to the degree of amino acid conservation between the two sequence (full bar: residues identity; empty bar: completely different residues). </p

Peptide mass fingerprints.

<p>Peptide mass fingerprinting of PtpB recorded by MS in absence (A) and presence of 300 µM KuwE (B). The tryptic peptide <i>m/z</i> 2224 corresponds to the complete sequence of the catalytic site ((R145)VVTLLAAGRPVLT<u>HCFAGKDR</u>(T167)) and the tryptic peptide <i>m/z</i> 1953 corresponds to a part of the catalytic site ((R145)VVTLLAAGRPVLT<u>HCFAGK</u>(D165)), which include the His159 and the catalytic Cys160 residues.</p

Structures of PtpB inhibitors.

<p>Chemical structure of PtpB inhibitors showing an IC₅₀ < 100 µM. Below the line are the two common chemical scaffolds: Scaffold A present in KuwE, Ega1, M2 and M2H; Scaffold B present in PirIII, Δ3, 6016 and Ac3.</p

Docking-based binding mode of KuwE.

<p>The binding mode of KuwE within the catalytic site of PtpB, as predicted by docking. KuwE is shown as cyan sticks; PtpB is represented as green cartoon and lines. Polar contacts between KuwE and PtpB are highlighted as black dotted lines. Residues Phe161, Lys164 and Asp165 that are not conserved in the human PTP1B are showed as green sticks. Residues numbering follows the PDB: 2OZ5 numbering scheme.</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

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

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

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