Figure 1.
Workflow overview for PTPσ inhibitor development.
In silico docking was performed using the crystal structure of the D1 active site of PTPσ as a target (PDB ID: 2fh7). Three structurally distinct scaffolds were chosen, along with 74 additional compounds identified by a sub-structure search of compounds in the ChemBridge, were tested in vitro to for the ability to inhibit PTPσ phosphatase activity. To eliminate oxidation-mediated inhibition, we optimized the biochemical assay and discovered one lead compound whose inhibition was not mediated by oxidation. Future efforts (dashed boxes and lines) will introduce selectivity into this lead compound through a detailed structural analysis of PTPσ and related PTPs.
Figure 2.
In silico docking identifies compounds which dock into the D1 active site of PTPσ.
(A) The D1 domain of PTPσ docked a phosphotyrosine (p-Tyr) substrate into the active site. Surface resonance of the active site is displayed with negatively (red) and positively (blue) charged residues shown and substrate drawn in ball-and-stick form. Structures were generated with ICM software (MolSoft). (B-D) In silico screening identified structurally distinct scaffolds (compounds 6, 48, and 49), which molecularly dock into the active site, similar to the pTyr peptide. These compounds were chosen as a platform for subsequent studies based on their structural diversity and ability to inhibit PTPσ activity by at least 70% in a pilot phosphatase assay (at 10 µM), comparable to the pan PTP inhibitor, sodium orthovanadate (data not shown). Chemical structures created in ChemDraw. Compound 6: N-[2-(1-cyclohexen-1-yl)ethyl]-2-(4-{[(1-phenylethyl)amino]sulfonyl}phenoxy)acetamide. Compound 48: 2-(1-naphthyl)-2-oxoethyl 9-oxo-9H-fluorene-3-carboxylate. Compound 49: N-(3-acetylphenyl)-3,5-bis(benzoylamino)benzamide.
Figure 3.
Optimization of biochemical screening conditions for PTPσ inhibition.
(A) Para-nitrophenyl phosphate (pNPP) is a generic phosphatase substrate whose dephosphorylated product, para-nitrophenol (pNP), yields an intense yellow color under alkaline conditions measurable at 405 nm absorbance on a spectrophotometer. (B) 20 µg purified recombinant GST-PTPσ-CTF (C-terminal fragment containing the active sites) protein was resolved by SDS-PAGE and stained with coomassie blue to demonstrate purity. (C) The linear formation of product by various quantities of recombinant GST- PTPσ was observed through time-course reactions. pNPP-phosphatase assays were completed with a saturating dose of 1 mM pNPP. Background-corrected absorbance of dephosphorylated product are plotted by time of reaction. Each plot stems from the quantities of PTPσ indicated in the legend. (D) 2 µg enzyme was chosen from (A) for analysis of activity with varying doses of pNPP substrate. Each plot represents a unique dose of pNPP (indicated in the legend). Background-corrected absorbance of dephosphorylated product are plotted by time of reaction. (E) Initial velocities of PTPσ phosphatase activity (Y-axis; in pNP product formed per minute) were derived from the slopes of the plots in (D) at each of the indicated pNPP substrate concentrations (X-axis). From this, a Km of 250 µM is observed (denoted by dashed line).
Figure 4.
In vitro screen identifies active compounds which inhibit PTPσ.
(A–C) The three in silico-identified scaffolds and 74 additional compounds identified by a sub-structure search of ChemBridge compounds for structural features relating to these scaffolds actives (A- similar to 6; B- similar to 48; C- similar to 49) were tested in vitro for potency of PTPσ. Compounds (at a final concentration of 100 µM) were pre-incubated with PTPσ for 30 minutes, then pNPP substrate added to a concentration of 200 µM and reactions continued for 30 minutes at 37°C. Dephosphorylated product was measured by its specific absorbance at 405 nm as a readout for PTPσ activity. Inhibition of PTPσ, expressed as a percent (normalized to vehicle, DMSO) is plotted for each compound. Sodium orthovanadate (Na3VO4) is a pan inhibitor of PTPs and was included as a positive control (white circles). Original scaffolds are indicated with black circles. Dashed lines denote a 90% inhibition threshold.
Figure 5.
Refined biochemical screen with oxidation constraints identifies a non-oxidative molecule.
(A) Catalase quenches hydrogen peroxide (H2O2), an oxidative species which inhibits PTP active sites through oxidation of the active site cysteine. (B) PTPσ activity towards pNPP was measured in the presence of DMSO vehicle, compound 48, or compound 49 as described previously. Bovine liver catalase (50 units per reaction) was included (+, gray bars) to degrade hydrogen peroxide, or excluded (−, black bars). Relative PTPσ activity was plotted (percent of activity in DMSO with or without catalase). (C) Sixty-six scaffolds identified in silico were evaluated using conditions optimized to minimize the effects of oxidation (H2O2). Compounds (10 µM) were pre-incubated with PTPσ for 10 min at 37°C then 225 µM substrate added for 15 minutes. PTPσ inhibition is plotted (normalized to vehicle, DMSO) and compounds are displayed by rank (increasing inhibition left to right). Most potent inhibitors, 36 and 38, are highlighted, as is the PTP inhibitor, Na3VO4, for reference. (D–E) PTPσ inhibition was measured as in (C) with compounds 38 (B) and 36 (C) with (+, gray bars) or without (−, black bars) the addition of catalase to quench hydrogen peroxide. Error bars represent standard deviation.
Figure 6.
Compound 36 is a µM inhibitor of PTPσ and binds the PTPσ active site in silico.
(A) Compound 36 was tested for inhibition of PTPσ. Compound 36 (at a final concentration from 0 to 100 µM) was pre-incubated with PTPσ for 10 min at 37°C. Reactions were then performed for 10 minutes at 37°C following the addition of 225 µM pNPP substrate. Relative PTPσ activity is plotted (percent of activity in DMSO). Dashed line indicates 50% inhibition (10 µM). Bars represent standard deviation from three experiments. (B) The structure of compound 36 is displayed. (C) Compound 36 was docked into the active site of PTPσ (PDB ID: 2FH7) using the D1 apo crystal structure. Molecular surface is colored by electrostatic potential. Red corresponds to negative potential and blue to positive potential. Docking was performed using the Schrödinger suite of software as described in Methods and figures generated using ICM (MolSoft).