Oxidation does not discriminate selectively for the PTPs active site and thus, is not an ideal mechanism of inhibition for a PTPs inhibitor. To address this, we optimized assay conditions to eliminate oxidative effects and found that compound 36 was able to inhibit PTPs by a mechanism largely independent of oxidation. This suggests that compound 36 functions as a competitive inhibitor of PTPs and in agreement with this, it docked favorably into the D1 active site of PTPs in silico. Although compound 36 proved potent and non-oxidative, we do not anticipate that it will be a selective inhibitor of PTPs in its current form, owing to the high degree of sequence conservation among phosphatase catalytic domains. In particular, the residues forming the active site predominantly lie within highly conserved motifs showing little sequence variability across the entire PTP family [47]. In fact, preliminary studies suggest compound 36 displays activity towards PTP1B, in addition to PTPs (unpublished data). This underscores the importance of our future efforts to identify and create modifications to the compound 36 scaffold which will favor selective inhibition of PTPs. We believe a combination of in silico methods and carefully optimized biochemical screening (i.e., an assay that minimizes inhibition by oxidative species) represents an useful approach to develop effective PTP inhibitors. Through the in silico approach described here, we were able to identify active phosphatase scaffolds while bypassing a primary assay that would entail a highthroughput biochemical screening of compounds in vitro. Coupling this effort with biochemical assays, we prioritized compound 36 as a lead molecule. Our future studies will include structure-based refinement of this scaffold in order to develop selective inhibitors of PTPs. In this approach, we will characterize the activity of compound 36 against related PTPs and following, use molecular docking and structural analyses of these counter-targets to identify chemical modifications that promote selectivity for PTPs.
Methods Structural Modeling and Phosphotyrosine Substrate Docking
The crystal structure of PTPs (PDB ID: 2FH7) was retrieved from the Protein Data Bank. The initial conformations of p-Tyr peptide (NPTpYS) were extracted from the CD45-p-Tyr peptide complex structure (PDB ID: 1YGU). The ICM program was used for protein and substrate preparation (MolSoft, La Jolla, CA). Phosphotyrosine peptide was docked into the active site of PTPs with default parameters implemented in the ICM program.
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) PTPs 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 (2, black bars). Relative PTPs 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 mM) were pre-incubated with PTPs for 10 min at 37uC then 225 mM substrate added for 15 minutes. PTPs 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) PTPs inhibition was measured as in (C) with compounds 38 (B) and 36 (C) with (+, gray bars) or without (2, black bars) the addition of catalase to quench hydrogen peroxide. Error bars represent standard deviation. Virtual Screening (VS)
We used the ZINC library (version 8; University of California San Francisco) of ChemBridge compounds for virtual screening with the D1 active site of PTPs (PDB ID: 2FH7). GOLD (Version 3.2) program was used for virtual screening and ChemScore scoring function was used to rank the top 200 hits with favorable binding energies (Cambridge Crystallographic Data Centre, Cambridge). Hydrogen bond restraints were used during molecular docking process. Molecules which formed at least one potential hydrogen bond with any of the residues, S1590,
A1591, V1593, G1594 or R1595, were given higher weight during the score calculation. We used ICM clustering analysis (MolSoft) to identify 66 representative compounds from unique clustering groups. A substructure and similarity search based on compounds 6, 48, and 49 was performed using the Canvas module in Schrodinger (Schrodinger, LLC, New York, NY, 2011) and the ?ChemBridge compound online search engine (ChemBridge, recombinant PTPs containing all residues C-terminal to the transmembrane domain (BC104812 cDNA; aa 883-1501) was generated in pGEXKG [48]. GST-tagged recombinant full-length PTP1B (BC018164) was generated with a 6xHIS tag in pGEXKG. Proteins were purified from BL21 Escherichia coli after isopropyl b-D-1-thiogalactopyranoside (IPTG) induction and purity was confirmed by SDS-PAGE and coomassie blue staining. Compounds were pre-incubated with recombinant enzymes in freshly prepared phosphatase buffer (50 mM sodium acetate, 25 mM Tris-HCl, 3 mM DTT, pH 6.5) for 10 to 30 minutes, as indicated in figure legends. Following, para-nitrophenyl phosphate (pNPP; Sigma S0942), initially diluted in assay buffer, was added to reactions for a final volume of 100 ml and reactions were carried out in a 37uC water bath for 15 to 30 minutes. Reactions were quenched with 100 ml 1N sodium hydroxide (NaOH) and 180 ml was transferred to flat-bottom clear 96-well plates. Absorbance of pNP product at 405 nm was measured on a spectrophotometer and plotted. Background absorbance values of compound-only wells were subtracted from the corresponding reactions. DMSO was included as a vehicle control. The IC50 value of compound 36 was calculated using the data from Figure 6A and BioDataFit (Chang Biosciences, Castro Valley, CA).
Docking of Compound 36 into PTPs
Compound 36 was docked to the open conformation of PTPs (PDB ID: 2FH7) which was retrieved from the Protein Data Bank. Docking was performed using Schrodinger’s graphical user ?interface Maestro (Maestro, version 9.2, Schrodinger, LLC, New ?York, NY, 2011). The protein was first processed using protein preparation wizard, which assigned bond orders, and added hydrogens and missing atoms, followed by minimization. Compound 36 was prepared in LigPrep (LigPrep, version 2.5, Schrodinger, LLC, New York, NY, 2011) module of Schrodinger ??in the OPLS-2005 force field [49] generating possible ionization states and stereoisomers for the ligand. Docking of the ligand was performed in Glide module (Glide, version 5.7, Schrodinger, LLC, ?New York, NY, 2011). A receptor grid was generated, defining the binding site of PTPs, and the prepared ligand was docked using extra precision scoring function while keeping the ligand flexible. Several poses were generated which were then minimized to optimize them further using MacroModel within the OPLS2005 force field (MacroModel, version 9.9, Schrodinger, LLC, New ?York, NY, 2011). For Figure 6C, complexes were exported into ICM (MolSoft) and surface representations generated.
