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PTP1B Inhibitor Screening Service
Summary
Before determining the IC50’s for ten potential PTP1b inhibitors, we performed some initial experiments to establish appropriate assay conditions. We assessed conditions for a full length human PTP1b and a truncated human PTP1b (Glu2-Asn321). First we titrated the PTP1b’s using 5 mM pNPP as the substrate. From these titrations we decided to use 50 ng/reaction for the full length PTP1b and 40 ng/rxn for the truncated PTP1b. We next titrated DMSO to determine the highest concentration that would be tolerated by each PTP1b. The Km of pNPP for each PTP1b was then measured in the presence of the highest tolerable DMSO concentration. Finally, the IC50 for a known tyrosine phosphatase inhibitor, Na3VO4 was measured for each PTP1b. For the full length PTP1b, we determined the maximum DMSO concentration to be 15 v% and the Km to be 0.7 ± 0.04 mM. For the truncated PTP1b, we determined the maximum DMSO concentration to be 7.5 v% and the Km to be 1.3 ± 0.1 mM. Using 0.7 mM pNPP, we observed an IC50 = 19.3 ± 1.1 µM for Na3VO4 for the full length PTP1b. Using 1 mM pNPP, we observed an IC50 = 54.5 ± 1.1 µM for Na3VO4 for the truncated PTP1b.
Results
PTP1b Titrations
In order to determine an appropriate amount of PTP1b to use for the IC50 determinations, we titrated each PTP1b. The full length PTP1b was titrated from 75 ng/rxn and the truncated PTP1b was titrated from 200 ng/rxn. Both titrations were performed using 5 mM pNPP as the substrate. The full length PTP1b titration was performed in a 384-well plate to maximize the absorbance signal. (Due to high variation between replicates, we decided to switch to 96-well plates for the IC50 analysis and all of the truncated PTP1b analyses.)
From these results we opted to use 50 ng/rxn for the full length PTP1b and 40 ng/rxn for the truncated PTP1b. At these concentrations the reaction would remain linear for at least 1 hr and the ∆OD should be significant (>0.2) even at the pNPP Km.
DMSO Titration
In order to determine tolerance for DMSO of each of the PTP1b’s, we ran DMSO titrations for each PTP1b (from 0–27 v% for the full length PTP1b and from 0-15 v% for the truncated PTP1b).
For these titrations we used 50 ng per reaction for the full length PTP1b and 40 ng truncated PTP1b. The reactions were initiated by adding 5 mM pNPP and were allowed to proceed for 60 min. We then computed the ∆OD (OD60min – OD0min), which is proportional to the rate of reaction, for each DMSO concentration. The ∆OD’s were then plotted versus DMSO concentration. From these results we discovered that DMSO actually enhanced the activity of each PTP1b up to a certain concentration. For the full length PTP1b, we decided to move forward with 15 v% DMSO and for the truncated PTP1b, we decided on 7.5 v% DMSO.
Km of pNPP
In order to determine the Km of pNPP for the PTP1b’s, we titrated pNPP from 0–20 mM with either 50 ng full length PTP1b or 40 ng truncated PTP1b and computed ∆OD (OD60min – OD0min), which is proportional to the rate of reaction, for each pNPP concentration. The ∆OD’s were then plotted versus pNPP and the Km was computed using Prism 4 (GraphPad Software Inc.). The full length PTP1b Km determination was performed in the presence of 15 v% DMSO and truncated PTP1b Km determination was performed in the presence of 7.5 v% DMSO. We found the Km = 0.7 ± 0.04 mM for the full length PTP1b and the Km = 1.3 ± 0.1 mM for the truncated PTP1b.
Sodium Vanadate Na3VO4 IC50 Determination
The IC50 determination required two steps: 1) 30 min pre-incubation of 25 µL PTP1b (50 ng full length PTP1b or 40 ng truncated PTP1b) with 15 µL Na3VO4 (in 100% DMSO for full length PTP1b or 50 v% DMSO for truncated PTP1b), and 2) phosphatase reaction with 60 µL pNPP (0.7 mM final [pNPP] for full length PTP1b and 1 mM final [pNPP] for truncated PTP1b) for 60 min. We titrated Na3VO4 from 0-1000 µM and computed the ∆OD for each Na3VO4 concentration. The ∆OD’s were then plotted versus the Log [Na3VO4] and the IC50 was computed using Prism 4 (GraphPad Software Inc.). We observed an IC50 = 19.3 ± 1.1 µM for the full length PTP1b and an IC50 = 54.5 ± 1.1 µM for the truncated PTP1b.
The IC50 determination required two steps: 1) 30 min pre-incubation of 25 µL PTP1b (50 ng full length PTP1b or 40 ng truncated PTP1b) with 15 µL Na3VO4 (in 100% DMSO for full length PTP1b or 50 v% DMSO for truncated PTP1b), and 2) phosphatase reaction with 60 µL pNPP (0.7 mM final [pNPP] for full length PTP1b and 1 mM final [pNPP] for truncated PTP1b) for 60 min. We titrated Na3VO4 from 0-1000 µM and computed the ∆OD for each Na3VO4 concentration. The ∆OD’s were then plotted versus the Log [Na3VO4] and the IC50 was computed using Prism 4 (GraphPad Software Inc.). We observed an IC50 = 19.3 ± 1.1 µM for the full length PTP1b and an IC50 = 54.5 ± 1.1 µM for the truncated PTP1b.
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