Journal: SLAS discovery : advancing life sciences R & D

Article Title: Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

doi: 10.1177/2472555218759267

Figure Lengend Snippet: Figure 1. Descriptive representation of workflows established to uncover PTP1B inhibitors. (A) Fundamental enzymatic reaction of PTP1B for the discovery of potential drug candidates. (B) Conventional AlphaScreen assay, which comprises the antibody binding competition between artificial biotinylated and physiological substrate peptides. A chemiluminescence signal is generated by singlet oxygen molecules diffusing from donor to acceptor beads solely when they are in close proximity to each other, induced by complex formation with the biotinylated substrate peptide. (C) For the MALDI-TOF screening assay, samples generated in the enzymatic reaction are supplemented with an isotope-labeled internal standard peptide containing 13C6,15N4-arginine at position 6, for accurate product quantification. Samples are transferred onto target plates in either 384- or 1536-well format and subsequently analyzed by the rapifleX MALDI-TOF instrument.

Article Snippet: The rapifleX MALDI PharmaPulse, recently launched by Bruker Daltonics (Bremen, Germany), offers HTS suitability by its ultrafast laser repetition rate (10 kHz) and direct compatibility with common automatization equipment.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Generated, Screening Assay, Labeling

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

doi: 10.1177/2472555218759267

Figure Lengend Snippet: Figure 2. MALDI-TOF analysis and buffer adjustment. (A) Zoomed region (1010–1130 m/z) of MALDI-TOF MS spectra for product (M1) and substrate (M3) of the PTP1B enzymatic reaction supplemented with the isotope-labeled standard peptide (M2) of the product peptide. (B) Representative mass spectra for the Tween 20–dependent interference of MALDI-TOF analysis. (C) Concentration- dependent signal suppression of the product and internal standard induced by the detergent Tween 20.

Article Snippet: The rapifleX MALDI PharmaPulse, recently launched by Bruker Daltonics (Bremen, Germany), offers HTS suitability by its ultrafast laser repetition rate (10 kHz) and direct compatibility with common automatization equipment.

Techniques: Labeling, Concentration Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

doi: 10.1177/2472555218759267

Figure Lengend Snippet: Figure 3. Verification of MALDI- TOF suitability for compound profiling of the PTP1B assay. (A) Linearity of the MALDI-TOF MS signal to verify dependency between the signal intensity and enzyme activity. Peak area variations of the substrate and product signals were balanced with the internal standard and plotted as a function of their concentration (mean ± 95% CI, n = 4). (B) Dose–response curves for PTP1B inhibition determined by MALDI-TOF for four reference compounds that were tested previously using AlphaScreen technology (n = 6). The ratio of the signal area of the product to the internal standard peptide is plotted against the concentration of the inhibitory compound, enabling the calculation of IC50 values. (C) Representative mass spectra (1010–1130 m/z) for the dose–response analysis applying ascending orthovandate concentrations to inhibit PTP1B phosphatase activity. The intensity decrease of the product peptide compared with the consistent internal standard offers a direct measure for enzyme inhibition.

Article Snippet: The rapifleX MALDI PharmaPulse, recently launched by Bruker Daltonics (Bremen, Germany), offers HTS suitability by its ultrafast laser repetition rate (10 kHz) and direct compatibility with common automatization equipment.

Techniques: Activity Assay, Concentration Assay, Inhibition, Amplified Luminescent Proximity Homogenous Assay, Enzyme Inhibition Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

doi: 10.1177/2472555218759267

Figure Lengend Snippet: Figure 4. Scatterplots to assess the consistency of MALDI- TOF-generated IC50 values. (A) Correlation between compound profiles for PTP1B inhibitors using the two respective technologies: AlphaScreen and MALDI-TOF. One hundred three compounds were tested. However, compounds triggering no enzyme inhibition at the highest concentration level (>100 µM) were excluded in this analysis. (B) Correlation of IC50 values generated in the MALDI- TOF PTP1B assay with and without on-target washing procedure. (C,D) Interday comparison of IC50 values generated by MALDI-TOF and AlphaScreen, respectively.

Article Snippet: The rapifleX MALDI PharmaPulse, recently launched by Bruker Daltonics (Bremen, Germany), offers HTS suitability by its ultrafast laser repetition rate (10 kHz) and direct compatibility with common automatization equipment.

Techniques: Generated, Amplified Luminescent Proximity Homogenous Assay, Enzyme Inhibition Assay, Concentration Assay, Comparison

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

doi: 10.1177/2472555218759267

Figure Lengend Snippet: Figure 5. Characterization of compound-dependent PTP1B modification. (A) To screen for compounds inducing irreversible covalent modifications at the catalytic cysteine (Cys215) of PTP1B, we established a secondary screening assay composed of tryptic digestion of the enzyme, addition of an isotope-labeled internal standard peptide, and subsequent MALDI-TOF analysis. This generates precious information to exclude compounds from drug candidate lists. (B) Representative mass spectra (2170–2230 m/z) illustrating the compound-dependent intensity loss of the peptide surrounding the catalytic cysteine (CatCysTide) that can be attributed to its covalent modification. We applied three controls for this instance: (1) low control, which lacks PTP1B; (2) high control, lacking compounds; and (3) H2O2 as positive control for the oxidation process. Three representative compounds were added for the potential effects: no. 2, a nonmodifying compound; no.17, a modifying but nonoxidizing compound; and no. 62, an oxidizing compound. (C) Scatterplot illustrating the CatCysTide quantification after compound incubation (n = 113) in two replicates. To assess potential CatCysTide modification, low (no PTP1B), high (no compound), and H2O2 controls are highlighted in the graph. The light gray square contains modifying compounds that have significantly reduced CatCysTide abundance (Ratio [CatCysTide/Int. Std.] Rep. 1 < 0.4591/ Rep. 2 < 0.4667—more than halved the high control mean ratio). (D) Scatterplot illustrating the quantification of oxidized CatCysTide (sulfonic acid generation at Cys215) after compound incubation (n = 113). The positive control is the H2O2-treated sample serving as the threshold to assign compounds to be oxidized in this context (Ratio [Ox. CatCysTide/Int. Std.] Rep. 1 > 0.0911/Rep. 2 > 0.0534).

Article Snippet: The rapifleX MALDI PharmaPulse, recently launched by Bruker Daltonics (Bremen, Germany), offers HTS suitability by its ultrafast laser repetition rate (10 kHz) and direct compatibility with common automatization equipment.

Techniques: Modification, Screening Assay, Labeling, Control, Positive Control, Incubation