Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a Testing Funnel for Identification of Small Molecule Modulators Targeting Secretin Receptors

doi: 10.1177/2472555220945284

Figure Lengend Snippet: (A) Pilot Screen of BioAscent library using LANCE Ultra cAMP kit and EC20 of Sec-FL as stimulant: primary screen and hit confirmation yielded 106 hits; 1,025 liquid stocks comprising of hits and corresponding analogs were purchased and subsequently validated in primary assay; counter screen with parental cells eliminated 11 compounds; 1,025 hits & analogs were screened with individual secretin peptides yielding 34 hits and 5 prevalent scaffolds. (B) Breakdown of 34 hits into 6 groups: confirmed hit by Sec-FL (yellow, 17), Sec(3–27) (light blue, 9), Sec(1–23) (orange, 4), all three peptides (dark blue, 2), Sec-FL & Sec(3–27) (red, 1) or Sec-FL & Sec(1–23) (green, 1). Scatterplot showing % activity with Sec(3–27) (x-axis) in correlation to % activity with Sec(1–23) (y-axis), squares indicating hit confirmation in first round of screen with Sec-FL; pie chart depicting fraction of hits and detected scaffolds per peptide/peptide mixture; scatterplot and pie chart created using TIBCO Spotfire. (C) Scaffold A, B and C contain closely related chemical structures. (D) Comparison of cAMP standard curves using LANCE Ultra (beige) or Cisbio GsD (blue) detection kit confirmed lower sensitivity but broader dynamic range for Cisbio GsD cAMP detection; assay dynamic range determined by IC10 – IC90 of cAMP standard curve: LANCE Ultra 0.18–3.52 nM cAMP; Cisbio GsD 0.22–38.2 nM cAMP. (E) cAMP formation of secretin analogs applying LANCE Ultra (dotted lines) compared to Cisbio GsD (solid lines) technology: Sec-FL (red), Sec(1–23) (orange) and especially Sec(3–27) (green) demonstrate greater responsiveness but lower reproducibility with LANCE Ultra (LANCE Ultra representative of three experiments, Cisbio GsD combined results of three experiments). Graphs plotted using GraphPad Prism. Data points shown as mean ± SEM.

Article Snippet: We decided that the small dynamic range of the LANCE Ultra kit is a major drawback for performing larger screens in PAM mode, since it is crucial to assure consistent baseline stimulation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4: caption a7 caption a8 Pilot Screen of BioAscent library employing LANCE Ultra cAMP assay revealed probe dependency of hits towards secretin or its truncated forms: (A) Pilot Screen of BioAscent library using LANCE Ultra cAMP kit and EC 20 of Sec-FL as stimulant: primary screen and hit confirmation yielded 106 hits; 1,025 liquid stocks comprising of hits and corresponding analogs were purchased and subsequently validated in primary assay; counter screen with parental cells eliminated 11 compounds; 1,025 hits & analogs were screened with individual secretin peptides yielding 34 hits and 5 prevalent scaffolds. (B) Breakdown of 34 hits into 6 groups: confirmed hit by Sec-FL (yellow, 17), Sec(3–27) (light blue, 9), Sec(1–23) (orange, 4), all three peptides (dark blue, 2), Sec-FL & Sec(3–27) (red, 1) or Sec-FL & Sec(1–23) (green, 1).

Techniques: Activity Assay, Comparison, Detection Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a Testing Funnel for Identification of Small Molecule Modulators Targeting Secretin Receptors

doi: 10.1177/2472555220945284

Figure Lengend Snippet: (A) Pilot Screens using LANCE Ultra cAMP (stimulant EC20 Sec-FL), Cisbio GsD Sec-FL (stimulant EC20 Sec-FL), Cisbio GsD 3-pep mix (stimulant EC10 Sec-FL, Sec(1–23) and Sec(3–27)) or CRELuc2P reporter (stimulant EC10 Sec-FL) technologies. Hit confirmation criteria of NZ-Score ≥ (≤ -) 3 was applied to all four formats. (B) Pie chart breaking down total confirmed hits (466): Confirmed via LANCE Ultra (light yellow, 76), Cisbio GsD Sec-FL (light blue, 10), Cisbio GsD 3-pep mix (dark blue, 210), CRELuc (light orange, 100) or via multiple assays (green, 70), whereby 68 of 70 compounds were confirmed by Cisbio GsD 3-pep mix. Pie chart created using Micrsoft Excel.

Article Snippet: We decided that the small dynamic range of the LANCE Ultra kit is a major drawback for performing larger screens in PAM mode, since it is crucial to assure consistent baseline stimulation. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4: caption a7 caption a8 Pilot Screen of BioAscent library employing LANCE Ultra cAMP assay revealed probe dependency of hits towards secretin or its truncated forms: (A) Pilot Screen of BioAscent library using LANCE Ultra cAMP kit and EC 20 of Sec-FL as stimulant: primary screen and hit confirmation yielded 106 hits; 1,025 liquid stocks comprising of hits and corresponding analogs were purchased and subsequently validated in primary assay; counter screen with parental cells eliminated 11 compounds; 1,025 hits & analogs were screened with individual secretin peptides yielding 34 hits and 5 prevalent scaffolds. (B) Breakdown of 34 hits into 6 groups: confirmed hit by Sec-FL (yellow, 17), Sec(3–27) (light blue, 9), Sec(1–23) (orange, 4), all three peptides (dark blue, 2), Sec-FL & Sec(3–27) (red, 1) or Sec-FL & Sec(1–23) (green, 1).

Techniques: