The kinase inhibitor library L1200 and the inhibitors for activity assays and co-crystallisation were purchased from Selleckchem. The peptides tested as LIMK1 substrates were purchased from Genscript (description in Supplementary Table S2 ).
L1200 kinase inhibitor library
Manufactured by Selleck Chemicals
Sourced in United States
The L1200 kinase inhibitor library is a collection of small-molecule compounds designed to inhibit the activity of various kinases. The library provides a diverse set of potential lead compounds for drug discovery and research related to kinase-mediated signaling pathways.
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Lab products found in correlation
Trichostatin a tsa, by Selleck Chemicals (2 mentions)
Mk 2461, by Selleck Chemicals (2 mentions)
Click it edu cell proliferation kit for imaging with alexa fluor 647 dye, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Akta prime system, by GE Healthcare (1 mentions)
Acquity ultra performance liquid chromatography, by Waters Corporation (1 mentions)
Auto itc200, by Malvern Panalytical (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Nanodrop nd 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
9 protocols using l1200 kinase inhibitor library
1
Kinase Inhibitor Library Evaluation
2
Recombinant Protein Purification Protocol
3
Screening Kinase Inhibitor Library for Anti-SARS-CoV-2 Activity
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The Kinase inhibitor library L-1200 (Selleckchem) containing 1796 compounds was tested in a proof-of-concept screen in Delta-infected Caco-2-F03 cells for the identification of antivirally active agents. Caco-2-F03 cells were seeded into 96-well plates (50,000 cells/well) and incubated at 37°C for 4 days. After the cells reached confluence, the supernatant was replaced by 25 μL/well of medium containing the ABCB1 inhibitor zosuquidar (final concentration 1 μM), 25 μL/well of medium containing kinase inhibitors (final concentration 10 μM) in singlets, and 50 μL/well SARS-CoV-2 suspension (MOI 0.01). Remdesivir (10 μM) was used as positive control. Plates were incubated at 37°C for 48h prior to the measurement of caspase 3/7 activity as described above. For each plate the Z′ score, a measure of statistical effect size and an index for assay quality control, was calculated by: Z′ = 1 − (3∗SD.signal +3∗SD.basal)/(Meansignal − Meanbasal). Only plates with Z'score ≥0.5 were further analyzed.
4
Kinase Inhibitor Screening in Pancreatic Cancer
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Kinase inhibitor library L1200 was purchased from Selleck Chemicals (Houston, TX, USA). AT 9283, abbreviated as AT, and WZ 3146, abbreviated as WZ in this manuscript, were identified as potent inhibitors from the kinase inhibitor library. A stock solution of 10 mM concentration of each compound was made in DMSO. For flow cytometry, Luminex Muse Cell analyzer and Muse assay kits were bought from EMD Millipore (Burlington, MA, USA).
Human pancreatic adenocarcinoma cell lines PANC-1 (CRL-1469) and BxPC-3 (CRL-1687) were purchased from ATCC (Manassas, VA, USA). The PANC-1 cells were grown in DMEM medium supplemented with 10% fetal bovine serum, 1% Penicillin-Streptomycin, and 2 mM L-glutamine. Cultures were incubated at 37 °C in an atmosphere of 5% CO2. BxPC-3 cells were grown under similar conditions as PANC-1 except that RPMI medium was used instead of DMEM.
Human pancreatic adenocarcinoma cell lines PANC-1 (CRL-1469) and BxPC-3 (CRL-1687) were purchased from ATCC (Manassas, VA, USA). The PANC-1 cells were grown in DMEM medium supplemented with 10% fetal bovine serum, 1% Penicillin-Streptomycin, and 2 mM L-glutamine. Cultures were incubated at 37 °C in an atmosphere of 5% CO2. BxPC-3 cells were grown under similar conditions as PANC-1 except that RPMI medium was used instead of DMEM.
5
Protein Purification and Kinase Inhibitor Assay
6
Kinase Inhibitor Screen in Zebrafish
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In our initial small molecule screen, dechorionated olig2:egfp larvae were treated with 10 μm kinase inhibitor in 1% DMSO in PTU egg water from 24 to 76 h post-fertilization (hpf). Kinase inhibitors used were 1 of 430 kinase inhibitors from the L1200 Kinase Inhibitor Library (Selleck Chem), MK-2461 (Selleck Chem), or Trichostatin-A (TSA) (Selleck Chem). Control siblings were treated with 1% DMSO in PTU egg water. The small molecule screen was conducted in triplicate.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5 °C then fixed for 1 h in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at −20°C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 h at RT and thoroughly washed overnight with PBSTX prior to imaging.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5 °C then fixed for 1 h in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at −20°C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 h at RT and thoroughly washed overnight with PBSTX prior to imaging.
7
Biochemical Reagents and Protein Quantification
8
Biochemical and Crystallographic Reagents
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Reagents and compounds for biochemical and crystallographic experiments
were purchased from Sigma-Aldrich and Hampton Research unless otherwise
indicated. The L1200 kinase inhibitor library was purchased from Selleck
Chemicals, and the GSK Published Kinase Inhibitor Set 1 (PKIS-1) was
kindly provided by Dr. David Drewry (GlaxoSmithKline). Protein concentration
was determined by A280 molar absorbance
using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies).
were purchased from Sigma-Aldrich and Hampton Research unless otherwise
indicated. The L1200 kinase inhibitor library was purchased from Selleck
Chemicals, and the GSK Published Kinase Inhibitor Set 1 (PKIS-1) was
kindly provided by Dr. David Drewry (GlaxoSmithKline). Protein concentration
was determined by A280 molar absorbance
using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies).
9
Kinase Inhibitor Screening in Zebrafish
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In our initial small molecule screen, dechorionated olig2:egfp larvae were treated with 10 μm kinase inhibitor in 1% DMSO in PTU egg water from 24 to 76 hours post fertilization (hpf). Kinase inhibitors used were 1 of 430 kinase inhibitors from the L1200 Kinase Inhibitor Library (Selleck Chem), MK-2461 (Selleck Chem), or Trichostatin-A (TSA) (Selleck Chem). Control siblings were treated with 1% DMSO in PTU egg water.
The small molecule screen was conducted in triplicate.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5°C then fixed for 1 hr in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at -20 °C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 hr at RT and thoroughly washed overnight with PBSTX prior to imaging.
The small molecule screen was conducted in triplicate.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5°C then fixed for 1 hr in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at -20 °C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 hr at RT and thoroughly washed overnight with PBSTX prior to imaging.
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