The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification

Abstract

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Keywords: KCGS; chemogenomic set; drug discovery; druggable genome; kinase inhibitor; phenotypic screening; protein kinase; small molecules; understudied kinase.

Figure 1

Analysis of kinase coverage in KCGS by publication frequency. The blue histogram indicates the number of publications for each kinase in PubMed (Table S2) [24,25] ordered by frequency with the top 25% noted by the horizontal bar. The red vertical bars indicate the 162 poorly studied dark kinases nominated by the NIH Illuminating the Druggable Genome (IDG) initiative [7]. The black vertical bars indicate the 215 kinases covered by an inhibitor in KCGS version 1.

Figure 2

KCGS contains inhibitors from 67 distinct chemotypes. (A) This graph displays the number of inhibitors in each SMILES arbitrary target specification (SMARTS) bin. The numbers above the left-most 9 bars represent the number of FDA approved kinase inhibitors in these chemotype bins at the time the manuscript was written. The inset provides names of the 9 most highly populated bins. (B) Tree plot of the human kinases with each subfamily uniquely shaded. Kinases covered by a member of the oxindole SMARTS bin are displayed as red dots, scaled by the number of compounds inhibiting a specific kinase. Representative chemical structures form the oxindole SMARTS bin are shown. (C) Kinases covered by the 4-anilino-quinazoline SMARTS bin with representative chemical structures. (# = number).

Figure 3

Calculated properties. (A) Predicted solubility of the KCGS compounds (black) compared to 52 FDA-approved kinase inhibitors (white) split into four categories and shown as percentage of the set. (B) Hydrophobicity analysis of the KCGS compounds (black) using SwissADME compared to the FDA-approved kinase inhibitors (white) split into six ranges of cLogP and shown as percentage of the set.

Figure 4

Cell toxicity assessment. (A) Effects of KCGS compounds at 10 µM on HeLa cells after 24 h. Measurements were made in triplicate with standard errors shown. Shown is the normalized healthy cell count with highlighted thresholds of 0.8 (80% healthy cells) and 0.5 (50% healthy cells). The panel on the right side displays the compounds with the greatest effect on healthy cell count in HeLa cells. (B) Averaged toxicity measured by normalized healthy cell count for every target covered by two or more chemotypes. Highlighted are target kinases that show a significantly lower healthy cell count than the DMSO control.

Figure 5

The kinase inhibitors in KCGS have different effects on cell growth across a wide range of lines. Normalized growth rate (GR) inhibition values were determined at 72 h post compound treatment. Each row displays data from a single cell line. Each column displays data from a single compound. GR is colored from −0.5 in black to 1.3 in yellow. The expanded views display data for compounds that showed cell-line selective decrease in GR and relatively cell-line independent effects, respectively.

Figure 6

Autophagic flux assay. Compounds (1 µM) were analyzed for their effect on autophagy flux in RPE1 cells stably expressing the general autophagy flux reporter GFP-LC3B-RFP-LC3∆C. Phase contrast as well as fluorescent images (GFP, RFP) were taken at indicated time points. (A) Ratio of the GFP/RFP signal correlating with high (Torin1; defined as 100%) and low (DMSO; defined as 0%) autophagy flux. Compound-induced changes in autophagic flux are represented as percentage of this difference. Small black arrows indicate assay workflow (B) Hits are defined as compounds showing > 20% aberration of the GFP/RFP ratio compared in five or more consecutive time points and categorized according to their cell proliferation rate as well as visual appearance. (# = number) (C) Examples of compounds with an effect on autophagic flux. Cell confluence is presented as percentage of covered growth area. Visual appearance of cells and cell confluence were ascertained by examining fluorescent images of the GFP channel at 96 h screen time. Graphs contain the individual average of experiments run in triplicate at two different times.