An efficient rapid system for profiling the cellular activities of molecular libraries

Abstract

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.

Fig. 1.

The components of the ACP system. (A) Three custom environmentally controlled (temperature, CO², and humidity) 486-plate or flask capacity incubators. (B) Combination flask to 1,536-well plate cell and reagent dispenser. (C) Control station consisting of a computer running custom scheduling software. (D) Perkin–Elmer ViewLux plate reader. (E) Olympus (Melville, NY) inverted microscope with an automated stage and Molecular Devices metamorph software. (F) Compound transfer station (typically 5- to 50-nl transfer volumes). (G) Proprietary tissue culture (TC) station. (H) BD Biosciences FACSArray Bioanalyzer with custom sampling tray. (I) Staubli RX130 anthropomorphic robot with a custom end effector (gripper).

Fig. 2.

Ba/F3 construction. (a) Retroviral construct for Ba/F3-TK library. pTK is based on the pMSCV (Clontech) backbone. The first 1 kb of ETV6, with an artificial myristoylation sequence (mTel), is located upstream of the Gateway reading frame B cassette (Invitrogen), which is followed by the Myc tag sequence EQKISEEDL and a stop codon. This is linked by the HCV IRES to puromycin in which the stop codon has been replaced by the luciferase coding sequence from pLucN2 (Clontech). The expressed protein, a functional dimer, is detailed to the left. Dimerization of the mTel domains bring the two fused kinase domains into proximity, leading to autophosphorylation and activation. (b) The tyrosine kinome. The tyrosine kinase-dependent Ba/F3 lines used in this screen are highlighted in green boxes against a phylogeny of all 90 tyrosine kinases reproduced courtesy of Cell Signaling Technology (Danvers, MA). (c) Cellular and biochemical assays. The Bcr-Abl-targeted kinase inhibitors Glivec and AMN107 were tested against the kinase cell line panel (Ba/F3) and the purified enzyme (in vitro). Data for Ba/F3 are listed as the IC₅₀ in μM, whereas the in vitro data are listed as the percent enzymatic activity remaining at 10 μM compound. The boxes are colored in green (potent inhibition), black (mild inhibition), or red (little to no inhibition).

Fig. 3.

Global data analysis. (a) Selectivity/potency correlation. The compounds were clustered based on chemical structure (Pipeline Pilot, Scitegic, San Diego) to yield 94 clusters. The test points for 14 clusters (those with 20 or more compounds) were plotted broken out by cluster to assess the specificity–potency relationships for focused structural classes. The clusters are numbered from 1 to 14, where blue dots represent compounds, and are distributed by specificity (abscissa) and potency (GI50; ordinate). (b) Relationship of structural similarity and biological similarity. Each marker represents a pair of compounds, with the pairwise chemical distance (Tanimoto coefficient) on the abscissa and the pairwise biological profile distance (Pearson correlation coefficient) on the ordinate. The line shown is the ordinary least-squares fit. The triangle encloses the outliers, which are discussed in the text. Markers are colored according to an alternative measure of biological profile similarity, the cosine (or “uncentered”) correlation coefficient.

Fig. 4.

New targets. (a) Heat map of ACP data. IC₅₀ values from the triplicate screens were averaged and clustered by using The Institute of Genome Research Multiple Array Viewer according to the average linking method of the Euclidian distance hierarchical clustering metric to generate the heat map shown in the lower left. The scale ranges from 0 (green) to 10 μM (red), as shown in the right-hand color triangle. The dendrogram associated with the heat map, showing kinase linkage, is enlarged in b, and selected inhibitors referred to in the text are expanded underneath. (b) Activities. Selected compounds from the ACP profiling experiment suggest alternate targets for several compounds. Here, the pan-p38 kinase inhibitor BIRB796 also inhibits Tie1 and Tie2. The dual src-abl inhibitor BMS-354825 also inhibits the Ephrins. Glivec (STI571) inhibits BCR-ABL, c-kit, and PDGF-RA/B.