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. 2017 Aug 2;139(30):10192-10195.
doi: 10.1021/jacs.7b04880. Epub 2017 Jul 20.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection

Alix I Chan  1 , Lynn M McGregor  1 , Tara Jain  1 , David R Liu  1
Affiliations

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection

Alix I Chan et al. J Am Chem Soc. .

Abstract

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

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Conflict of interest statement

The authors declare the following competing financial interest(s): D.R.L. is a founder of Ensemble Therapeutics, a company that uses DNA-templated synthesis for drug discovery.

Figures

Figure 1
Figure 1
Overview of IDUP. DNA-barcoded small molecules and proteins are combined in cell lysate. Primer extension, PCR and DNA sequencing reveal the identity of protein:ligand pairs.
Figure 2
Figure 2
Results of seven replicate IDUP experiments of combined protein and small molecule libraries. Eight DNA barcodes corresponding to bona fide interactions (Bak:Bcl-xL, PKI:PRKX, JQ1:BRD2, JQ1:BRD3, bisX:GSK3α, EA:MAP2K6) enriched (red) out of 32 096 possibilities. KD and IC50 values are for compounds and proteins not linked to DNA. Nonspecific amplification across some protein barcodes may arise from poor expression of those targets (see Figure S2).
Figure 3
Figure 3
Inhibition of MAP2K6 by EA is dependent on the Michael acceptor. The nonelectrophilic analogs shown are >30-fold less potent.
Figure 4
Figure 4
Kinases were incubated with 100 µM EA (+EA) or DMSO (−EA), then dialyzed to remove free EA. Activity was assayed as a function of kinase concentration. Ethacrynic acid inhibits MAP2K6 to a greater degree when Cys38 is present (left) than when this residue is mutated to an alanine (right).
Figure 5
Figure 5
EA has higher affinity for MAP2K6 than the related MAP2K family members, as measured in both LanthaScreen Eu assays and our initial IDUP assay. n.d. = not determined.
See this image and copyright information in PMC

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