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

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.

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

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. K_D 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

Inhibition of MAP2K6 by EA is dependent on the Michael acceptor. The nonelectrophilic analogs shown are >30-fold less potent.

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

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.