Bivalent inhibitors of protein kinases

Abstract

Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described.

Figure 1

Bivalent inhibition of a protein kinase. (a) A single inhibitor is generated by fusing an ATP-competitive ligand to a secondary binding ligand. The ATP-competitive ligand interacts with the kinase active site, directing the bivalent inhibitor to the kinase. The secondary binding ligand introduces selectivity for a particular kinase. In the case of bisubstrate inhibitors, this ligand targets the protein substrate site of the protein kinase. (b) Regulatory domains of protein kinases present an avenue for bivalent inhibition where secondary-binding ligands can target domains distal to the kinase domain.

Figure 2

Reported bisubstrate/bivalent inhibitors of protein kinases. (a) Crystallographic representation of secondary binding ligands used in previously described bisubstrate/bivalent inhibitors superimposed on a protein kinase. [PDB codes 1GAG, 2CPK, 1UKI, 1Y57] (b) Crystal structure of cIRK in complex with a bisubstrate inhibitor. [PDB code 1GAG]

Figure 3

A SNAP-tag-based bivalent inhibitor approach. (a) Schematic depiction of a representative SNAP-tag-based bivalent inhibitor. SNAP-tag self-labels an active site cysteine with a quinazoline-based kinase inhibitor conjugated to a benzylguanine (BG) group. The genetically encoded SNAP-tag fusion contains a polyproline peptide ligand, which recognizes the SH3 domain of Src. The two ligands of the bivalent inhibitor interact with distinct Src tyrosine kinase binding sites. [PDB codes 3L00 and1Y57] (b) The sequences of peptide ligands that have been successfully used in SNAP-tag-based bivalent/bisubstrate inhibitors, and the kinases they target, are shown. The binding sites that have been targeted with SNAP-tag-based bivalent inhibitors are shown. These sites are superimposed on a crystal structure of Src tyrosine kinase. [PDB code 1Y57]

Figure 4

Crystallographic representation of the engineered protein SNAP-tag. The N- and C-termini, as well as the active site cysteine, are shown. Lengths (in Å) represent the distances between the N-terminus and the active site or the C-terminus and the active site. [PDB code 3L00]

Figure 5

Schematic representation of the strategy for intra-cellular assembly and use of SNAP-tag-based bivalent inhibitors. Mammalian cells express a genetically encoded SNAP-tag fusion protein. A CLP-derivatized ATP-competitive kinase inhibitor (CLP-KI) passively diffuses into cells and labels the monovalent SNAP-tag fusion. Excess monovalent CLP-KI is removed through several wash steps. Upon stimulation of the signaling pathway, kinase activity can be determined by monitoring downstream effects. As a control, a CLP-derivatized small molecule lacking inhibitory activity is used to label (block) the SNAP-tag active site before addition of CLP-KI. Any effects observed are due to CLP-KI and not the SNAP-tag-based bivalent inhibitor.