Reactivity-based chemical-genetic study of protein kinases

Abstract

The human protein kinase superfamily comprises over 500 members that operate in nearly every signal transduction pathway and regulate essential cellular processes. Deciphering the functional roles of protein kinases with small-molecule inhibitors is essential to enhance our understanding of cell signaling and to facilitate the development of new therapies. However, it is rather challenging to identify selective kinase inhibitors because of the conserved nature of the ATP binding site. A number of chemical-genetic approaches have been developed during the past two decades to enable selective chemical perturbation of the activity of individual kinases. Herein, we review the development and application of chemical-genetic strategies that feature the use of covalent inhibitors targeting cysteine residues to dissect the cellular functions of protein kinases.

Fig. 1. General structure of the catalytic domain of protein kinases. (A) Ribbon representation of the kinase domain of the epidermal growth factor receptor (EGFR) in its active state with key structural elements highlighted in different colors as follows: activation loop (A-loop), yellow; αC helix, red; phosphate-binding loop (P-loop), blue; catalytic loop (C-loop), pink; hinge region, green; DGF motif, dark green. Adenylyl-imidodiphosphate (AMP-PNP) is bound to the ATP-binding pocket (PDB ID 2GS6). (B) Hydrogen bonds between the ATP-binding site of EGFR and AMP-PNP are indicated by black dashed lines.

Fig. 2. Bump-hole approach allows for selective inhibition of a single engineered protein kinase. (A) Partial sequence alignment of different kinase domains highlighting the conserved gatekeeper residue. (B) Mutation of this residue to glycine or alanine creates an extra pocket (“hole”) in the active site of the analog-sensitive kinase (AS-allele) that can be specifically targeted by a bulky (“bumped”) inhibitor. (C) Chemical structures of non-selective kinase inhibitor PP1 and rationally designed PP analogs.

Fig. 3. Cysteine-targeting covalent approaches to selectively inhibit protein kinases. (A) Covalent chemical-genetic approaches achieve selectivity by relying on the covalent complementarity between a native or engineered cysteine and an electrophilic inhibitor. (B) Cysteine residues at different positions can be used instead of relying on just the gatekeeper. A few previously reported positions are highlighted in and near the ATP-binding pocket in the kinase domain of EGFR (PDB ID 2GS6). (C) Covalent complementarity makes it possible to develop orthogonal pairs of engineered kinases and electrophilic inhibitors that allow the study of signaling pathways involving multiple kinases in the same cell.

Fig. 4. Covalent chemical-genetic approach to specifically target tyrosine kinases. (A) Partial sequence alignment of several protein kinases showing the chosen sites for the two-point mutations: the gatekeeper and a rare cysteine residue (Cys797 in EGFR). (B) By introducing two mutations into its active site, only the sensitized kinase (AS + ES) will bind to the rationally designed inhibitor since these features are not found together in any WT kinase. (C) Chemical structures of 6-acrylamido-4-anilinoquinazoline irreversible inhibitors used in the studies. The 4-anilinoquinazoline core is common in many EFGR inhibitors, such as erlotinib. (D) Chemical structure of covalent affinity probe to measure target engagement and study the downstream signaling of EGFR allele in cells. A nitrobenzoxadiazole (NBD) fluorophore is linked to the quinazoline scaffold at C7 via a PEG chain.

Fig. 5. Constitutively active BRAF(V600E) is inhibited by ATP-competitive drugs (A) but not BRAF(WT) (B). (C) CRAF kinase was engineered with a cysteine residue analogous to EGFR Cys797 to achieve isoform selectivity through covalent inhibition by electrophilic quinazolines. (D) Covalent chemical genetics explained the paradoxical activation of BRAF(WT). Binding of the covalent inhibitor to the electrophile-sensitive protomer* within a RAF dimer produced both abolition of the catalytic activity of the inhibitor-bound RAF and transactivation of the drug-free RAF. (E) Reversible and irreversible ATP-competitive inhibitors that were employed in the chemical-genetic study.

Fig. 6. Covalent chemical-genetic approach exploiting two selectivity filters to target RSK kinases. (A) RSK2 is amongst the few protein kinases bearing a reactive cysteine at the P-loop C-terminal end and a small threonine gatekeeper residue. (B) Structures of the rationally designed halomethylketone pyrrolopyrimidines, cmk and fmk. (C) The two filters combined afforded selective targeting of RSK2 with the bulky electrophilic inhibitor.

Fig. 7. Cysteine installation for modulating allostery and targeted inhibition of kinases (CystIMATIK) method to probe kinase conformation. (A) Compound 1 was used as a starting point to generate conformation-selective probes 2 and 3. By changing the C5 substituent on the pyrrolopyrimidine scaffold, CystIMATIK probes stabilize different ATP-binding site conformations. (B) Cysteine residue at the P-loop C-terminal end (V284 in Src) and a threonine gatekeeper residue (T341 in Src) were chosen as the two selectivity filters to generate CystIMATIK-sensitive kinases. (C) CystIMATIK was used to modulate Src's global conformation to investigate phosphotransferase-independent activity. Mutant Src* (V284C), that has a small gatekeeper residue, was selectively targeted by the inhibitors while sparing most wild-type protein kinases, allowing for the specific dissection of the engineered kinase's cellular activities.

Fig. 8. Covalent chemical-genetic approach named Ele-Cys to identify selective covalent inhibitors of protein kinases. (A) A position six residues C-terminal to the gatekeeper was chosen to install the cysteine, which is only found in a single human kinase, EphB3. (B) Chemical structures of the inhibitors utilized in the study. Electrophilic quinazoline 1 and bumped inhibitor 3-MB-PP1 serve as a comparison. (C) Both AS and Ele-Cys chemical-genetic approaches afforded orthogonal inhibition of two kinases in the same cell. (D) The two strategies were employed together to examine the communications between two distinct Eph kinases: EphB3 and EphB1. Selective inhibition of EphB3-WT and EphB1-AS upon treatment with the two inhibitors shown in (B) caused selective abolishment of autophosphorylation of only the respective Eph kinase, indicating that there is little trans-phosphorylation between them.

Fig. 9. Summary of the protein kinases that have been studied by covalent chemical-genetic methods according to the targeted cysteine positions (either native or mutated).

Renata Rezende Miranda

Chao Zhang