Controlling the Covalent Reactivity of a Kinase Inhibitor with Light

Abstract

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and become effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Keywords: JNK3; covalent inhibitors; kinase inhibitors; photopharmacology; photoswitchable affinity labels; photoswitches.

Figure 1.

A) Representative FDA-approved covalent kinase inhibitors binding irreversibly to specific cysteine residues. B) Photoactivation through caging. C) Photoactivations through photoswitching.

Figure 2.

Design and synthesis of the covalent photoswitchable inhibitors. A) Azologization of the known covalent inhibitor 1 and sign inversion to yield diazocine 3. B) Additional photoswitchable JNK3 inhibitors prepared. C) Synthesis of diazocine 3. D) Absorption spectra of inhibitors 2 and 3 in the dark or after 400 nm irradiation for 3 minutes in DMSO and thermal characterization of 2 and 3 in a 1:1 mixture of DMSO and PBS (pH 7.4).

Figure 3.

In vitro characterization. Quantitative ELISA assay of differential ATF-2 phosphorylation by JNK3 for 50 min at 37 °C in the dark or with pulsed irradiation (100 ms every 5 s) and inhibitors: A) 1 B) 2 C) 3 D) 4 E) 5 F) 6.

Figure 4.

X-ray analysis of 4 bound to JNK3. A) Structural overlay of 1 and 4 covalently bound to JNK3. B) The bent cis isomer cannot reach Cys154 and only binds non-covalently. C) The extended isomer binds covalently to Cys154. The crystal structures have been deposited to the wwPDB with accession codes 7ORE and 7ORF.