Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis

Abstract

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

Figure 1

Reversible covalent binding of activated Michael acceptors to cysteine thiols. (a) RSK2 inhibitors, 1 and 2. A cocrystal structure (PDB code: 4D9U) reveals hydrophobic interactions between the tert-butyl group of 2 and the glycine-rich loop of RSK2. (b) Reversible covalent binding of an activated acrylonitrile to a hypothetical cysteine-containing protein. In this example, the electron-withdrawing group (EWG) and the β-substituent (R) both contribute to the free energy of binding by providing favorable noncovalent interactions with the protein.

Figure 2

Brønsted-type plot of computed proton affinity (ΔΔG_aq for syn-diastereomers) vs β-elimination rate (log k, min^–1) for BME/acrylonitrile adducts 5a–12a (Table 1). Adducts 3a and 4a are not shown, as only the upper limit of their t_1/2 values (<1 min) could be determined. BME/acrylonitrile adducts with higher proton affinity (more negative ΔΔG_aq) undergo β-elimination at slower rates. Plotting ΔΔG_aq for the anti-diastereomers affords a similar correlation (Supplementary Figure S11).

Figure 3

Targeting RSK2 kinase with aryl/heteroaryl-activated acrylonitriles. (a) Half-maximal inhibitory concentration (IC₅₀, mean ± s. d.) of acrylonitriles 13–18 in kinase assays with wild-type RSK2 (WT) and the Cys436Val mutant (C436V). (b) Binding of acrylonitriles 13–18 (0.1 and 1.0 μM) and FMK (1.0 μM, positive control) to endogenous RSK1 and RSK2 in MDA-MB-231 breast cancer cells, assayed by competitive labeling with the irreversible fluorescent probe, BODIPY-FMK. Cells were incubated with acrylonitriles 13–18 for 2 h prior to treatment with BODIPY-FMK for 1 h. Cells were lysed and proteins were analyzed by gel electrophoresis, followed by fluorescence scanning and immunoblotting for RSK1/2. (c) Dose response for RSK1/2 occupancy in MDA-MB-231 cells by 13 and 14 (RSK1/2 immunoblot: green and red respectively).

Figure 4

Potent, selective, and durable inhibition of RSK2 by a 1,2,4-triazole-activated acrylonitrile, 20. (a) Dose–response curves for 20 in kinase assays with RSK2, NEK2, and PLK1 (IC₅₀ values for compound 19 reproduced from ref (22)). (b) RSK1/2 occupancy was determined 2 and 24 h after treating cells with 19 and 20 (1 μM) by competitive labeling with BODIPY-FMK (RSK1/2 immunoblot: red and green, respectively). (c) Overlay of 20/RSK2 cocrystal structure (this work) with NEK2 (red, PDB code: 2WQO), highlighting Leu546 of RSK2 and the structurally homologous residue Phe148 of NEK2. The side chain of NEK2 Phe148 would clash with the 1,2,4-triazole.