Structural bioinformatics-based design of selective, irreversible kinase inhibitors

Abstract

The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.

Fig. 1

Structural bioinformatics guides the design of electrophilic inhibitors of RSK family protein kinases. (A) Sequence alignment of the 11 human kinases with a cysteine selectivity filter at the C-terminal end of the glycine-rich loop. Of these 11, RSK1, RSK2, and RSK4 are the only kinases with a threonine selectivity filter in the gatekeeper position. Src, which has a threonine gatekeeper but lacks the cysteine, is shown for comparison. (B) Chemical structures of adenine and the rationally designed halomethylketone pyrrolopyrimidines, cmk and fmk.

Fig. 2

Selective, irreversible targeting of RSK family kinases by fmk. (A) Covalent labeling of WT, but not mutant RSK2 by biotin-fmk. RSK2 with a hexahistidine tag (His₆-RSK2) CTDs were treated with 1 μM biotin-fmk in the presence of His₆-ERK2 for 1 hour. Proteins were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blot with streptavidin-HRP or antibodies to His₆. (B) Targeting of two ∼90-kD proteins in human epithelial cell lysates by biotin-fmk. HEK-293 cell lysates were treated with the indicated concentrations of unlabeled fmk and then with 1 μM biotin-fmk. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and stained with Ponceau S (right). Proteins labeled with biotin-fmk were detected with streptavidin-HRP (left). (C) Identification of RSK1 and RSK2 as biotin-fmk targets. HEK-293 lysates were treated with 1 μM biotin-fmk. Proteins were immunoprecipitated with control IgG, RSK1-specific polyclonal antibodies, or a RSK2-specific monoclonal antibody (Santa Cruz Biotechnology). Immunoprecipitates (P) and supernatants (S) were analyzed by Western blot with streptavidin-HRP and antibodies to RSK1 and RSK2.

Fig. 3

Effect of fmk on EGF-activated RSK2 or MSK1. (A) Inhibition of EGF-stimulated RSK2 autophosphorylation by fmk. COS-7 cells were deprived of serum for 20 hours then treated with the indicated concentrations of fmk. Cells were stimulated for 10 min with EGF (1 ng/mL). RSK2 was immunoprecipitated and analyzed by Western blot with antibodies to phospho-Ser³⁸⁶ RSK (Cell Signaling Technology) and total RSK2. (B) Failure of fmk to inhibit EGF-stimulated activation of ERK1 and ERK2. Serum-starved COS-7 cells were treated with or without 10 μM fmk, then stimulated with EGF as in (A). Doubly phosphorylated and total ERK1 and ERK2 were detected by Western blot (both antibodies from Cell Signaling Technology). (C) Inhibition of EGF-stimulated H3 phosphorylation in cells expressing WT RSK2, but not T493M RSK2. COS-7 cells were transfected with hemagglutinin (HA)-tagged WT or T493M RSK2. Twenty-four hours after transfection, cells were serum-starved for 3 hours then treated with the indicated concentrations of fmk. Cells were stimulated with EGF (150 ng/mL) for 25 min and subsequently lysed in Laemmli sample buffer. Proteins were resolved by SDS-PAGE and detected by Western blot with antibodies specific for phospho-Ser³⁸⁶ RSK, the HA epitope (Roche), or phospho-Ser¹⁰ H3 (Upstate). (D) Inhibition of MSK1 by fmk after mutation of Met⁴⁹⁸ to Thr. COS-7 cells were transfected with HA-tagged WT or M498T MSK1. MSK1 autophosphorylation and H3 phosphorylation were assessed as in (C).