Identification and SAR of squarate inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)
Graphical abstract
A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. An MK-2 co-structure was obtained and a structure based approach was followed to optimize potency and selectivity.
Introduction
Tumor Necrosis Factor-alpha (TNF-α is an important cytokine with pro-inflammatory properties that has been associated with various inflammatory diseases such as rheumatoid arthritis (RA),1 psoriasis,2 inflammatory bowel disease3 and others.4 Anti-TNF-α therapies, such as TNF-α monoclonal antibodies (Remicade™ and Humira™) and TNF-α soluble receptors (Enbrel™)5, 6 have been shown to be efficacious for the treatment of various inflammatory diseases such as RA.
In the resting state, MAPKAP kinase 2 (MK-2) resides in the nucleus and upon stimulus it is phosphorylated by p38α and subsequently exported to the cytoplasm.7, 8, 9 Once in the cytoplasm it is responsible for the induction of TNF-α production, as well as other pro-inflammatory cytokines.10, 11 Recent studies have helped to elucidate the role MK-2 plays in the upregulation of TNF-α. Induction of the translation of TNF-α mRNA is achieved by the phosphorylation of tristetraprolin (TTP), a protein which binds the adenosine/uridine-rich element (ARE).12, 13 MK-2 deficient mice have been shown to be protected against brain ischemic injury,14 heart reperfusion injury15 and are resistant to collagen-induced arthritis.16
Recently, there have been several reports of MK-2 inhibitors (Fig. 1). Anderson et al. have reported on two scaffolds, the aminocyanopyridines 117 and the pyrrolopyridine inhibitors 3.18 The latter were shown to be potent and reasonably selective against a panel of kinases. More recently Trujillo et al. have reported on the tetrahydro-β-carboline carboxylic acids 2 as MK-2 inhibitors.19 While not as potent as the former, they did demonstrate good selectivity for MK-2. Wu et al. of Boehringer–Ingelheim also reported a series of carboline (4) analogs as MK-2 inhibitors.20 Interestingly, both the pyrrolopyridine 3 analogs as well as the carboline 4 analogs gain much of their affinity by interactions with the specificity surface.21 Herein we wish to report our own progress on the identification of a series of MK-2 inhibitors.
Compound 17 was identified as an inhibitor of MK-2 (IC50 = 8.9 μM) as part of a high throughput screen. In order to take a structure based approach we obtained a co-crystal of 17 with MK-2 (Fig. 2, PDB code 3FPM). The key hinge binding interaction is between Leu141 and the pyridine nitrogen. One of the squarate oxygens makes a hydrogen bond with the side chain nitrogen of Lys93. The aryl ring tucks up under the glycine rich loop.
While 17 was selective against a number of kinases, it did inhibit several in our screening panel (see Table 7). It also proved to be an inhibitor of the Cytochrome P450 (Cyp450) isozymes 3A4 and 2C9 (IC50’s of 1.3 and 0.6 μM respectively).22 Thus as part of our routine screening paradigm, we needed to address potency and selectivity as well as identify approaches to reduce Cyp450 inhibition.
Section snippets
Chemistry
Compound 17 was originally prepared as part of an investigation into a previous project and much of the chemistry herein has been described.24, 25 Investigations on the right hand side of the molecule began with the addition of amines to compound 524 (Scheme 1) in ethanol. If reactions were sluggish at room temperature or reflux, they were heated at 140 °C under microwave irradiation. Non-commercial amines were prepared by the condensation of ketones with hydroxylamine and the resulting oximes
Results and discussion
Examination of 17 in the protein crystal (Fig. 2) reveals the key interaction of these squarate inhibitors. The squarate ring is the central scaffold which interacts with the conserved Lys93 via one of the squarate oxygens (Fig. 2). These oxygens carry a significant negative charge due to a resonance form wherein the butadiene is aromatic with a +2 charge and each of the oxygens carry a −1 charge.33, 34 The 4-pyridine provides the interaction with the NH of Leu141 in the hinge region. The aryl
Materials and methods for the MAPKAP kinase-2 kinase assay
MK-2 kinase activity was assessed using human recombinant MK-2 containing residues 41–353 used at 5 nM in an ELISA based assay. The kinase reaction was performed on 96-well streptavidin coated plates using a biotinylated 13-mer peptide derived from LSP1 at 200 nM in 20 mM Hepes pH 7.4, 10 mM MgCl2, 3 mM DTT, 1 μM ATP. The reaction was stopped after 30 min incubation at room temperature and washed in PBS 0.05% Tween 20. Polyclonal Anti phospho-LSP1 antibodies was then added to the plate along with Goat
Acknowledgements
The authors would like to thank Li Di and Susan Petusky for the Cyp450 data and Jennifer Gale and Jenny Togias for kinase selectivity data. The authors would also like to thank Steve Tam for helpful discussions. Protein crystallization data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID (or 22-BM) beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon
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