An angle on MK2 inhibition – Optimization and evaluation of prevention of activation inhibitors
Introduction
The mitogen-activated protein kinases (MAPK) p38α MAPK (p38α) and MAPK-activated protein kinase 2 (MK2) are attractive drug discovery targets for the treatment of inflammatory conditions such as rheumatoid arthritis (RA).[1],[2],[3] Several p38α inhibitors have been tested in the clinic for rheumatoid arthritis including BIRB796 (BI),[4] VX-745[5] and VX-702 (Vertex)[6], but were discontinued due to concerns over their toxicological profile and/or lack of efficacy at the doses that could be used.[7]
Selectively targeting MK2 downstream of p38α has been suggested to provide p38α-like efficacy with downregulation of TNF-α production along with an improved safety profile.[3],[8] The interest in selectively targeting MK2 also resides in the fact that p38α not only activates MK2 but also mitogen- and stress- activated protein kinase 1 (MSK1). The inhibition of MSK1 leads to downregulation of the anti-inflammatory cytokines interleukin (IL)-10 and IL-1 receptor antagonist (IL-1RA) and hence reduction of the overall efficacy.[8] We hypothesized that a ligand selectively binding to the heterodimeric complex of p38α/MK2 could block the production of pro-inflammatory TNF-α, whilst maintaining the IL- 10 and IL-1RA levels.
Furthermore, we aimed at inhibiting the phosphorylation of MK2 by binding to the heterodimeric complex of p38/MK2 via a prevention of activation (PoA) mechanism.[9],[10] Hence, the inhibition should be substrate selective since it wouldn’t interfere with the p38-MSK1 signaling as described previously.[10] High throughput screening of the AstraZeneca compound collection afforded two distinct lead series represented by hit compounds 1 (series 1) and 2 (series 2) , showing a promising separation of p38-MSK1 substrate selectivity (Figure 2).[10] In this paper we describe our efforts to further explore the pyrazole amide series 2, evolved from compound 2.
Results and Discussion
The p38α inhibitor 3, previously disclosed by Das et al,[11] is structurally similar to compound 2, but while 2 is 98x selective for MK2 inhibition over MSK1, 3 is not selective (Figure 2). In order to understand the selectivity profile of 2, it was crystallized in complex with p38α/MK2, and its binding interactions compared to 3. The X-ray of 2 bound to p38α/MK2 (pdb code: 4THY) shows that it can bind to the ATP binding pocket of p38α (Figure 3a).
The binding of 2 to the p38α/MK2 complex displays hydrogen bond interactions between the sulfonamide oxygen and the Asp168 backbone NH, and between the pyrazole nitrogen and the hinge residue Met109. The n-propyl substituent does not appear to make any specific interaction and leaves room for further modification of this substituent. The pyrazole phenyl group reaches towards the MK2 protein surface in the protein complex however, the distance is too long for direct interactions (closest distance to Gln369 is 3.7 Å).
The structurally analogous compound 3 displays similar binding interactions to p38α (Figure 3b), but without the sulfonamide oxygen interaction to Asp168, potentially responsible for driving the selectivity for PoA-MK2 versus PoA-MSK1. Compound 2 is a moderately potent inhibitor of MK2 but with good selectivity towards MSK1 through its specific interactions with the p38α/MK2 complex, but with low aqueous solubility and low stability in human liver microsomes.
Hence, our design strategy was to improve the MK2-PoA potency and the drug-like properties, while improving selectivity towards MSK1 by building on the interactions made by 2 to the p38α/MK2 complex, and further extending towards the MK2 protein surface.
Initial structure-activity relationship (SAR) investigation focused on identifying alternatives to the pyrazole core. The imidazole- based compound 4 (Table 1) offered an opportunity to maintain the key interaction with Met109 while exploring lipophilicity- dependent effects, e.g. solubility, and intrinsic clearance, and thereby improve the overall pharmacokinetic profile. The potency for the imidazole analogue 4 improved 2-fold in the MK2-PoA assay compared to its pyrazole analogue 2.
Despite equivalent LogD, the solubility of imidazole 4 was improved 70-fold compared to the corresponding pyrazole 2. The importance of the sulfonamide in the aniline 5-position was confirmed by introducing different polar groups, preserving the possibility for a hydrogen bond interaction with Asp168 in the p38α/MK2 complex, as previously shown in the pyrazole series.[10]
Changing the tertiary sulfonamide 4 to the primary sulfonamide 5 increased MK2-PoA potency, but with an overall drop in the MK2-PoA/MSK1-PoA selectivity. Replacing the sulfonamide with primary or secondary amides 6 and 7, resulted in sub-nanomolar MK2-PoA potencies, but also led to a complete loss of selectivity over MSK1. The carboxylic acid 8 lost 6-fold in MK2-PoA potency with a 70-fold gain of selectivity over MSK1-PoA compared to amide 6.
Unfortunately, the carboxylic acid analogue 8 did not inhibit TNF- α production in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) (data not shown). To further improve potency and selectivity, we extended the molecules towards the MK2 protein surface of the p38α/MK2 complex in an attempt to identify novel interactions (Figure 3a and 3b). With the X-ray structure of 2 bound to the p38α/MK2 complex as starting point, compounds reaching towards the MK2 protein surface were designed and synthesized.
Structure based design suggested that the terminal phenyl acetamide substituent had the possibility to generate a hydrogen bond to the Asp366 from MK2 protein as can be seen in docking shown in figure 4, which lead us to include this substituent in a library to explore SAR. As predicted, these meta-substituted phenyl acetamides showed promising activity and selectivity profiles. The primary amide 9, as well as the secondary amide 10, showed slightly improved potency compared to unsubstituted 4, but more importantly the selectivity over MSK1-PoA increased more than 3-fold.
However, both compounds suffered from poor metabolic stability and low solubility. Compound 9 was tested in a pan kinase selectivity panel of 273 kinases and was not active against any kinase tested at 1 µM (Table S1 in the Supporting Information). The left hand R1 substituent was revisited to investigate if the structure–activity relationship (SAR) for the R2 = phenyl acetamide subseries is parallel to that of the R2 = H subseries.
The secondary sulfonamide 11 showed low nanomolar MK2-PoA potency, but with decreased MSK1 selectivity compared to the tertiary sulfonamide 9. A similar trend was seen for the primary sulfonamide 12, which was 10-fold less selective against MSK1 compared to compound 10, although an improved potency was obtained. The primary amide 13 showed excellent MK2-PoA potency (IC50 = 2 nM), but displayed no selectivity over MSK1, in comparison with 6. Compounds 11 and 13 suffered from low solubility but showed improved metabolic stability in human liver microsomes compared to compound 9.
In summary, parallel SAR was observed for the two different sub- series. Nonetheless, comparing compound 9 to the compound 4, the combination of tertiary sulfonamide and primary m- phenylacetamide substitutions afforded an apparent cooperative effect to improve MK2 selectivity over MSK1. This selectivity data supports the idea that substrate selectivity can be improved by extending towards the MK2 surface in the protein dimer complex, as suggested by docking studies.[12] These studies suggest the formation of a possible hydrogen bond between the NH of the phenylacetamide (R2-substituent) and Asp366 in the MK2 protein (Figure 4).
Conclusions
In summary, by replacing the core pyrazole of compound 2 with imidazole, potency and solubility were improved within the series of compounds. The presence of dialkyl sulfonamide was essential to balance MK2 potency and selectivity, through its interaction with Asp168 in the p38 part of the p38/MK2 complex.
We discovered the highly selective compound 9 with a more than 200- fold selectivity of MK2-PoA over MSK1-PoA by extending towards the MK2 interface of the binding pocket. This unfortunately at the cost of oral drug properties, and thus our focus moved to other parts of the molecule. Supported by analysis of the central torsional angle, compound 18 was discovered, representing a novel series with increased potency and favorable physical chemistry properties.
When we attempted to confirm this selectivity in the human PBMC cell assay, we found that selective inhibition of the MK2 activation did not preserve anti-inflammatory IL-10 while inhibiting pro-inflammatory TNF-α production. Using protein immunoblotting to explore the pathways around p38 confirmed that the majority of PoA MK2 compounds, despite their biochemical selectivity against MSK1 phosphorylation, behaved similarly to p38 inhibitors.
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