Protein kinases, including protein kinases of the MAP kinase pathways are attractive therapeutic targets for development of specific inhibitors because they often display perturbed activity in clinical conditions such as cancer, cardiac conditions, neurological and inflammatory diseases [69
]. The search for highly specific MAP kinase inhibitors is therefore a major challenge for pharmaceutical companies. Unfortunately, many of the protein kinase inhibitors developed today display little specificity and have therefore not entered the clinic [73
MK5 is a compound of the MAP kinase pathways, but studies aimed at elucidating the role of MK5 have been hampered by the lack of a specific inhibitor. DAs are a group of complex plant chemicals with similar structure to that of terpene. A large number of these alkaloids were isolated from Aconitum
The DAs demonstrate plethora of pharmacological properties, such as analgesic, anti-arrhythmic, anti-inflammatory, arrhythmogenic, curariform, hypotensive, local anesthetic, neurotropic, psychotropic, and spasmolytic (reviewed in [36
]). Therefore, we investigated whether they possess the potential to inhibit MK5. Our screening showed that some of the DAs can suppress the catalytic activity of MK5. Of the tested compounds, noroxoaconitine (compound 11
) displayed the strongest MK5 inhibition with an IC50
= 37.5 μM, and a very favorable interaction energy predicted by docking studies. The IC50
value is in the same range as that of the two previously described unspecific MK5 inhibitors: epigallocatechin gallate (EGCG) from green tea (IC50
< 10 μM) and flavokavain A (IC50
= 10 μM) from kava (Piper methysticum
]. In accordance with flavokavain A, noroxoaconite inhibited MK5 and MK3 kinase activity, but had no effect on MK2 [78
]. The effect of EGCG on the enzymatic activity of MK2 and MK3 was not tested [74
]. Both EGCG and flavokavain, however, are rather unspecific. EGCG at a concentration of 10 μM was shown to inhibit the in vitro kinase activity, by ~90%, of ERK2, DYRK1A, ROCK-II, and PDK1 [74
]. The same concentration of flavokavain A also inhibited the protein kinases Aurora B, DYRK1A, IKKβ, and MK3 more or to a similar extent, while approximately 40–70% inhibition was measured for ERK8, GSK3β, RSK2, MK2, MSK1, p70S6K
, PDK1, PHK, PKA, PKB2, PLK1, p38δ, and smMLCK [78
]. While our studies suggest that 20 μM noroxoaconitine can inhibit the kinase activity of MK5 in cells, the effect of EGCG and flavokavain on MK5 in vivo has not been investigated.
The theoretical docking studies indicated that noroxoaconite fitted nicely into the ATP binding pocket of MK3 and MK5 in agreement with the experimental studies. The docking indicated that noroxoaconite binds a bit differently in MK3 and MK5. In MK3, the phenyl ring of noroxoaconite binds in between Leu52 in a β-sheet region, and Glu172 located four amino acids C-terminal of the catalytic RD region. In MK5, the phenyl ring of noroxoaconite interacted in a quite hydrophobic region of the binding pocket consisting of Leu28, Ala49, Met102, Met105 and Leu155 (Fig. ). In MK5, a more bulky region of the inhibitor interacts inbetween Ala30 and Glu152 that corresponds to Leu52 and Glu172 in MK3. The main reason for the differences in orientation seems to be that the cleft between Leu52 and Glu172 in MK3 is much narrower than the corresponding cleft between Ala30 and Glu152 in MK5. The atomic distance between the Cα-atoms of Leu52 and Glu172 in MK3 is 8.6 Å, while the corresponding distance between Ala30 and Glu152 in MK5 is 12.1 Å, which might explain our docking predictions indicating that noroxoaconite interacts with MK3 with the phenylring inbetween Leu52 and Glu172, while it interacts with MK5 with a more bulky region between Ala30 and Glu152.
The experimental studies showed that noroxoaconite did not prevent MK2-mediated phosphorylation of recombinant Hsp27 at serine residue 78. However, this was not predicted by the docking studies since noroxoaconite bound MK2 similar to the binding mode in MK5. At present, we cannot explain the specificity of compound 11
for MK3 and MK5, but flavokavain A also inhibited MK3 and MK5, but not MK2, despite the higher degree of overall identity between MK2 and MK3 than between MK3 and MK5 [55
]. Similarly, the MK2 inhibitor 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H
= 8.5 nM) is a more potent inhibitor for MK5 (IC50
= 81 nM) than for MK3 (IC50
= 210 nM), even though the ATP binding pockets of MK2 and MK3 show most identity [68
In an effort to determine the specificity of noroxoaconite, a screen was performed by the company ProQinase (Freiburg, Germany) with 25 μM of this compound against 252 different protein kinases. While significant inhibition of only two other protein kinases was detected (GRK2 and S6Kβ), no inhibition was found for MK3 and MK5 (see Electronic supplementary material, Table S1). At present, we cannot explain why this company failed to detect inhibition of MK5, while we clearly observed an inhibition using different experimental approaches. Differences in assay, source of MK5, conditions, and substrate may account for this discrepancy. While we used Praktide or Hsp27 as specific substrates for MK5, ProQinase used the peptide RBER-CHKtide, which was also used for 56 other protein kinases in their screen.
In conclusion, we have identified a potent novel MK5 inhibitor, noroxoaconitine (compound 11
), which most probably acts by competing with ATP for the ATP binding pocket in the enzyme. Noroxoaconitine also reduced MK3 but not MK2 kinase activity towards Hsp27 in vitro. As MK3 is less expressed and less active than MK2 [22
], the use of this inhibitor may allow testing the individual involvement of MK2 and MK5 in cellular responses to stress which are know to activate both enzymes [15
]. Rationally designed derivatives of noroxoaconitine may then be synthesized and experimentally tested for their potency and specificity to inhibit MK5, and may enable us to elucidate the biological roles of MK5. In addition, such inhibitors may prove therapeutic values in diseases with aberrant MK5 functions.