The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor
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The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC50 = 37.5 μM; Ki = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.
KeywordsMitogen-activated protein kinase-activated protein kinase MK5 Kinase activity Diterpenoid alkaloid Noroxoaconitine MK5 MK3 ATP binding site Molecular docking
Extracellular signal-regulated kinase
Internal coordinate mechanics
c-Jun N-terminal kinase
- MAP2K, MEK or MKK
MAP kinase kinase
- MAP3K, MEKK or MKKK
Mitogen-activated protein kinase kinase kinase
Mitogen-activated protein kinase
MAPK-activated protein kinase
Mitogen- and stress-activated protein kinases
Predicted structure quality score
Root mean squared deviation
p90 ribosomal S6 kinase
Structural Analysis and Verification Server
Mitogen-activated protein kinase (MAPK) signal transduction pathways are activated by many different stimuli, and they regulate diverse processes ranging from proliferation and differentiation to apoptosis. To date, seven distinct MAPKs pathways have been discovered in mammalian cells, including: extracellular signal-regulated kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2 and JNK3); p38MAPK (p38α, p38β, p38γ and p38δ); ERK3 and ERK4; ERK5; and ERK7 and ERK8 [1, 2, 3, 4, 5, 6, 7, 8, 9]. The typical MAPKs family consists of at least three-tiered cascades: upstream MAPK kinase kinase (MAP3K, MEKK or MKKK), a MAPK kinase (MAP2K, MEK or MKK), and a downstream MAPK. Each MAPK can phosphorylate a variety of substrates, including protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs or MKs) [3, 5, 10, 11, 12, 13, 14]. The human MK family comprises 11 different members: 4 p90 ribosomal S6 kinases (RSK1-4), 2 mitogen- and stress-activated protein kinases (MSK1 and MSK2), 2 MAPK-interacting kinases (MNK1 and MNK2), and 3 MAPK-activated protein kinases (MK2, MK3 and MK5) . According to their sequence, all MKs can be classified into five subgroups: RSKs, MSKs, MNKs, MK2 and MK3, and MK5 [2, 16, 17, 18, 19, 20].
MK5 or the human analogue p38-regulated/activated kinase, PRAK, was originally discovered as a sequence tag by the searching of sequences homologous to MK2 [21, 22]. Their catalytic domain displays 48% amino acid identity and their overall sequence identity is 42%. MK3, which possesses 75% overall identity with MK2, is 41% identical with MK5. All three proteins seem to be ubiquitously expressed [16, 23], but the expression level and activity of MK2 are significantly higher than MK3 in the cells and tissues examined . The expression level and activity of MK2 and MK5 in different cells has not been compared. MK2, MK3, and MK5 exert different biological roles as is reflected by their substrate specificity and differences in phenotypes of knock-out mice [15, 24, 25, 26]. Nevertheless, they may participate in common functions such as F-actin remodeling (MK2 and MK5) and stimulation of tumor necrosis factor synthesis and p38MAPK stabilization (MK2 and MK3) . Studies in MK5-deficient mouse models and cells have demonstrated that depletion promoted oncogenic transformation by c-Ha-RAS and facilitated dimethylbenzanthrance-induced skin carcinogenesis . Furthermore, MK5 overexpression inhibited proliferation of NIH3T3 cells . MK5 also seems to be involved in PKA-induced F-actin rearrangement and cell migration through Hsp27 phosphorylation, which is suppressed by 14-3-3ε [28, 29, 30]. Moreover, transgenic mice expressing constitutive active MK5 displayed anxiety-related traits and locomotor differences compared to wild-type littermates, suggesting the involvement of MK5 in anxiety behavior . In spite of these described functions, the exact biological role of MK5 is still incompletely understood because MK5 knock-out mice do not display any obvious phenotype under normal conditions [24, 25].
The physiological function of protein kinases can be investigated by multiple approaches using targeted gene knock-out and knock-in animal models, RNA interference, overexpression of constitutive active or dominant negative mutants, and by the use of specific inhibitors (reviewed in [4, 32]). In recent years, there has been a globally increasing interest in herbal compounds because of their beneficial effect on human health, but also as potential specific protein kinase inhibitors [33, 34]. One group of them, the diterpenoid alkaloids (DAs), has been tested, but due to their toxic nature there are very few reports about their pharmacological effects. Tyrosinase inhibition and structure–activity relationship (SAR) of 15 DAs, with lycoctonine skeleton, and semisynthetic derivatives have been reported . Some of the DAs were also reported to be active chemotherapeutic agents by inhibiting American trypanosomiasis . To date, no specific MK5 inhibitors have been reported. This prompted us to test several DAs for their ability to suppress enzymatic activity of MK5. One of them, noroxoaconitine, specifically inhibited MK5 but not MK2 with an IC50 = 37.5 μM. This compound may therefore be a valuable tool in MK5 studies.
Materials and methods
The DAs were generously provided by Prof. M. Sultankhudzaev, Uzbek National Academy of Science, Tashkent, Uzbekistan. The compounds are HPLC grade pure and structures have been confirmed by different NMR techniques. The compounds were earlier cited elsewhere , and the structures are shown in Fig. 7 (see below, under “Docking of inhibitors at the ATP binding site of MK5”). Compounds 1–8 and 12–14 are natural alkaloids, while compounds 9–11 are semi-synthetic derivatives [37 and references therein]. Forskolin was purchased from Sigma-Aldrich (St. Louis, MO, USA), 32P γ-ATP was obtained from Perkin Elmer (Waltham, MA, USA), while recombinant MK2, MK3, and MK5 were from Invitrogen (Carlsbad, CA, USA). The expression plasmid encoding EGFP-MK5 fusion protein has been previously described . Recombinant Hsp27 was purchased from Signal Chem (Richmond, BC, Canada). The MK5 substrate peptide PRAKtide (KKLRRTLSVA) was purchased from Millipore (Billerica, MA, USA).
Screening and kinase activity
In vitro phosphorylation of Hsp27 by MK2, MK3, and MK5 was performed as previously described . The pSer-78 Hsp27 specific antibodies were purchased from Millipore (cat. no. #04-447).
Enzyme inhibition reactions were performed to study the kinetic trends and ATP competitions of the active compounds (11 and 8). The biochemical method was similar to the screening described above. The difference was that multiple inhibitor (0.025, 0.125, 0.25, 1.25, and 2.5 mM) and ATP concentrations (1, 8, 16, and 32 mM) were used.
Cell culture and transfection
PC12 cells, a kind gift from Dr. Jaakko Saraste (University of Bergen, Norway), were maintained in F-12K Medium (Kaighn’s Modification of Ham’s F-12 Medium) supplemented with 2 mM l-glutamine, 1.5 g/l sodium bicarbonate, 15% Horse serum (Gibco) and 2.5% fetal calf serum, penicillin (110 U/ml) and streptomycin (100 μg/ml). Cells were transfected using the Nucleofection kit (Amaxa) according to the manufacturer’s instructions. The EGFP-MK5 expression plasmid has been previously described . The plasmid that encodes the EGFP-NES fusion protein with the NES motif of the Rev protein of human immunodeficiency was constructed by cloning the complementary oligonucleotides 5′-CCG GAG ACG CTC TAC CAC CGC TTG AGA GAC TTA CTC TTG ACC GAG CT-3′ and 5′-CGG TCA AGA GTA AGT CTC TCA AGC GGT GGT AGA GCG TCT-3′ into the BspEI/SacI sites of pEGFP-C1 (Clontech, Mountain View, CA, USA).
Cells were fixed for 10 min with 4% formaldehyde. Then cells were washed with PBS and examined using a confocal laser-scanning Zeiss LSM 510 META microscope. Several hundred cells were monitored and representative pictures are presented.
Homology modeling of MK5
The X-ray crystal structure of MK5 is not known. A model of MK5 was therefore constructed by homology with MK2 using the traditional homology modeling approach. The PDB co-ordinates of MK2 (chain A, 2OZA ) was obtained from the Protein Databank (PDB, http://www.pdb.org) and used as template for constructing the MK5 model based on the amino acid sequence of MK5 . The model was built using the ICM™ Bioinformatics module. The ICM program constructs the molecular model by homology from core sections defined by the average of Cα atom positions in conserved regions.
Evaluations of model quality
The model quality check was performed using Structural Analysis and Verification Server (SAVS, http://nihserver.mbi.ucla.edu/SAVS/). PROCHECK [44, 45], WHAT_CHECK, ERRAT , VERIFY_3D [47, 48], and PROVE, were used to calculate quality factors of both the template (2OZA) and the generated 3D model of MK5. The total, local, burial and contact energies of the predicted models were calculated using predicted structure quality score (PSQS) (http://www1.jcsg.org/psqs) [49, 50, 51, 52].
Compounds 8 and 11 were docked into the MK5, while compound 11 was also docked into the X-ray structures of MK2 and MK3 . The docking studies of the compounds were performed as previously described . The Internal Coordinate Mechanics (ICM) program from Molsoft (http://www.molsoft.com/) was used for the docking studies.
Compounds 8 and 11 were docked into the predicted binding site of MK5, which corresponds to the ATP binding site of MK2 and MK3, while compound 11 was also docked into the ATP binding site of MK2 and MK3. The best docking conformations were selected based on docking energies, RMSD of ligand from initial ligand conformation, and similarities with ATP binding in known X-ray crystal structures (2PHK  and 2JBO ). The potent MK5 inhibitor 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridine-4-one was also docked into the predicted binding site of the MK5 model and the X-ray structures of MK2 and MK3 using the approach as for compounds 8 and 11.
Biochemical studies of DAs against MK5
Next, we performed dose–response studies using multiple concentrations of compounds 8 and 11, and determined the IC50 values. While the IC50 value for compound 8 was in the mM range, compound 11 had an IC50 of 37.5 μM (data not shown). These results demonstrate that compound 11 is a more potent MK5 inhibitor than compound. 8.
DA compounds prevent PKA-induced nuclear export of MK5
Compound 8 and 11 are ATP but not substrate competitors
Compound 11 abrogates MK3- and MK5-, but not MK2-mediated phosphorylation of Hsp27
Homology modeling of MK5
The structural quality of the model was checked and compared with the template structure (MK2). The Ramachandran plot of the model (panel b of Fig. 5) revealed that 88.7% of the residues were in most favored regions, which is better than the template X-ray structure (86.9%). Verify_3D showed that 71.6% of the residues had an averaged 3D–1D score of more than 0.2, whereas the corresponding value for the template was 82%. During the assessment of PROVE scores for both the model and template, the mean Z score was found to be 0.086 and 0.278, respectively, which also indicates a structurally high quality model.
Finally, the 3D MK5 model was superimposed onto the 3D structure of the template 2OZA (shown in panel c of Fig. 5), giving a RMSD of 0.68 Å between backbone Cα atoms of template and model. The structural deviations between the model and the template were mainly seen in some loop regions, but secondary structure elements (not shown) and the ATP binding site (GXGXXG) were highly conserved (Fig. 5).
Docking of inhibitors at the ATP binding site of MK5
The docking indicated that compound 8 has several hydrophobic interactions with amino acids in the ATP binding pocket. Leu28, Gly29, Gly31 Leu155 and Met105 of MK5 all interacted with compound 8 after docking. A hydroxyl oxygen of compound 8 formed a hydrogen bond with a side chain oxygen of Asp169. The hydroxyl group of compound 11 (Fig. 7) had a hydrogen bond with the backbone carbonyl oxygen of Glu152. Compound 11 also formed a hydrogen bond with the backbone carbonyl group of Leu28.
Compound 11 was also docked into the ATP binding sites of the X-ray structure of MK2 (PDB id: 2OZA)  and MK3 . The docking indicated that compound 11 may bind both MK2 and MK3, but with weaker affinity than for MK5. The docking energy for MK2 was −79.1 kcal/mol, while the calculated binding energy was −5.2 kcal/mol. The corresponding values for MK3 were −72.1 kcal/mol and −4.2 kcal/mol, respectively.
To validate our docking procedure, the potent inhibitor of MK2, MK3 and MK5 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridine-4-one was docked into the ATP binding pockets of MK2, and MK3 and the predicted pocket of the MK5 model. This inhibitor has IC50 values of 0.008.5 μM for MK2, 0.21 μM for MK2 and 0.081 μM for MK5 . The calculated binding energies (ΔG) for MK2, MK3 and MK5 were −10.3 kcal/mol, −3.8 kcal/mol and −7.3 kcal/mol, respectively. The ΔG and the IC50 values were compared using linear regression and found to be highly correlated (R2 = 0.93).
The calculated binding energy (ΔG) of compound 11 is overestimated compared with the experimental observation. Compound 11 contains functional groups with strong hydrogen bonding capacity, and a reason for the overestimations may be imperfect parameterization of these reactive groups. However, the results obtained for the potent binder 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridine-4-one show the validity of our docking approaches.
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, 70, 71, 72]. 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, 74, 75].
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, Delphinium and Consolida. 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, 76, 77]). 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) [74, 78]. In accordance with flavokavain A, noroxoaconite inhibited MK5 and MK3 kinase activity, but had no effect on MK2 . The effect of EGCG on the enzymatic activity of MK2 and MK3 was not tested . 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 . 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 . 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. 6). 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 . Similarly, the MK2 inhibitor 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (IC50 = 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 .
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 , 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 . 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.
We are grateful to Prof. M. Sultankhudzaev, Uzbek National Academy of Science, Tashkent, Uzbekistan, for kindly supplying us the compounds reported here. We are also grateful to Dr. Thomas Hesterkamp Evotec AG, Germany, and Dr. John Barker Evotec Ltd., UK, for providing the X-ray structure coordinates of MK3. We are also grateful to Dr. Mona Johannessen, University of Tromsø. S.K and M.T.H.K. were fellows of the PhD School for Molecular and Structural Biology, University of Tromsø, Norway. This work was supported by the Norwegian Cancer Society (A01037) and the Mohn Foundation.
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