A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

<div><p>A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC₅₀ of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 <i>in vitro</i>. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (<i>K_d</i>) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH₂-terminal protein kinases <i>in vivo</i>. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.</p></div

MKK4 and MKK7 homology in mammalian MAPK pathways.

<p>A) Phylogenetic tree for human MKK4, MKK7, MKK3, MKK6, MEK1, p38, JNK, and ERK. B) Amino acid sequence alignment starting from the hinge region to the activation loop are shown for MKK3 (UniProtKB accession code: P46734), MKK6 (UniProtKB accession code: P52564), MKK4 (UniProtKB accession code: P45985), and MKK7 (UniProtKB accession code: O14733). Green denotes a highly conserved region among these MKKs. Residues highlighted in yellow designate sequence variations in the activation loop. C, D) Three-dimensional structure of MKK4 suggests that HWY336 interacts with the activation loop through hydrogen bonding. C) (top) Amino acid sequence variations within the activation loop of MKKs. (bottom) Proposed docked pose of ATP in MKK4. The arrow designates different amino acids that may determine MKK selectivity. The MKK4 structure is shown in the background with the activation loop (white) containing the varying amino acids (Arg²⁶²) at the respective positions (generated with the Pymol program; <a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). D) Hydrophobic interactions between HWY336 and the MKK4 active site are shown. Hydrophobic residues within the active site are designated by the cap-stick model and HWY336 is displayed using transparent hydrophobic surfaces. The hydrophobicity index is displayed on the left, where brown and blue denote highly hydrophobic and hydrophilic areas, respectively. Pro²⁶⁸, Phe³⁰⁵, Pro³⁰⁸, and Val³¹³ interact with HWY336 side chains. HWY336 interacts with the activation loop of MKK4 through hydrogen bonding via the hydroxyl group of Thr²⁶¹.</p

Identification of HWY336 as an inhibitor of MKK4 and MKK7 by screening a protoberberine derivative chemical library.

<p>A) Chemical modification of berberine chloride produced 80 protoberberine derivatives. B) Scheme for screening protoberberine derivatives for inhibition of various MKKs (MKK4/MKK7, MKK3/MKK6, and MEK1/MEK2) and MAPKs (JNKs, p38, and ERKs). Each MKK and MAPK was immunoprecipitated from HEK293 or CHO cells. C) Chemical structure and simplified synthetic process of HWY336 from berberine chloride.</p

SPR detects the interaction of HWY336 with MKK4.

<p>The concentration of HWY336 was varied from 100-MKK4 complex with time was measured over the course of the interaction between HWY336 and MKK4.</p

Three-Dimensional Superlocalization Imaging of Gliding <i>Mycoplasma mobile</i> by Extraordinary Light Transmission through Arrayed Nanoholes

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding <i>Mycoplasma mobile</i> (<i>M. mobile</i>). The results analyzed with multiple <i>M. mobile</i> bacteria show individual characters and reveal that <i>M. mobile</i> undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision

HWY336 does not inhibit the activity of MKK4 and MKK7 mutants of the proposed docking residues.

<p>Flag-tagged MKK4, MKK4-Q253Y I258V R262M, HA-tagged MKK7, and MKK7-R283Y K288V R292M were immunoprecipitated from activated HEK293 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091037#s2" target="_blank">Materials and Methods</a>. Usage of the same amount of MKK4, MKK4 mutant, MKK7, and MKK7 mutant was confirmed by western blots with anti-flag antibody and anti-HA antibody, respectively. A, B) Kinase activity of immunoprecipitated (A) MKK4 and the MKK4 mutant, (B) MKK7 and the MKK7 mutant was assayed by measuring γ-P³² phosphorylation of JNK as a substrate. C–F) Effect of HWY336 on the kinase activity of (C, D) wild-type (top) and mutant (bottom) MKK4, (E, F) wild-type (top) and mutant (bottom) MKK7 was assayed after treatment with increasing concentrations of HWY336 (10, 30, 50 µM) or DMSO. D, F) Kinase activity of wild-type and mutant MKK4 and MKK7 with increasing concentrations of HWY336 shown in (C, E), was quantified by NIH ImageJ software.</p

Three-Dimensional Superlocalization Imaging of Gliding <i>Mycoplasma mobile</i> by Extraordinary Light Transmission through Arrayed Nanoholes

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding <i>Mycoplasma mobile</i> (<i>M. mobile</i>). The results analyzed with multiple <i>M. mobile</i> bacteria show individual characters and reveal that <i>M. mobile</i> undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision

Three-Dimensional Superlocalization Imaging of Gliding <i>Mycoplasma mobile</i> by Extraordinary Light Transmission through Arrayed Nanoholes

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding <i>Mycoplasma mobile</i> (<i>M. mobile</i>). The results analyzed with multiple <i>M. mobile</i> bacteria show individual characters and reveal that <i>M. mobile</i> undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision

Three-Dimensional Superlocalization Imaging of Gliding <i>Mycoplasma mobile</i> by Extraordinary Light Transmission through Arrayed Nanoholes

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding <i>Mycoplasma mobile</i> (<i>M. mobile</i>). The results analyzed with multiple <i>M. mobile</i> bacteria show individual characters and reveal that <i>M. mobile</i> undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision