Data_Sheet_1_Dual-Specificity Phosphatase CDC25B Was Inhibited by Natural Product HB-21 Through Covalently Binding to the Active Site.docx

Cysteine 473, within the active site of the enzyme, Cdc25B, is catalytically essential for substrate activation. The most well-reported inhibitors of Cdc25 phosphatases, especially quinone-type inhibitors, function by inducing irreversible oxidation at this active site of cysteine. Here, we identified a natural product, HB-21, having a sesquiterpene lactone skeleton that could irreversibly bind to cys473 through the formation of a covalent bond. This compound inhibited recombinant human Cdc25B phosphatase with an IC50 value of 24.25 μM. Molecular modeling predicted that HB-21 not only covalently binds to cys473 of Cdc25B but also forms six hydrogen bonds with residues at the active site. Moreover, HB-21 can dephosphorylate cyclin-dependent kinase (CDK1), the natural substrate of Cdc25b, and inhibit cell cycle progression. In summary, HB-21 is a new type of Cdc25B inhibitor with a novel molecular mechanism.</p

A possible model showing the last two steps of biosynthesis and the molecular actions of the three sesquiterpenoids in <i>Drosophila</i>.

<p>In the CA, FA is the common precursors for JHB3, JH III, and MF biosynthesis. In the hemolymph and peripheral tissues, MF either directly acts through Met/Gce or is converted to JHB3. JHAMT only accounts for JHB3 biosynthesis; and other methyltransferases and P450 epoxidase with question marks have not been identified. Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005038#sec008" target="_blank">Discussion</a> for details on the model. Text and arrow sizes convey magnitude of treatment and response. The gray line separates CA from hemolymph and peripheral tissues.</p

MF plays a dual role: as a JHB3 precursor and as a hormone.

<p>(A) Percentage of rescuing <i>Aug21-GAL4>UAS-Grim</i>, <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i>, and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} to adults by topical application of methoprene, JHB3, JH III and MF (0.5×10^-2 μmol per larva) at 96h AEL. (B) Percentage of rescuing <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i> to adults by topical application of a dose gradient of methoprene, JHB3, JH III, and MF (0.5×10^-9~-2 μmol per larva) at 96h AEL. (C) qPCR measurements of fold-changes of relative <i>Kr-h1</i> mRNA levels in Kc cells treated with methoprene, JHB3, JH III, and MF (1×10^-10~-6 M) for 30 min. (D) qPCR measurements of relative <i>Kr-h1</i> mRNA levels in fat body tissues isolated from <i>w</i>^<i>1118</i> and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} at 96h AEL after treatments with methoprene, JHB3, JH III, and MF (1×10^-6 M) for 30 min. (E) qPCR measurements of the relative <i>Kr-h1</i> mRNA levels in the fat body tissues isolated from <i>w</i>^<i>1118</i>, <i>jhamt</i>^<i>2</i>, <i>Met</i>^<i>27</i>, <i>gce</i>^{<i>2</i>.<i>5k</i>}, and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} at 3h AIW. (F) MF promotes interaction of Met and SRC in mouse embryonic fibroblast 3T3 cells. 3T3 cells were transiently transfected with GAL4:TcMet and TcSRC. And the transfected cells were cultured in the medium containing different concentrations of MF and JH III (DMSO as control). After 24 hours exposure to the ligands, cells were assayed for luciferase reporter activity. The luciferase activity was normalized based on the total protein concentration determined for cells in each well. (G-G”) Measurements whole body titers of JHB3 (G), JH III (G’), and MF (G”) in <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i> at 3hAIW after topical application of MF (0.5×10^-2 μmol per larva; dissolved in acetone) at 96h AEL (about 24 hours after treatments).</p

Mutation of <i>jhamt</i> decreases JHB3 but not JH III and MF biosynthesis.

<p>(A) Measurements of methyltransferase activity in the brain-RG complexes in <i>w</i>^<i>1118</i> and <i>jhamt</i>^<i>2</i> at 3hAIW using FA or JHA as the substrate. (B) Measurements of JH biosynthesis in the brain-RG complexes in <i>w</i>^<i>1118</i> and <i>jhamt</i>^<i>2</i> at 3hAIW using the RCA-TLC method. (C-C”) Quantitative measurements of whole body titers of JHB3 (C), JH III (C’), and MF (C”) in <i>w</i>^<i>1118</i> and <i>jhamt</i>^<i>2</i> at 3hAIW using the HPLC-FD protocol.</p

Reduction of <i>hmgcr</i> expression in the CA of <i>jhamt</i> mutant results in complete lethality.

<p>(A and A’) The brain-RG complex in <i>jhamt-GAL4>UAS-GFP</i>. BR, brain; CA, corpus allatum. Observed under bright-field (A) or fluorescence (A’) using the same microscope. The CA cells expressing JHAMT were labeled with GFP. (B and B’) (B) Lethality of <i>jhamt-GAL4</i>><i>UAS-hmgcr dsRNA</i> during the embryonic, larval, and pupal stages. <i>jhamt-GAL4/+</i> and <i>UAS-hmgcr dsRNA/+</i> were used as the controls. (B’) Lethality of <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i>; <i>jhamt-GAL4>UAS-hmgcr dsRNA</i> during the embryonic, larval, and pupal stages. <i>jhamt</i>^<i>2</i>/+; <i>jhamt-GAL4/+</i> and <i>jhamt</i>^<i>2</i><i>/+; UAS-hmgcr dsRNA/+</i> were used as the controls. (C) Images of various pupal lethal phenotypes of <i>jhamt</i>^<i>2</i><i>/jhamt</i>^<i>2</i>; <i>jhamt-GAL4>UAS-hmgcr dsRNA</i>. (1–6) the abdominal sides; (1’-6’) the dorsal sides. The black asterisks point to empty portions of the pupae; the white asterisks, eye defects showing no pigmentation; the red asterisks, wing defects showing a unilateral wing loss.</p

Generation and characterization of the <i>jhamt</i> mutants.

<p>(A) Scheme for <i>jhamt</i> targeting. <i>jhamt</i> (black box) is replaced with <i>white</i> (gray box) by homologous recombination of the flanking sequences (white boxes). Red bars represent the primer pairs <i>jhamt-1/jhamt-2</i>; green bars, <i>jhamt-3/jhamt-4</i>; purple bars, <i>jhamt-1/jhamt-5</i>; blue bars, <i>jhamt-6/jhamt-4</i>. (B) Genomic DNA PCR to detect <i>white</i> and <i>jhamt</i> DNA using the above-mentioned primer pairs and genomic DNA extracted from <i>pw25-jhamt</i> and <i>jhamt</i>^<i>1</i>, <i>jhamt</i>^<i>2</i>, and <i>jhamt</i>^<i>3</i> lines. (C) Reverse transcription PCR to detect <i>jhamt</i> mRNA (<i>rp49</i> as the internal control) from <i>pw25-jhamt</i> and <i>jhamt</i>^<i>1</i>, <i>jhamt</i>^<i>2</i>, and <i>jhamt</i>^<i>3</i>. All of the mRNAs were isolated from the brain-RG complexes at 3 hours after initiation of wandering (3hAIW). (D and D’) Immunohistochemistry to detect JHAMT in the CA of <i>w</i>^<i>1118</i> (D) and <i>jhamt</i>^<i>2</i> (D’) at 3hAIW. The red arrow points to the CA showing JHAMT expression. (E) The lethality of <i>w</i>^<i>1118</i> and <i>jhamt</i>^<i>2</i> homozygous mutant during embryonic, larval and pupal stages.</p