Hyae-Kyeong Kim

Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell‐based experiments, we used SAA (normal hEGFR‐overexpressed NIH3T3) cells and B104‐1‐1 (neu*‐transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D‐treated cell lysates with polyclonal anti‐Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti‐Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose‐dependent manner in both B104‐1‐1 and EGF‐stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104‐1‐1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras‐MAP kinase signaling pathway. These results suggest that actinomycin D could be a non‐phosphorylated natural and cellular membrane‐permeable SH2 domain antagonist.

Actinomycin D, C2 and VII, inhibitors of Grb2-SHC interaction produced by Streptomyces.

Actinomycin D, C2 and VII, cyclic peptides, inhibited Grb2 SH2 domain association (IC50 5-7 microM) with a phosphotyrosine containing peptide derived from the Shc protein (pTyr317). Actinomycins are the first examples of nonphosphorylated natural ligands of SH2 domain.

5-Demethylovalicin, as a Methionine Aminopeptidase-2 Inhibitor Produced by Chrysosporium

5-Demethylovalicin was isolated from the fermentation broth Chrysosporium lucknowense and the structure was identified by spectroscopic methods. 5-Demethylovalicin inhibited the recombinant human MetAP-2 (IC(50)=17.7 nM) and the growth of human umbilical vein endothelial cells (HUVEC; IC(50)=100 nM) in cell proliferation assay without cytotoxicity on the transformed and cancer cell lines.

Natural and synthetic analogues of actinomycin D as Grb2-SH2 domain blockers

Natural analogues (D, C2, and VII) of actinomycin inhibit Grb2 SH2 domain binding with phosphopeptide-derived from Shc in vitro and in intracellular system. To study structure-activity relationships, 13 actinomycin analogues were synthesized and we found that the inhibition activity depended on the substituents of cyclic peptide groups in actinomycin and two analogues with Tyr residue are the most potent inhibitors with IC50 value of 0.5 and 0.8 microM, respectively.

Isolation of farnesyltransferase inhibitors from herbal medicines.

Ras proteins (H, K, and N) are small guanine nucleotide binding proteins that undergo a series of posttranslational modifications including farnesylation onto cysteine 186 at C-terminal of Ras by farnesyl protein transferase (FPTase). This is a mandatory process before Ras anchoring to plasma membrane, which is critical for its biologic activity, such as cell proliferation and tumorigenesis. Evidence now exists that specific inhibitors of FPTase will have an antitumorigenic effect. Many research teams are working on the isolation of natural inhibitors to give chemical leads to develop effective therapeutic agents for the treatment of cancers.1,2 In the course of our screening for potent inhibitors of FPTase from herbal medicines, we isolated 2-hydroxycinnamaldehyde, rhombenone, and arteminolide as inhibitors of FPTase