trichostatin-a and chlamydocin

Chlamydocin, a cyclic tetrapeptide containing aminoisobutyric acid (Aib), l-phenylalanine (l-Phe), d-proline (d-Pro), and a unique amino acid l-2-amino-8-oxo-9,10-epoxydecanoic acid, inhibits the histone deacetylases (HDACs), a class of enzymes, which play important roles in regulation of gene expression. Sulfur containing amino acids can also inhibit potently, so zinc ligand, such as sulfhydryl group connected with a linker to the so-called capping group, corresponding to cyclic tetrapeptide framework in case of chlamydocin is supposed to interact with the surface of HDAC molecule. Various changes in amino acid residues in chlamydocin may afford specific inhibitors toward HDAC paralogs. To find out specific inhibitors, we focused on benzene ring of l-Phe in chlamydocin framework to shift to various parts of cyclic tetrapeptide. We prepared and introduced several aromatic amino acids into the cyclic tetrapeptides. By evaluating inhibitory activity of these macrocyclic peptides against HDACs, we could find potent inhibitors by shifting the aromatic ring to the Aib site.

Topics: Aminoisobutyric Acids; Cells, Cultured; Circular Dichroism; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Histone Deacetylases; Molecular Structure; Peptides, Cyclic; Structure-Activity Relationship

Histone deacetylases (HDAC) are associated with a variety of transcriptional repressors that control cellular differentiation and proliferation. HDAC inhibitors such as trichostatin A, trapoxin and chlamydocin could be useful tools to modulate these cellular processes. We investigated their effect on the self-renewal of hematopoietic stem cells (HSC) during ex vivo culture.. Purified murine HSC with the phenotype c-Kit+,Thy-1.1(lo), Lin(-/lo), Sca-1+ were cultured for 4 days with IL-3, IL-6 and c-Kit ligand without or with HDAC inhibitors, after which their degree of phenotypic differentiation in culture was assessed by flow cytometric analysis. To explore whether HDAC inhibitors could have a beneficial role in human HSC transplantation, mobilized peripheral blood CD34+ cells were cultured with thrombopoietin mimetic peptide, flt3 ligand, and c-Kit ligand, without or with various HDAC inhibitors. The fluorescent dye, carboxyfluorescein-diacetate succinimidylester (CFSE), was used to track division of cell subsets, and engrafting ability was evaluated in a non-obese diabetic (NOD) -SCID xenotransplantation model.. Murine HSC cultured with HDAC inhibitors maintained a more primitive phenotype than control cultures. The number of human HSC expressing Thy-1 increased up to seven-fold during a 5-day culture with HDAC inhibitors compared with control cultures. Chlamydocin was the most effective of the HDAC inhibitors tested at promoting Thy-1 expression on human cells. CFSE tracking showed that the increase in Thy-1+ cells resulted from cell division. In a NOD-SCID repopulation assay, cells exposed to chlamydocin for 24 h displayed an average four-fold higher engrafting ability over control cells.. Our studies suggest that HDAC inhibitors can induce ex vivo expansion of human HSC, and may improve engraftment in hematopoietic transplant patients when cell dose is limiting.

Topics: Animals; Antigens, CD34; Cell Differentiation; Cell Proliferation; Cells, Cultured; Flow Cytometry; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Interleukin-3; Interleukin-6; Mice; Mice, SCID; Peptides, Cyclic; Phenotype; Stem Cell Factor; Thy-1 Antigens