We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34-, CD10 + , CD19 + , and cyto-plasmic ? chain (C ?)-negative surface phenotype from human CD34 + cells purified from umbilical cord blood (CB). When 10 3 CB CD34 + cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34 + population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a C ? + phenotype, and a small number of cells had immunoglobu-lin ? chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34 + cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34 + cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.

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We have developed a method for converting a transforming growth factor-β-activated kinase 1 (TAK1) type I inhibitor into a type II or c-helix-out inhibitor by structure-based drug design (SBDD) to achieve an effective strategy for developing these different types of kinase inhibitor in parallel. TAK1 plays a key role in inflammatory and immune signaling, and is therefore considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). We have already reported novel type I TAK1 inhibitor, so we utilized its X-ray information to design a new chemical class type II and c-helixout inhibitors. To develop the type II inhibitor, we superimposed the X-ray structure of our reported type I inhibitor onto a type II compound that inhibits multiple kinases, and used SBDD to design a new type II inhibitor. For the TAK1 c-helix-out inhibitor, we utilized the X-ray structure of a b-Raf c-helix-out inhibitor to design compounds, because TAK1 is located close to b-Raf in the Sugen kinase tree, so we considered that TAK1 would, similarly to b-Raf, form a c-helix-out conformation. The X-ray crystal structure of the inhibitors in complex with TAK1 confirmed the binding modes of the compounds we designed. This report is notable for being the first discovery of a c-helix-out inhibitor against TAK1.Key words transforming growth factor-β-activated kinase 1 (TAK1); inhibitor; type I; type II; c-helix-out; structure-based drug design Of over 500 kinases in human that maintain the functions of cells, 1) it is not completely clear which kinase inhibition causes an adverse event; therefore, it is important for kinase inhibitors to have a highly selective profile. However, in many cases, kinase inhibitors possess undesired off-target kinase activity, so multiple development candidates with different kinase selectivity profiles need to be prepared, and then a compound with a wide therapeutic window that has no severe adverse effects can be selected as a development candidate. In general, kinase inhibitors are classified into three different types 2,3) : a type I inhibitor binds to the active conformation (DFG-in conformation) of a kinase at the ATP-binding site and competes with ATP; a type II inhibitor binds to both the ATP binding site and its adjacent binding pocket in an inactive conformation (DFG-out conformation) of a kinase; a type III inhibitor is an allosteric inhibitor that has a binding site distinct from the ATP-binding site. Discovery of a type I inhibitor with high kinase selectivity is additionally challenging because amino acid residues in the ATP-binding site are highly conserved in many kinases. On the other hand, it is considered easier to identify a selective type II inhibitor because a type II inhibitor is able to utilize the lipophilic binding pocket derived from the DFG-out conformation, which is less conserved in terms of amino acid residues (though it has recently been reported that not all type II inhibitors possessed high kinase selectivity, 4) so this topic is...

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4− BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4− cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.