Phosphodiesterase 4 (PDE4), the primary cAMP-hydrolyzing enzyme in cells, is a promising drug target for a wide range of conditions. Here we present seven co-crystal structures of PDE4 and bound inhibitors that show the regulatory domain closed across the active site, thereby revealing the structural basis of PDE4 regulation. This structural insight, together with supporting mutagenesis and kinetic studies, allowed us to design small-molecule allosteric modulators of PDE4D that do not completely inhibit enzymatic activity (I(max) approximately 80-90%). These allosteric modulators have reduced potential to cause emesis, a dose-limiting side effect of existing active site-directed PDE4 inhibitors, while maintaining biological activity in cellular and in vivo models. Our results may facilitate the design of CNS therapeutics modulating cAMP signaling for the treatment of Alzheimer's disease, Huntington's disease, schizophrenia and depression, where brain distribution is desired for therapeutic benefit.

Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene, SMN2 that produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by two-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-quinazoline probe, we identified the scavenger decapping enzyme, DcpS as a potential binder. We show that the C5-quinazolines potently inhibit DcpS decapping activity, and that the potency of inhibition correlates with potency for SMN2 promoter induction. Binding of C5-quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m7GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.

No abstract

Enforced expression of IntroductionHematopoiesis relies on the unique abilities of relatively few hematopoietic stem cells to self-renew and generate progenitors that will differentiate into the mature cells forming the blood system. This dynamic process is tightly regulated by a complex of internal and external signals, such as transcription factors, growth factors, and cell cycle regulators (for reviews, see Orkin 1 and Verfaillie 2 ). Many transcription factors, including homeobox (Hox) transcription factors, have been shown to be key players in the proliferation and differentiation of early progenitor cells. 3,[4][5][6] Specific expression patterns of multiple Hox genes have been detected in normal and leukemic hematopoiesis. 7,8 Enforced expression of Hox genes has been shown to affect the ability of progenitors and stem cells to proliferate and differentiate. [9][10][11][12][13][14][15][16][17] One of these genes, Hoxb4, has been implicated in the regulation of hematopoietic stem cell regeneration, 8 and retrovirally engineered overexpression in murine bone marrow cells dramatically increases the stem cell pool ex vivo and in vivo, resulting in faster, more complete recovery of the stem cells in transplantation studies with no adverse effect on differentiation or lineage distribution. 14,[18][19][20][21] This is in contrast to the overexpression of other Hox genes, which can perturb the proliferation and lineage commitment of primitive progenitors and can give rise to hematopoietic malignancies. 10,11,13,15,16,[22][23][24] However, recent studies have suggested that the effect of Hoxb4 is concentration dependent and is not necessarily restricted to proliferation. [25][26][27] Thus, the level of Hoxb4 expression has to be within a specific range for Hoxb4 to increase stem cell proliferation without adverse effects on differentiation. Although enforced expression of Hoxb4 in hematopoietic cells has been studied in detail, its physiologic role in hematopoiesis is poorly understood. Recently, we described a mouse model deficient in Hoxb3 and Hoxb4, showing reduced proliferative capacity of the stem cell pool without otherwise perturbing hematopoiesis. 28 Here we report a novel mouse model in which the Hoxb4 gene alone has been completely removed through the Cre/loxP technique. Hoxb4-deficient mice have a phenotype similar to that of double Hoxb3/Hoxb4 knockout (KO) mice, although the effects are milder in the Hoxb4 Ϫ/Ϫ mice. The phenotype observed seems mainly confined to the stem cell pool, resulting in reduced proliferative capacity of bone marrow and fetal liver hematopoietic stem cells (HSCs) without affecting differentiation or lineage choice. Deficiency of Hoxb4 or Hoxb3 and Hoxb4 affects the expression of other Hox genes and the expression of cell cycle regulators, indicating a complex regulatory role of these Hox genes. Collectively, these findings indicate that Hoxb4 improves proliferative recruitment of HSCs in settings demanding high proliferation, such as transplantation, but that it has a less pro...

The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hemato-poietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dick-kopf-1 [Dkk-1], growth factor independent -1 [Gfi-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfi-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development. (Blood. 2007;109:3687-3696)