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.

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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)

Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin ؊ ScaI ؉ c-kit ؉ hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.Class I Homeobox (Hox) genes encode a family of 39 transcription factors sharing a highly conserved DNA-binding domain. In mammals they play a major role in specifying position and tissue fate in the embryo, as has been demonstrated by several lack-of-function Hox gene mutants that exhibit various developmental abnormalities (see, for example, references 6, 29, 30, 38, 41, 49, and 58). Hox genes are also expressed postnatally, and several of them are expressed in primitive hematopoietic cells and committed progenitors but downregulated upon differentiation to mature cells (44).Murine models have been generated where enforced expression of Hox genes is used to determine the effect of overexpression on self-renewal, differentiation, and other cell fate decisions during hematopoiesis (for reviews, see references 10 and 55). Such models include overexpression of HOXA10, as well as HOXA9, which both affected myelo-and lymphopoiesis and ultimately lead to myeloid leukemia (5, 9, 23, 52, 53). Expression of HOXB3 and HOXB4 is found in the primitive CD34 ϩ population that is highly enriched for human hematopoietic stem cells (HSCs) but is rapidly downregulated as the cells differentiate into committed progenitors (44). Despite very similar expression pattern of HOXB3 and HOXB4, suggesting a common role or collaboration between these factors, the consequences from overexpressing these genes are very different. Although enforced expression of HOXB3 blocks both T-and B-cell development and causes a myeloproliferative disorder (46), overexpression of HOXB4 greatly...

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by death of motor neurons in the spinal cord that is caused by deletion and/or mutation of the survival motor neuron gene ( SMN1). Adjacent to SMN1 are a variable number of copies of the SMN2 gene. The two genes essentially differ by a single nucleotide, which causes the majority of the RNA transcripts from SMN2 to lack exon 7. Although both SMN1 and SMN2 encode the same Smn protein amino acid sequence, the loss of SMN1 and incorrect splicing of SMN2 have the consequence that Smn protein levels are insufficient for the survival of motor neurons. The therapeutic goal of our medicinal chemistry effort was to identify small-molecule activators of the SMN2 promoter that, by up-regulating gene transcription, would produce greater quantities of full-length Smn protein. Our initial medicinal chemistry effort explored a series of C5 substituted benzyl ether based 2,4-diaminoquinazoline derivatives that were found to be potent activators of the SMN2 promoter; however, inhibition of DHFR was shown to be an off-target activity that was linked to ATP depletion. We used a structure-guided approach to overcome DHFR inhibition while retaining SMN2 promoter activation. A lead compound 11a was identified as having high potency (EC50 = 4 nM) and 2.3-fold induction of the SMN2 promoter. Compound 11a possessed desirable pharmaceutical properties, including excellent brain exposure and long brain half-life following oral dosing to mice. The piperidine compound 11a up-regulated expression of the mouse SMN gene in NSC-34 cells, a mouse motor neuron hybrid cell line. In type 1 SMA patient fibroblasts, compound 11a induced Smn in a dose-dependent manner when analyzed by immunoblotting and increased the number of intranuclear particles called gems. The compound restored gems numbers in type I SMA patient fibroblasts to levels near unaffected genetic carriers of SMA.

Myocardial infarction and stroke are caused by blood clots forming over a ruptured or denuded atherosclerotic plaque (atherothrombosis). Production of prostaglandin E(2) (PGE(2)) by an inflamed plaque exacerbates atherothrombosis and may limit the effectiveness of current therapeutics. Platelets express multiple G-protein coupled receptors, including receptors for ADP and PGE(2). ADP can mobilize Ca(2+) and through the P(2)Y(12) receptor can inhibit cAMP production, causing platelet activation and aggregation. Clopidogrel (Plavix), a selective P(2)Y(12) antagonist, prevents platelets from clotting but thereby increases the risk of severe or fatal bleeding. The platelet EP(3) receptor for PGE(2), like the P(2)Y(12) receptor, also inhibits cAMP synthesis. However, unlike ADP, facilitation of platelet aggregation via the PGE(2)/EP(3) pathway is dependent on co-agonists that can mobilize Ca(2+). We used a ligand-based design strategy to develop peri-substituted bicylic acylsulfonamides as potent and selective EP(3) antagonists. We show that DG-041, a selective EP(3) antagonist, inhibits PGE(2) facilitation of platelet aggregation in vitro and ex vivo. PGE(2) can resensitize platelets to agonist even when the P(2)Y(12) receptor has been blocked by clopidogrel, and this can be inhibited by DG-041. Unlike clopidogrel, DG-041 does not affect bleeding time in rats, nor is bleeding time further increased when DG-041 is co-administered with clopidogrel. This indicates that EP(3) antagonists potentially have a superior safety profile compared to P(2)Y(12) antagonists and represent a novel class of antiplatelet agents.