In addition to being an important receptor in leukocyte activation and mobilization, CCR5 is the essential coreceptor for human immunodeficiency virus (HIV). A large number of smallmolecule CCR5 antagonists have been reported that show potent activities in blocking chemokine function and HIV entry. To facilitate the design and development of next generation CCR5 antagonists, docking models for major classes of CCR5 antagonists were created by using site-directed mutagenesis and CCR5 homology modeling. Five clinical candidates: maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220 were used to establish the nature of the binding pocket in CCR5. Although the five antagonists are very different in structure, shape, and electrostatic potential, they were able to fit in the same binding pocket formed by the transmembrane (TM) domains of CCR5. It is noteworthy that each antagonist displayed a unique interaction profile with amino acids lining the pocket. Except for TAK-779, all antagonists showed strong interaction with Glu283 in TM 7 via their central basic nitrogen. The fully mapped binding pocket of CCR5 is being used for structure-based design and lead optimization of novel anti-HIV CCR5 inhibitors with improved potency and better resistance profile.Human immunodeficiency virus (HIV) enters the host cell via the interaction of the viral envelope protein gp160 and the receptor/coreceptors on host cell surface. The majority of primary HIV-1 strains use CCR5 as coreceptor (termed R5 virus), whereas some viruses are able to use another chemokine receptor, CXCR4, as coreceptor (termed X4 virus) or use both CCR5 and CXCR4 as coreceptors (termed R5X4 virus). Because CCR5 is the predominant coreceptor for clinical HIV isolates, and the normal physiology within the human genetic knockout population, CCR5 has become a very attractive target for anti-HIV therapy. A number of small molecule CCR5 antagonists have been identified that demonstrated potent antiviral effects both in cell culture and in clinical trials.TAK-779, a quaternary ammonium anilide, was the first small molecule CCR5 antagonist reported (Baba et al., 1999). This compound was terminated as a result of poor oral availability. Two structurally diverse followers TAK-220 and TAK-652 are both in clinical trials (Imamura et al., 2006;Seto et al., 2006). Several other small molecule CCR5 antagonists with good potency and/or pharmacological properties have also been reported by other pharmaceutical companies. These include SCH-C (SCH-351125), vicriviroc (VVC, SCH-D, SCH-417690), aplaviroc (APL, AK602, GW873140), and maraviroc (MVC, UK-427,857). SCH-C is an oximino-piperidino-piperidine amide (Palani et al., 2002) that showed potent antiviral activity in vivo. However, its clinical development was terminated as a result of HERG inhibitory activity. SCH-D is the next generation compound of SCH-C, which is in late stage clinical development. SCH-D showed better oral availability, potency, safety, and pharmacological properties than SCH-C Article, publication d...

Glucocorticoid in excess suppresses bone formation in vivo and disrupts bone matrix protein synthesis by osteoblasts in vitro. In contrast, transforming growth factor ␤ (TGF-␤) potently enhances bone matrix apposition. The rat TGF-␤ type I receptor gene promoter contains cis-acting elements for transcription factor CBFa1, which increases in parallel with osteoblast differentiation. Here we present molecular data linking these events. We show that previously unexplained effects of glucocorticoid on bone loss may be mediated in part by suppression of CBFa1, with a resultant decrease in the expression and activity of the TGF-␤ type I receptor on matrix-producing bone cells.Glucocorticoid-dependent bone loss by disregulated hormone expression or pharmacologic excess causes clinically significant osteoporosis in approximately 50% of affected individuals. Although changes in calcium absorption and effects on nonskeletal tissues contribute to the disease, striking effects occur directly on osteoblasts and at sites of active skeletal matrix deposition and remodeling (1, 2). A chronic reduction in osteoblast activity without corresponding changes in resorption would uncouple normal bone remodeling and decrease skeletal durability. Important genes targeted by glucocorticoid and molecular mediators for these events remain uncertain.Transforming growth factor ␤ (TGF-␤) 1 enhances bone matrix synthesis and repair, and bone contains perhaps the largest store of TGF-␤ in the body (reviewed in Ref. 3). Bone cells exhibit conventional type II and type III TGF-␤ receptors (TRII and TRIII) that influence TGF-␤ binding to type I receptor (TRI) or its activation, both essential for TGF-␤-dependent events (4 -6). There are few systems where regulation of TRI expression has been examined in detail and where functional changes correlate with these variations. We found TRI levels specifically maintained on differentiated bone cells in vitro, despite decreases in TRII and TRIII in response to bone morphogenetic protein 2 (5). In contrast, high levels of glucocorticoid rapidly reduce the proportion of TGF-␤ binding to TRI on bone cells and correspondingly decrease TGF-␤ activity (6).To understand these events further, we cloned the rat TRI promoter and observed higher promoter activity in osteoblastlike cells compared with undifferentiated bone cells or dermal fibroblasts (7). The TRI promoter includes a CpG island, several transcription factor Sp1 binding sites consistent with constitutive expression by many cells, and six cis-acting elements for transcription factors, termed CBFa (7-8).2 Whereas CBFa2 and CBFa3 are important gene regulators in lymphoid cells (9), CBFa1 expression increases in parallel with osteoblast differentiation in vitro (10).2 Moreover, targeted disruption of the CBFa1 gene eliminates osteogenesis in mice, and insertion, deletion, or missense mutations in CBFa1 occur in humans with the skeletal disorder cleidocranial dysplasia (11). Genes directly affected by CBFa1, especially those important for skeletal developme...

Transcription factor CCAAT/enhancer-binding protein ␦ (C/EBP␦) is normally associated with acute-phase gene expression. However, it is expressed constitutively in primary osteoblast cultures where it increases insulin-like growth factor I synthesis in a cAMP-dependent way. Here we show that the 3 proximal region of the C/EBP␦ gene promoter contains a binding sequence for Runt domain factor Runx2, which is essential for osteogenesis. This region of the C/EBP␦ promoter directed high reporter gene expression in osteoblasts, and specifically bound Runx2 in osteoblast-derived nuclear extract. C/EBP␦ gene promoter activity was reduced by mutating the Runx binding sequence or by co-transfecting with Runx2 antisense expression plasmid, and was enhanced by overexpression of Runx-2. Exposure to prostaglandin E 2 increased Runx-dependent gene transactivation independently of Runx2 binding to DNA. Runx2 bound directly to the carboxyl-terminal region of C/EBP␦ itself, and its ability to drive C/EBP␦ expression was suppressed when C/EBP␦ or its carboxyl-terminal fragment was increased by overexpression. Consistent effects also occurred on C/EBP␦-dependent increases in gene expression driven by synthetic or insulin-like growth factor I gene promoter fragments. These interactions between Runx2 and C/EBP␦, and their activation by prostaglandin E 2 , provide new evidence for their importance during skeletal remodeling, inflammatory bone disease, or fracture repair.

Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E 2 (PGE 2 ) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) ␦ is a major component of a PGE 2 -stimulated DNA-protein complex involving HS3D and find that C/EBP␦ transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a ϳ 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBP␦ comprised most of the PGE 2 -activated gel-shifted complex. C/EBP␦ was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE 2 . An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Cotransfection of osteoblast cell cultures with a C/EBP␦ expression plasmid enhanced basal and PGE 2 -activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBP␤, did not alter basal IGF-I gene activity but did increase the response to PGE 2 . In osteoblasts and in COS-7 cells, C/EBP␦, but not C/EBP␤, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast nuclear proteins. These results identify C/EBP␦ as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a high affinity binding site for this protein.

Maximal gene expression driven by the promoter for the transforming growth factor ␤ type I receptor (TGF-␤RI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBF␣ binding sites among other possible cis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBF␣-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-␤RI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-␤RI levels that parallel changes in bone cell differentiation or activity.Transforming growth factor-␤ (TGF-␤) 1 receptors occur on most cells, and a functional TGF-␤ type I receptor (TGF-␤RI) is required for all known TGF-␤-dependent effects. In some situations its activity is controlled by complex interactions with other cell surface components (1-3). However, in contrast to TGF-␤RII and the cell surface proteoglycan also termed TGF-␤RIII or betaglycan, expression of TGF-␤RI is maintained on differentiated bone cells (4). For these reasons, and because little is known about the molecular control of TGF-␤RI expression, we cloned the rat TGF-␤RI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats. The rat TGF-␤RI promoter lacks a typical TATA box, but initiates transcription at multiple sites within a 220-bp span upstream of the initial methionine codon in differentiated bone cells. The 3Ј-terminal 300-bp sequence encompassing this region contains a GC-rich CpG island, seven consensus Sp1 binding sites, and two CCAAT boxes. Transfection studies using different fragments of TGF-␤RI promoter cloned upstream of the reporter gene luciferase demonstrated maximal activity by a 1.0-kb fragment that encompassed these and other possible cis-acting elements. Importantly, several dispersed elements appeared to cooperate for maximal reporter gene expression in osteoblast-enriched cultures (5). Coincident with this work, the human TGF-␤RI promoter was cloned, and its sequence reveals a similar organization with identically spaced CCAAT box motifs (6).These features suggested that the TGF-␤RI gene is driven by a constitutively active promoter that maintains expression of TGF-␤RI in many cells. Nevertheless, this promoter is part...

Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E 2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein ␦ (C/EBP␦) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBP␦ and HS3D. C/EBP␦, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBP␦. C/EBP␦ bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBP␦ also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBP␦ with similar affinity. We also found that prostaglandin E 2 -induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBP␦ expression plasmid. Taken together, our results provide evidence that C/EBP␦ is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.

Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.

The proportion of transforming growth factor-␤ (TGF-␤) binding among conventional membrane receptors on bone cells can vary with hormone or growth factor treatment or with the state of osteoblast-like activity and appears to determine the nature of its biological effects. Therefore, functional TGF-␤ receptor stability could be an important aspect of regulation. Suppression of protein synthesis reduced TGF-␤ binding to types I and II receptors with t 1/2 of 2 h and to betaglycan with t 1/2 of 6 h. In contrast, suppression of mRNA transcription reduced TGF-␤ binding at least 3-fold more slowly at each receptor site. Preexposure to TGF-␤ decreased its binding at all three sites within 4 h in osteoblast-enriched cultures. This effect was transient with lower TGF-␤ concentrations, where the receptor profile was nearly fully restored within 24 -48 h. In contrast, less differentiated bone cells were less sensitive to ligand-dependent receptor down-regulation. Agents that alter protein kinase and phosphatase activity also modified the TGF-␤ binding profile in specific ways. Together, these results indicate that cell surface TGF-␤ receptors turn over rapidly by ligand-independent and ligand-dependent mechanisms, demonstrate that the binding capacity of TGF-␤ receptors is less stable than their mRNAs, and that functional receptor levels may be determined in part by post-transcriptional events.Transforming growth factor type ␤s (TGF-␤s) 1 are potent, ubiquitous growth regulators that are abundant in skeletal tissue. Their expression is controlled in temporal and spatial ways during skeletal tissue development, and they produce complex stimulatory and inhibitory changes in osteoblast function in vitro. The presence of TGF-␤s in bone and their importance as local skeletal growth regulators were first described in fetal rat calvariae, and cells derived from various bone tissues have provided much information about TGF-␤ activity and receptor expression during osteogenesis (reviewed in Ref. 1).Similar to cells found in many tissues, fetal rat bone cells express type I (53 kDa) and type II (73 kDa) glycoprotein receptors, as well as type III proteoglycans (Ͼ250 betaglycans) that could directly or indirectly influence TGF-␤ binding to the signaling types I and II sites (2-5). In this model, the association of TGF-␤ with each receptor varies with the basal state of bone cell differentiation, and relative mRNA abundance levels generally reflect the cell surface binding profiles found in untreated cells (5). Rapid changes in the proportion of TGF-␤ binding among receptors, induced by certain growth factors and hormones, are consistent with specific changes in its biological effects (5-8). For example, glucocorticoid decreases TGF-␤ binding to types I and II receptors, increases its binding to type III sites, and reduces its overall effectiveness (6). In contrast, bone morphogenetic protein (BMP)-2 reduces TGF-␤ binding to type II receptors and betaglycan, whereas it enhances binding to type I receptors. After BMP-2 treatment, the m...