Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular tar-gets of androgens in skeletal muscle. In rodents, the skeletal (cid:1) -actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens andfocusedonandrogen-dependentup-regulationoftheskel-etal (cid:1) -actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the (cid:1) -actin promoter were used. The human skel- etal (cid:1) -actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo . Using a series of de- letion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal (cid:1) -actin promoter was demonstrated by the correlation betweenmuscleandthe (cid:1) -actinpromoteractivityforanarray of selective androgen receptor modulators (SARMs), includ- ing an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal (cid:1) -actin by andro- gens/SARMs may represent an important model system for understanding androgen androgen receptor modulator is efficacious on bone, muscle sex function with reduced impact on prostate.

Compounds in the diazo/hydrazino acid class require expression of Tpo receptor (TpoR) for activity. These compounds have demonstrated a remarkable species specificity in their Tpo receptor agonist activity, i.e., the activation of signalling pathways by this class of compounds has only been demonstrated in human and chimpanzee platelets. Platelets of other species demonstrate signalling in response to rhTpo, however, no signalling in response to SB-497115 is detectable by electrophoretic mobility shift assays using platelets of cynomolgus macaques, cat, mouse, mouse, rag, pig, ferret or tree shrew. Additionally, HepG2 cells transiently transfected with human TpoR, but not murine or cynomolgus monkey receptor results in STAT activated-reporter gene activity. To elucidate the mechanism by which this occurs, HepG2 cells were transiently transfected with a STAT-activated reporter gene and various chimeric and mutated receptors and treated with SKF-57626, a tool compound in this diazo class. A series of cyno and human TpoR chimeric receptors were constructed in which the complement receptor homology region 1 (CRH1), CRH2 and the transmembrane (TM) and cytoplasmic domains were interchanged. The functionality of all the chimeric receptors was confirmed by response to rhTpo. The minimal human composition of the chimeric TpoR activated by SKF-57626 was composed of human sequence within the membrane proximal region of the CRH2 domain and the TM domain. Two amino acids in this region are different between cyno and human, a Thr to Ala change in the extracellular CRH2 domain and a Leu at residue 499 in cynomolgus monkeys rather than His in humans in the TM domain. Sequencing of TpoR transmembrane domains of mice, dogs and ferrets, species in which these compounds are inactive, confirmed that the receptor contains Leu499, similar to cynomolgus macaques, while chimpanzee TpoR is similar to human with His at residue 499. To verify the requirement for His499 in the TM domain, a point mutation replacing only Leu499 with His in the cyno TpoR conferred activity when treated with compound. Replacement of His499 with Leu in the human TM domain resulted in an inactive human TpoR in response to compound, but not rhTpo. Further experiments involved mutations in the murine G-CSF receptor (mGCSFR). Human TpoR and mGCSFR have little homology in their TM domains and there is no detectable signalling, proliferation or differentiation responses following treatment of GCSFR expressing cells with either compound or rhTpo. A mGSFR point mutation was tested that contained a His residue nine amino acids into the hydrophobic TM domain of GCSFR, corresponding to His499 in the human and chimpanzee TpoR. HepG2 cells transiently transfected with this receptor and the reporter gene construct responded to compound. A double mutation in which an additional residue three amino acids N-terminal to the His was replaced with Thr, as in the hTpoR exhibited an increase in activity over the single mutation. These results suggest a model in which these TpoR agonist compounds interact with His499, in addition to Thr496, to either change conformation of TpoR or induce dimerization, resulting in activation of the signal transduction pathways of TpoR and imparting biologically relevant function.

Steroidal glucocorticoids are commonly used due to their powerful antiinflammatory activity. However, despite their excellent efficacy, severe side effects frequently limit the use of these drugs. The search for novel glucocorticoids with reduced side effects has been intensified by the discovery of new molecular details regarding the function of the glucocorticoid receptor. These new insights may pave the way for novel, safer therapies that retain the efficacy of currently prescribed steroids.

Drugs such as tamoxifen, which act at the estrogen receptor (ER), have very different in vitro and in vivo effects from those of the native hormone. Previous research has established that different ligands induce distinct conformational changes in the ER, thus affecting the interactions of the receptor with cell-specific coactivating or corepressing proteins (cofactors) and estrogen response elements (EREs), thus potentially driving differing biological effects. Affinity-selected peptides have been used to probe the conformational changes that occur within the ER upon binding various ligands. In this study, the authors characterize the ability of several peptides to be recruited to liganded ER under cellular conditions. Approximating ER conformation via recruitment of this peptide to the ER is concluded to be a better predictor of the agonist nature of an ER ligand under these different cellular contexts than is a canonical cotransfection transactivation assay.

SB-497115 is a selective, low molecular weight, non-peptidyl thrombopoietin receptor (TpoR) agonist. identified by its ability to activate the JAK/STAT signalling pathway. SB-497115 is being developed for the treatment of thrombocytopenias, such ass, immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. SB-497115 requires TpoR to activate the JAK/STAT signalling pathway and stimulates transcription through the STAT based (IRF-1) and megakaryocyte specific (gpIIb) promoters. An analysis of the receptor selectivity of SB-497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon -alpha or Interferon-gamma, were active. SB-497115 was inactive over a three-fold concentration range in proliferation, reporter gene, or STAT activation assays performed on cell lines that did not express TpoR. To characterize the kinetics and specificity of SB-497115 in cells, multiple molecular markers for Tpo activity were measured. Western blot analysis for activation of the STAT and MAPK pathways was performed using phospho-specific antibodies on lysates of UT7-Tpo cells treated with SB-497115. In addition, mRNA expression of several early response genes associated with proliferation and Tpo activation (i.e., Fos, EGR-1 and thyroid-like receptor 3 ), was measured in response to SB-497115 treatment. The kinetics and level of induction for pathway phosphorylation events and gene expression were similar to that seen with Tpo. A proliferative response in the human Tpo-dependent cell line, UT7-Tpo, by SB-497115 was assayed by thymidine incorporation and an EC50 of 30 nM was demonstrated. An analysis of the receptor selectivity of SB 497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon-alpha or Interferon-gamma, were active. SB 497115 demonstrated a complete lack of activity over a three-fold concentration range in proliferation, reporter gene or STAT activation assays performed on cell lines that did not express TpoR. SB-497115 was shown to be equal to or better than rhTpo in the ability to induce differentiation of normal human bone marrow progenitors (CD34) into CD41+ cells of the megakaryocyte lineage, with an EC50 of 100 nM. These latter activities of SB-497115 were similar to or greater than the level of maximal activity seen with Tpo in these assays. SB-497115 demonstrates specificity for human and chimpanzee TpoR and increases platelet counts in both chimpanzees and normal human volunteers when dosed orally. In conclusion, SB-497115 is the first non-peptide small molecule TpoR agonist to demonstrate activity in human in vitro bone marrow assays and demonstrate pharmacological activity in humans.

Thrombopoietin (TPO) is a 332 amino acid cytokine that plays a key role in the regulation of megakaryopoiesis and platelet production. Over the past 10 years, recombinant forms of TPO have shown various degrees of effectiveness in the treatment of thrombocytopenias associated with chemotherapy or various disease states, although therapy with these agents may be associated with a risk of producing neutralizing antibodies. A small-molecule nonpeptidyl TPO receptor agonist is expected to offer a safer alternative to these protein agents and also offer advantages in terms of lower cost of production and an easier oral route of administration. The mode of action of TPO involves oligomerization of its specific cell-surface receptor followed by activation of multiple signaling proteins such as the JAK1/JAK2 kinases and the STAT transcription factors. Using a high-throughput reporter-gene assay based on activation of STATs in BAF-3/TPO-R cells, screening of a library of 260,000 small-molecule compounds resulted in the identification of diazo naphthalenesulfonic acids as a novel series of TPO-R agonists. Modification of the core structure and adjustment of unwanted functionality resulted in the development of hydrazino naphthalenesulfonates which displayed efficacies equivalent to those of TPO in several cell-based assays, such as luciferase reporter gene and proliferation in a TPO receptor-dependent way (i.e., no effect on TPO-insensitive cell lines). Furthermore, these compounds elicited signal-transduction responses, such as JAK2 and STAT-5 activation, in TPO-receptor-expressing cells similar to those in TPO itself, and promoted megakaryocyte differentiation in cultures of human bone marrow cells. Potencies for the best compounds in the series were quite high for such small molecules, with EC50 values in the 10–100 nM range. However, the compounds were devoid of oral bioavailability, presumably due to the highly polar sulfonic acid functionality. Molecular modeling studies of three different series of agonists suggested a number of potential replacements of the sulfonate group, which resulted in the development of the biphenyl carboxylates, a series of compounds that showed excellent pharmacokinetic parameters, including oral bioavailability. Out of a total of over two hundred analogs, SB-497115 was selected as the candidate for clinical studies due to its optimum biological and PK properties: the compound showed full maximal efficacy of TPO both in the proliferation of BAF-3/TPO-R cells (EC50 = 30 nM) as well as in the increase of the number of CD41+ cells, a marker of megakaryocyte differentiation, in cultures of human bone marrow cells (EC50 = 100 nM). Oral bioavailabity of SB-497115 was determined to be 26%, 83% and 89%, for rat, dog and monkey, respectively. The identification of SB-497115 (MW of 442) as a TPO mimetic provides proof of principle for drug discovery using JAK/STAT based assays, and shows for the first time that a small non-peptidyl molecule can trigger the selective activation of a cytokine receptor which is confirmed by the elevation in platelet numbers in human volunteers, as demonstrated in Phase I clinical studies. Evaluation of the potential of SB-497115 as an agent for the treatment of thrombocytopenia in humans is ongoing.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

High-throughput screening has resulted in the discovery of thiosemicarbazone thrombopoietin mimics. A shared pharmacophore hypothesis between this series and a previously identified class, the pyrazol-4-ylidenehydrazines, led to the rapid optimization of both potency and efficacy of the thiosemicarbazones. The application of high-throughput chemistry and purification techniques allowed for the rapid elucidation of structure-activity relationships.

The invention of a new class of naphtho[1,2-d]imidazole thrombopoietin mimics based on a pharmacophore hypothesis for small-molecule thrombopoietic agonists is discussed. Parallel array synthesis and purification techniques allowed for the rapid exploration of structure-activity relationships within this class and for the improvement in TPO mimetic potencies and efficacies.

High-throughput screening for the induction of a luciferase reporter gene in a thrombopoietin (TPO)-responsive cell line resulted in the identification of 4-diazo-3-hydroxy-1-naphthalenesulfonic acids as TPO mimics. Modification of the core structure and adjustment of unwanted functionality resulted in the development of (5-oxo-1,5-dihydropyrazol-4-ylidene)hydrazines which exhibited efficacies equivalent to those of TPO in several cell-based assays designed to measure thrombopoietic activity. Furthermore, these compounds elicited biochemical responses in TPO-receptor-expressing cells similar to those in TPO itself, including kinase activation and protein phosphorylation. Potencies for the best compounds were high for such low molecular weight compounds (MW < 500) with EC(50) values in the region of 1-20 nM.

Objective. Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line.Methods. STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro.Results. Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey.Conclusions. SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo. (C) 2005 International Society for Experimental Hematology. Published by Elsevier Inc.

Regulation of cytokine activity has application in the treatment of numerous diseases, as illustrated by the successful clinical use of recombinant cytokines. The discovery of two key families of signaling - proteins the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs)-has greatly increased our understanding of cytokine signal transduction. This review describes some of the key interactions between JAKs. STATs and other components of the signal transduction cascade, and the potential for developing high-throughput biochemical screens for the detection of small molecules that target these interactions. Novel compounds of this type have potential as agonists or antagonists of cytokine action.

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

The mouse mutation viable motheaten (me(v)) results in defects in the expression and catalytic activity of the cytoplasmic protein tyrosine phosphatase known as hematopoietic cell phosphatase (HCP). This reduction in HCP activity leads to the aberrant regulation of several myeloid and lymphoid cell lineages, including substantial increases in numbers of granulocytes. The differentiation, proliferation, and survival of cells in this lineage are normally supported by granulocyte-colony stimulating factor (G-CSF). In this study we have determined the consequences of the loss of HCP activity in me(v)/me(v) mice on the response of bone marrow cells to G-CSF. Bone marrow from these mice exhibited substantial increases in clonogenic and proliferative responses to G-CSF. These enhanced activities of G-CSF correlated with an increase in the level of immature granulocytic, G-CSF receptor positive cells in the bone marrow. These results suggested the possibility that HCP may regulate the G-CSF receptor by a direct interaction. However, under conditions where the previously described interaction between the erythropoietin receptor and HCP was readily observed, HCP did not detectably associate with the G-CSF receptor.

Cytokines and non-peptidyl small molecules, such as steroid hormones, exert many of their effects on cells through rapid regulation of gene expression. This is achieved by the activation of different families of latent transcription factors, which bind to specific sequences in the promoters of regulated genes. High throughput assay systems have been developed based on a detailed molecular understanding of these transcriptional regulation processes, and are being used as screens for both agonists and antagonists of specific cytokines and hormones. The opportunities for the discovery of novel and selective compounds using these systems is discussed.

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