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Biotech / Medical : Ligand (LGND) Breakout! -- Ignore unavailable to you. Want to Upgrade?

To: WTDEC who wrote (16670)	3/6/1998 10:47:00 PM
From: Henry Niman	Read Replies (1) \| Respond to of 32384

Here's a broad STATs patent issued last month (to Rockefeller - which LGND has rights to): United States Patent 5,716,622 Darnell, Jr., et. al. Feb. 10, 1998 Functionally active regions of signal transducer and activators of transcription Inventors: Darnell, Jr.; James E. (Larchmont, NY); Wen; Zilong (New York, NY); Horvath; Curt M. (New York, NY); Zhong; Zhong (New York, NY). Assignee: The Rockefeller University (New York, NY). Appl. No.: 369,796 Filed: Jan. 6, 1995 Intl. Cl. : A61K 39/385, C07K 14/715, C07K 17/02 Current U.S. Cl.: 424/185.1; 424/193.1; 530/350; 530/403 Field of Search: 530/350, 403; 424/185.1, 193.1 References Cited \| [Referenced By] Foreign Patent Documents WO 93/19179 Sept., 1993 WO Other References Darnell et al., 1994, Science 264:1415-1421. Improta et al., 1994, Proc. Natl. Acad. Sci. USA 91:4776-80. Shuai et al., 1994, Cell 76:821-28. Zhong et al., 1994, Proc. Natl. Acad. Sci. USA 91:4806-4810. Zhong et al., 1994, Science 264:95-98. Eck et al., 1993, Nature 362:87-91. Felder et al., 1993, Mol. Cell. Biol. 13:1449-55. Khan et al., 1993, Proc. Natl. Acad. Sci. USA 90:6806-10. Muller et al., 1993, EMBO J. 12:4221-28. Muller et al., 1993, Nature 366:129-35. Pearse et al., 1993, Proc. Natl. Acad. Sci. 90:4314-18. Sadowski et al., 1993, Nature 362:79-83. Sadowski et al., 1993, Science 261:1739-44. Shuai et al., 1993, Nature 366:580-83. Shuai et al., 1993, Science 261:1744-46. Songyang et al., 1993, Cell 72:767-78. Watling et al., 1993, Nature 366:166-70. Wegenka et al., 1993, Mol. Cell. Biol. 13:276-88. Schindler et al., 1992, Proc. Natl. Acad. Sci. USA 89:7836-39. Schindler et al., 1992, Science 257:809-13. Decker et al., 1991, EMBO J. 10:927-32. Lew et al., 1991, Mol. Cell. Biol. 11:182-91. Fu et al., 1990, Proc. Natl. Acad. Sci. USA 87:8555-59. Wagner et al., 1990, EMBO J. 9:4477-84. Primary Examiner: Chan; Christina Assistant Examiner: VanderVegt; F. Pierre Attorney, Agent or Firm: Klauber & Jackson Abstract The present invention relates generally to intracellular receptor recognition proteins or factors, termed Signal Transducers and Activators of Transcription (STAT), to methods and compositions utilizing such factors, and to the antibodies reactive toward them, in assays and for diagnosing, preventing and/or treating cellular debilitation, derangement or dysfunction. More particularly, the present invention relates to particular functional domains of molecules that exhibit both receptor recognition and message delivery via DNA binding in receptor-ligand specific manner, i.e., that directly participate both in the interaction with the ligand-bound receptor at the cell surface and in the activity of transcription in the nucleus as a DNA binding protein. The invention likewise relates to the antibodies and other entities that are specific to the functional domain of a STAT protein and that would thereby selectively modulate its activity. 16 Claims, 31 Drawing Figures The research leading to the present invention was supported by National Institute of Health Grant Nos. AI34420 and AI32489. Accordingly, the Government may have certain rights in the invention.

To: WTDEC who wrote (16670)	3/7/1998 9:30:00 AM
From: Henry Niman	Read Replies (1) \| Respond to of 32384

wtd, I have not been able to get a publication date on the Science paper yet. LGND's STAT program is designed to identify small orally available molecules that will mimic the signal initiated by various polypeptide hormones that use STAT pathways. LGND exclusively licensed the technology in 1992, when it was very young (was called RAFTs back then), and I would expect a series of extremely broad patents to be issued.. Here's the 1992 press release (oral delivery was indicated back then): LIGAND PHARMACEUTICALS LICENSES GENETIC TECHNOLOGY FROM ROCKEFELLER UNIVERSITY SAN DIEGO (OCT. 8) BUSINESS WIRE - Ligand Pharmaceuticals has licensed a novel transcription factor technology from Rockefeller University which the company believes will be important in its research efforts to discover drugs to control cellular gene expression, according to David E. Robinson, Ligand's president and chief executive officer. The agreement gives Ligand exclusive worldwide rights to any patents and other proprietary rights covering the technology discovered by James Darnell, M.D., at Rockefeller University, and his colleague David Levy, Ph.D., now at New York University. Darnell, a member of the National Academy of Science, has been a key figure in explaining how cells control gene expression. He serves as a member of Ligand's scientific advisory board and as an exclusive consultant to the company. Darnell and Levy have discovered, cloned and characterized the first members of a novel transcription factor superfamily. These related proteins transmit signals from interferon receptors on the cell surface to the cell nucleus, bringing about specific changes in the pattern of gene activity in the cell and therefore in how the cell functions. These new receptor-activated factors of transcription (RAFTs) were recently described in Science (Volume 257, Aug. 7, 1992). The licensed technology affords the potential to develop small organic molecules which can be administered orally to mimic or block the actions of the polypeptides interferon-alpha and interferon-gamma. Possible therapeutic applications for such drugs include cancer, inflammation and various infectious diseases. Robert B. Stein, M.D., Ph.D., Ligand's vice president of research and preclinical development, said Darnell and Levy unraveled a central problem in biology -- explaining for the first time how interferons make cells resistant to viral infection and control cell growth. Other polypeptide bioregulatory molecules, including cytokines, lymphokines, interleukins and various growth factors may also control gene expression through related members of the RAFT superfamily, he said. Ligand's current drug discovery efforts focus on intracellular receptors (IRs), a superfamily of transcription factors activated by small organic hormones such as sex steroids, glucocorticoids and retinoids. "The work of Drs. Darnell and Levy is widely acclaimed to be a scientific tour de force," Stein said. "The RAFT technology is highly complementary to our core technology from both a technical and biological perspective. The IR and RAFT technologies provide Ligand significant, novel

To: WTDEC who wrote (16670)	3/7/1998 9:38:00 AM
From: Henry Niman	Respond to of 32384

Here's the abstract for the 1992 Science paper: Science 1992 Aug 7;257(5071):809-813 Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Schindler C, Shuai K, Prezioso VR, Darnell JE Jr Laboratory of Molecular Cell Biology, Rockefeller University, New York, NY 10021. The interferon-alpha (IFN-alpha)-stimulated gene factor 3 (ISGF3), a transcriptional activator, contains three proteins, termed ISGF3 alpha proteins, that reside in the cell cytoplasm until they are activated in response to IFN-alpha. Treatment of cells with IFN-alpha caused these three proteins to be phosphorylated on tyrosine and to translocate to the cell nucleus where they stimulate transcription through binding to IFN-alpha-stimulated response elements in DNA. IFN-gamma, which activates transcription through a different receptor and different DNA binding sites, also caused tyrosine phosphorylation of one of these proteins. The ISGF3 alpha proteins may be substrates for one or more kinases activated by ligand binding to the cell surface and may link occupation of a specific polypeptide receptor with activation of transcription of a set of specific genes. Comments: Comment in: Science 1992 Aug 7;257(5071):744-5 PMID: 1496401, UI: 92358244

To: WTDEC who wrote (16670)	3/7/1998 9:41:00 AM
From: Henry Niman	Respond to of 32384

Here's a Nature paper on JAKs: Nature 1993 Dec 9;366(6455):583-585 Interferon-induced nuclear signalling by Jak protein tyrosine kinases. Silvennoinen O, Ihle JN, Schlessinger J, Levy DE Kaplan Comprehensive Cancer Center, New York, New York. Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling. PMID: 7504785, UI: 94077184

To: WTDEC who wrote (16670)	3/7/1998 9:42:00 AM
From: Henry Niman	Respond to of 32384

Here's a JAK/STAT review: Science 1994 Jun 3;264(5164):1415-1421 Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Darnell JE Jr, Kerr IM, Stark GR Laboratory of Molecular Cell Biology, Rockefeller University, New York, NY 10021. Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction. Publication Types: Review Review, tutorial PMID: 8197455, UI: 94255764

To: WTDEC who wrote (16670)	3/7/1998 9:45:00 AM
From: Henry Niman	Read Replies (1) \| Respond to of 32384

Here's a 1993 Nature paper with some of the molecular details: Nature 1993 Dec 9;366(6455):580-583 Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins. Shual K, Ziemiecki A, Wilks AF, Harpur AG, Sadowski HB, Gilman MZ, Darnell JE Laboratory of Molecular Cell Biology, Rockefeller University, New York 10021-6399. Binding of interferons IFN-alpha and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). Interferon-alpha activates both Stat91 (M(r) 91,000; ref. 1) and Stat113 (M(r) 113,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs 3, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-alpha and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-alpha response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs alpha and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-alpha and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91. PMID: 7504784, UI: 94077183

To: WTDEC who wrote (16670)	3/7/1998 9:48:00 AM
From: Henry Niman	Read Replies (1) \| Respond to of 32384

Here's a more recent review: Science 1997 Sep 12;277(5332):1630-1635 STATs and gene regulation. Darnell JE Jr Laboratory of Molecular Cell Biology, Rockefeller University, New York, NY 10021, USA. STATs (signal transducers and activators of transcription) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Biochemical and molecular genetic explorations have defined a single tyrosine phosphorylation site and, in a dimeric partner molecule, an Src homology 2 (SH2) phosphotyrosine-binding domain, a DNA interaction domain, and a number of protein-protein interaction domains (with receptors, other transcription factors, the transcription machinery, and perhaps a tyrosine phosphatase). Mouse genetics experiments have defined crucial roles for each known mammalian STAT. The discovery of a STAT in Drosophila, and most recently in Dictyostelium discoideum, implies an ancient evolutionary origin for this dual-function set of proteins. Publication Types: Review Review, tutorial PMID: 9287210, UI: 97433375

To: WTDEC who wrote (16670)	3/7/1998 10:02:00 AM
From: Henry Niman	Respond to of 32384

Here's a recent review by Ihle (another Ligand consultant) that specifically mentions EPO: Stem Cells 1997;15 Suppl 1:105-111 Jaks and Stats in cytokine signaling. Ihle JN, Nosaka T, Thierfelder W, Quelle FW, Shimoda K Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. Hematopoiesis is regulated through the binding of cytokines to receptors of the cytokine receptor superfamily. Although lacking catalytic domains, members of the cytokine receptor superfamily mediate ligand-dependent activation of protein tyrosine phosphorylation through their association and activation of members of the Janus kinase (Jak) family of protein tyrosine kinases. The activated Jaks phosphorylate the receptors which creates docking sites for SH2-containing signaling proteins which are tyrosine phosphorylated following their association with the complex. Among the substrates of tyrosine phosphorylation are members of the signal transducers and activators of the transcription family of proteins (Stats). Various cytokines induce the tyrosine phosphorylation and activation of one or more of the seven family members. The pattern of Stat activation provides a level of cytokine individuality that is not observed in the activation of other signaling pathways. The role of various Stats in the biological responses to cytokines has been assessed through the analysis of receptor mutations which disrupt Stat activation and more recently by disruption of the genes in mice. Our results have demonstrated that the activation of Stat5a and Stat5b by erythropoietin is critical for the activation of a number of immediate early genes but is not required for a mitogenic response. Mice in which the genes for Stat4 and Stat6 are disrupted are viable but lack functions that are mediated by interleukin 12 (IL-12) or IL-4, respectively, suggesting that these Stats perform very specific functions in immune responses. Publication Types: Review Review, tutorial PMID: 9368330, UI: 98034700

To: WTDEC who wrote (16670)	3/7/1998 10:05:00 AM
From: Henry Niman	Respond to of 32384

Here's a Ihle paper that describes STATs involved with G-CSF signaling: Blood 1997 Jul 15;90(2):597-604 Jak1 plays an essential role for receptor phosphorylation and Stat activation in response to granulocyte colony-stimulating factor. Shimoda K, Feng J, Murakami H, Nagata S, Watling D, Rogers NC, Stark GR, Kerr IM, Ihle JN Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN 38105, USA. The proliferation and differentiation of neutrophils is regulated by granulocyte-specific colony-stimulating factor (G-CSF). G-CSF uses a receptor of the cytokine receptor superfamily and, in common with all members of the family, induces the tyrosine phosphorylation and activation of members of the Janus protein tyrosine kinase (Jak) family. In both myeloid cells and a human fibrosarcoma cell line expressing the G-CSF receptor, G-CSF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. In addition, G-CSF induces the tyrosine phosphorylation of the receptor and members of the signal transducers and activators of transcription (Stat) family, including Stat3, as well as Stat1 and Stat5, depending on the cells involved. Using mutant cell lines lacking various Jaks, we show here that Jak1 is critical for G-CSF-mediated Stat activation, whereas Jak2 or Tyk2 are either not required or play redundant or ancillary roles. In the absence of Jak1, G-CSF induces activation of Jak2 and Tyk2, but fails to induce receptor tyrosine phosphorylation and induces dramatically reduced levels of Stat activation. A kinase-inactive Jak2, when overexpressed in cells lacking endogenous Jak2, can suppress Jak1 activation, receptor phosphorylation, and Stat activation, suggesting competition in the receptor complex either for Jak1 binding or substrates. Because the requirement for Jak1 is very similar to that previously shown for interleukin-6 signaling, the data support the concept that the G-CSF receptor and gp130 are both structurally and functionally similar. PMID: 9226159, UI: 97369718

To: WTDEC who wrote (16670)	3/7/1998 10:29:00 AM
From: Henry Niman	Respond to of 32384

Here's an abstract defining some of the FAT STATs targeted by LGND's leptin signaling program: Proc Natl Acad Sci U S A 1996 Jun 25;93(13):6231-6235 Defective STAT signaling by the leptin receptor in diabetic mice. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC Department of Pharmacology, Biozentrum, University of Basel, Switzerland. Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin. PMID: 8692797, UI: 96270520

To: WTDEC who wrote (16670)	3/7/1998 10:33:00 AM
From: Henry Niman	Respond to of 32384

Here's a report on leptin levels and TZDs: Proc Natl Acad Sci U S A 1996 Jun 11;93(12):5793-5796 Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes. Kallen CB, Lazar MA Division of Endocrinology, Diabetes, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA. Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma. PMID: 8650171, UI: 96234042